2_test

PMC:6120343 / 25193-25195 JSON TXT

Structural Basis of Mitochondrial Receptor Binding and Constriction by DRP1 Abstract Summary Mitochondrial inheritance, genome maintenance, and metabolic adaptation depend on organelle fission by Dynamin-Related Protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogs Mitochondrial Dynamics 49 and 51 (MID49/MID51) and Mitochondrial Fission Factor (MFF), but the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryoEM structure of human, full-length DRP1 coassembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a remarkable elongation and rotation of the G-domain, Bundle-Signaling Element (BSE) and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes polymerization of a linear DRP1 filament with MID49/MID51. Following coassembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery. Introduction Fragmentation of the mitochondrial reticulum disperses units of the organelle during cell division1,2, coordinates morphological adaptation with metabolic demand3,4, and quarantines damaged units for turnover5,6. Recent work also led to the discovery of the role mitochondrial fission plays in regulated cell death pathways7–9, brain development and synaptic function10,11, and how certain pathogens disrupt these processes and hijack mitochondrial resources12,13. Finally, there is a growing understanding of how inter-organelle contacts between the ER and mitochondria initiate mitochondrial fission14,15, and how this process impacts mitochondrial genome duplication and integrity16,17. The master regulator that unites these processes across eukaryotic evolution is the membrane-remodeling GTPase DRP12,18,19. DRP1 is necessary but not sufficient for mitochondrial fission because receptors must recruit the enzyme to the Outer Mitochondrial Membrane (OMM). In mammals, these receptors include the paralogs MItochondrial Dynamics proteins MID49 and MID51 and the Mitochondrial Fission Factor, MFF20–24. Following receptor-dependent recruitment, DRP1 assembles into polymers that encircle mitochondria and, via poorly understood mechanisms, channels energy from GTP binding, hydrolysis, and nucleotide exchange into a mechanochemical constriction8,23,25–28. In addition to DRP1 and its OMM receptors, a recent study revealed that a second member of the dynamin-family of GTPases, dynamin-2/DNM2, may enact the final fission event downstream of DRP1-driven constriction of a mitochondrial tubule29. Thus, mitochondrial division is a stepwise reaction regulated by DRP1 receptor binding, oligomerization and guanine nucleotide-dependent conformational dynamics. We and others have reported that the OMM receptors MFF or MID49/51 are independently sufficient to recruit DRP1 to divide mitochondria20,21,23,30. Previous work has suggested that the mitochondrial receptor in yeast, Mdv1p, can coassemble with the DRP1 homolog Dnm1p31. We observed that MID49/51 coassembled with DRP1 to form a copolymer with altered properties, compared with DRP1-only oligomers23. While these results suggest that organelle receptors could nucleate and alter the architecture of a dynamin polymer, the organization and functions of such a coassembly in organelle constriction remain unclear. Here, we report structural snapshots of DRP1-driven constriction through a MID49/51-dependent reaction. We used cryoEM to observe how nucleotide binding to the G-domain induces conformational changes that allosterically propagate through the BSE to open and elongate DRP1 and expose multiple surfaces required for receptor-binding and polymerization. 3D reconstruction revealed how MID49/51 binding stabilized an arrangement of GTP-bound DRP1 tetramers and nucleated polymerization of a linear co-filament. Then, we reconstituted a path-dependent reaction to observe how GTP hydrolysis and nucleotide exchange lead to conformational constriction by the DRP1 polymer. Specifically, when DRP1 subunits within the MID49/51 cofilament were allowed to exchange and hydrolyze GTP, they released the OMM receptors and the polymers shortened while curling into closed rings. Finally, we designed phosphomimetic, structure-based and disease-causing mutations to validate our structural models and to dissect the allosteric rearrangements that govern curling of linear strings into closed and constricted rings following receptor dissociation. Results and Discussion To date, many structural studies of dynamin-family proteins have relied upon mutated or truncated constructs to facilitate crystallization. We purified wild-type, full-length human DRP1 including the N-terminal GTPase domain (G-domain), Bundle Signaling Element (BSE), and four-helix bundle known as the stalk (Fig. 1a). This construct also contained the lipid binding ~100 amino acid region referred to as the variable domain (VD) that resides between the third and fourth α-helices of the stalk, analogous to the Pleckstrin Homology (PH) domain found in endocytic dynamin proteins. A crystal structure of a nucleotide-free and truncated DRP1 mutant revealed the organization of these domains and an overall similarity with the structure of nucleotide-free endocytic dynamin25,32,33. We also purified soluble truncations of MID49 and MID51 engineered to lack their N-terminal transmembrane anchors but include the cytoplasmic nucleotidyltransferase-like domain and the “Dynamin Recruitment Region” (DRR) required for DRP1 binding (Fig 1a)20,34–37. Structure of the DRP1-MID49 cofilament Incubating equimolar ratios of DRP1 with soluble MID49126-454, MID51132-463, or both proteins together, in the presence of Mg2+, GTP or GTP analogs GMPPCP/GTPγS, but not other nucleotides, resulted in cofilament assembly (Extended Data Fig. 1). We focused on the filaments formed with MID49126-454 in the presence of GMPPCP and determined their structure from cryoEM images to an average resolution of 4.2Å (from ~3.5Å to ~8Å, Extended Data Figs. 2–6 and Supplementary Table 1). 3D reconstruction revealed a polymer comprised of three equivalent faces that meet through defined vertices to form a triangular assembly (Extended Data Figs 2–3). A combination of helical reconstruction, segmentation, and single-particle alignment and averaging resolved the elongated DRP1 subunits bound stoichiometrically to MID49126-454, but without assignable density for the majority of the variable domain (Fig. 1b, Extended Data Fig. 2–6). Surprisingly, each chain of DRP1 bound MID49 via four different surfaces, and each MID49 in turn bound four DRP1 molecules to yield a vast interaction network (Figs. 1b-c, Extended Data Figs. 2, 3a-c). MID49 binding to four DRP1 molecules stabilized a linear arrangement of inter-DRP1 interfaces reminiscent of those observed for other dynamin-family proteins (Fig. 1b, Extended Data Figs. 2c, 3d)25,32,33,38–40. We refer to the four distinct surfaces of DRP1 that contribute to MID49/51 binding as receptor interfaces 1 through 4 (Fig. 1c). Structure-based mutants disrupt DRP1-MID49 assembly MID49’s DRR motif occupied the space between two neighboring G-domains and contacted both via receptor interfaces 1 and 2 (buried surface areas of ~530Å2 and ~200 Å2, respectively, Figs. 1b-c, 2a). The precise spacing required for this bivalent G-domain interaction explains why prior mutagenesis efforts suggested that the size and topology of the β4–α4 loop, rather than its exact sequence (which differs between MID49 and MID51) are critical determinants of binding34,35,37. Nevertheless, the structure indicated that R235 of MID49 within the DRR (analogous with R243 in MID51, Extended data Fig. 7) makes key contacts between two neighboring G-domains (Fig. 2a). Accordingly, the MID49R235E point mutant could not coassemble with DRP1 (Fig. 2d-f). In addition, we mutated conserved DRP1 residues involved in receptor interface 1—the largest interface. Both the D190A mutation, which should neutralize a salt bridge with the receptor, and the D221A mutation, which should alter the conformation of a key loop within receptor interface 1, prevented coassembly with MID49 (Fig. 2a). These substitutions also altered DRP1’s self-assembly properties, suggesting pleiotropic effects on nucleotide handling and receptor binding (Extended Data Fig. 8)41. Specifically, D190 and D221 are also involved in nucleotide-dependent G-domain dimerization (PDB:3W6O), leading to the intriguing possibility that MID49 and MID51 may modulate GTP-dependent conformational dynamics. Unexpectedly, MID49 also made contact with the stalk loops of a third and fourth DRP1 molecule through receptor interfaces 3 and 4 (buried surface areas of ~450Å2 and ~230Å2, respectively, Fig. 1c, 2b-c). The DRP1 loops involved in these receptor interfaces, the L1NS and L2S loops, are key determinants of assembly for other dynamin-family oligomers25,32,33,38,40. Receptor interface 3, in particular, involves the conserved loop L1NS and is the site of multiple disease alleles that correlate with elongated mitochondrial morphologies42–44, including G362D and G363D (Fig. 2b, Extended Data Figs. 6b, 6e, 7a). Prior work established that this loop comprises part of the intra-molecular PH domain binding site for the soluble state of endocytic dynamin tetramers (Extended Data Fig. 6f-h)40, and is a determinant of conformational heterogeneity for these and other dynamin-family proteins38,40. The presence of disease alleles near this interface suggests that these mutations may compromise receptor binding and that defects in the recruitment of DRP1 to mitochondria may contribute to pathogenesis. Accordingly, we found that the disease-associated G362D mutant of DRP1 (Fig. 2b) failed to coassemble with MID49 and displayed altered assembly and conformational properties (Extended Data Figs. 6e, 8a, 8i-l). Receptor interface 4 includes Ser611 (equivalent to Ser637 of isoform 1, Extended data Fig. 7c), an intensively studied phosphorylation site for protein kinase A (PKA) or Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα)45–47. Phosphorylation at this site, as well as phosphomimetic alleles of DRP1, have been reported to inhibit mitochondrial fission, although the mechanism of inhibition has been unclear45,48. Our structure suggests that phosphorylation should inhibit MID49 binding, and accordingly we found that the phosphomimetic mutant, S611D, failed to coassemble with MID49126-454 under the same solution conditions that bound the wild type protein (Fig. 2g-i). Nucleotide-induced conformational changes enable receptor binding Understanding the allosteric coupling between nucleotide binding, hydrolysis and exchange and the conformational repertoire of dynamin-family GTPases remains an unmet challenge. We observed that the GMPPCP-bound G-domains and the BSE of DRP1 adopt strikingly different conformations in the cryoEM density compared to the nucleotide-free crystal structure25. In addition to other nucleotide-induced conformational changes within the G-domain, the most salient are the closing of the G2/switch-1 loop to form a closed “lid” over the nucleotide (Figs. 3a-c). Analogous to the conformational change reported for Dynamin-149, the closure of the switch-1 lid propagates through the adjacent beta sheet to push the α-helices of the BSE into an orthogonal position (Supplementary Video 1). When evaluated in the context of an inter-DRP1 stalk interface, the interface-2 “X-shaped” DRP1 dimer, this conformational change is a 90° rotation of the G-domain and a 40Å translation toward the stalk (Fig. 3d, Supplementary Video 2). Two of the four DRP1 surfaces that engage the DRR of MID49/51 (receptor interfaces 1 and 2, Fig. 2a) are inaccessible in the nucleotide-free state but become available for binding upon nucleotide-driven elongation (Fig. 3d, Supplementary Video 2). GTP-hydrolysis induces filaments to curl into rings We next evaluated the dynamics of the DRP1+MID49 filaments in the presence of hydrolyzable GTP, rather than the non-hydrolyzable analog used for 3D reconstruction. Following copolymerization in the presence of the non-hydrolyzable analog, we exchanged GMPPCP for GTP through dialysis and followed the reaction using negative stain TEM at sequential time points until the GTP was exhausted. We observed that the linear, three-sided DRP1-MID49 cofilaments disassembled into shorter, single-sided filaments before disassembling entirely upon complete hydrolysis to GDP (Fig. 4). The thinner single-sided filaments seen at intermediate time points resembled the single-sided filaments we observed after mutagenizing a salt bridge that appears to hold the triangular structure together (Extended data fig. 3e-i). The single-sided filaments, moreover, exhibited intriguing dynamics at intermediate time points (Fig. 4b-c). Specifically, upon reaching a reproducibly narrow range of lengths, the nearly linear single-sided filaments spontaneously curled into closed rings of remarkably uniform diameter (Fig. 4c). A model for closed DRP1 rings In a separate but related experiment, we also evaluated the assembly properties of the DRP1 mutant G362D—which disrupts receptor interface 3—with and without MID49126-454. As described above, this disease-associated residue sits at the base of the L1NS loop and this loop is a key inter-stalk interaction between adjacent DRP1 molecules in the linear filament (Figs. 1c, 2b, Extended Data Fig. 6b, e). We found that DRP1G362D purified as a nearly monodisperse and stable dimer, rather than a mix of tetramers and higher order species observed for the wild-type, full-length protein (Extended Data Fig. 8a). In addition, DRP1G362D exclusively formed rings, not filaments, with or without MID49126-454 and in the presence of GTP or GMPPCP (Fig. 5a, Extended Data Figs. 8i-l). These rings resembled those observed with wild-type DRP1 in all respects except that the wild-type protein only formed closed rings from the linear MID49 copolymer through the path-dependent reaction described above (Fig. 4c versus Fig. 5a). We also observed that these apparently closed DRP1G362D rings could constrict liposomes into membrane tubules and circumscribe lipid nanotubes (Extended Data Fig. 9). Switching to the non-hydrolyzable GTP analog GMPPCP with the DRP1G362D rings improved structural homogeneity, presumably because the rings remain dynamic and eventually disassembled upon complete hydrolysis to GDP (Fig. 4). We imaged the GMPPCP-bound Drp1G362D rings using cryoEM and used 2D class averages of the predominant 12-dimer closed ring to model the differences between the linear filaments and the closed rings (Fig. 5, Extended Data Fig. 10). To account for the projected ring density, the G-domain and the BSE of DRP1 must move even further down toward the stalks. Stalk interface-2 remains constant, as revealed by the “X-shaped” dimer seen in projection (Fig. 5d, e). The curvature of the ring, however, dictates that stalk interfaces 1 and 3, and the conformations of the L1NS and L2S loops, must be extensively remodeled to allow a ~30 degree bending per dimer in comparison with the linear DRP1-MID49 copolymer (Fig. 5d-e, Extended Data Fig. 10e-f, Supplementary Videos 2–3). We did not observe any density for MID49 in the wild-type rings that form by curling of the MID49-DRP1 cofilament in the presence of GTP, nor in our higher-resolution analysis of the Drp1G362D rings that form with or without MID49 present (Fig. 5b-c, Extended Data Fig. 8i-l, 10). This suggests that MID49 binding is incompatible with the curled state of the ring-shaped oligomer, and that constriction therefore requires receptor dissociation (Supplementary Video 3). Discussion We note that with an inner diameter of ~16nm, the closed ring may be sufficient to sever a double-membrane mitochondrion if both the outer and inner membranes are compressed together. Alternatively, if inner membrane fission is separable and precedes outer membrane fission, a 16nm diameter suggests that a single membrane tubule would be stabilized by these rings. The structures we observe in vitro may therefore correspond with a highly-constricted but pre-fission state observed in vitro50 and in living cells when another dynamin-family protein, dynamin-2, is depleted29. Constriction by DRP1, therefore, may stabilize the high degree of membrane curvature that is suitable for the recruitment and final fission event catalyzed by additional dynamin-family enzymes. Together, these findings establish four advances. First, our cryoEM structure and mutagenesis studies revealed how receptor proteins like MID49/MID51 recruit and stabilize a specific nucleotide-bound conformation of DRP1 and initiate polymerization of a cofilament. We speculate that the nearly linear properties of this polymer have adapted to encircle low-curvature mitochondrial tubules. Second, analysis of the MID49-DRP1 copolymer revealed how guanine nucleotide binding induces a conformational rearrangement to expose an avid network of receptor binding sites. We now understand these nucleotide-driven allosteric transformations in the context of both full-length and oligomeric DRP1. Third, a path-dependent constriction reaction revealed GTP-dependent conformational dynamics. In this reaction, nucleotide exchange and hydrolysis led to MID49/51 receptor dissociation, disassembly from the ends of the linear filament, and concomitant curling of the shortening filaments into closed rings. The requirement for MID49/51 receptor dissociation before constriction may explain how overexpression of the MID receptors inhibits mitochondrial fission21. Fourth and finally, analysis of a disease mutant in the L1NS loop, Drp1G362D, highlights this loop as a cardinal determinant of receptor binding as well as the inter-stalk interactions that govern oligomer geometry. Together, these observations teach us how DRP1 performs mechanical work by curling from linear filaments into closed rings around mitochondria. Methods Construct design Wild type (WT) DRP1 isoform 2 sequence was purchased from DNASU (sequence ID HsCD00043627, UNIPROT identifier: O00429-3, also known as Dlp1a) and was cloned into pET16b plasmid (Novagen) between the Nde1 and BamH1 sites. The vector was kindly provided by the laboratory of Wesley I. Sundquist with a 10X-His tag followed by a PreScission protease site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro). Wild-type MID49126-454 sequence was PCR amplified and cloned into pGEX6p1 vector having an N-terminal GST tag followed by a PreScission protease site. Site directed mutagenesis was performed on pET16b-DRP1 and pGEX6p1-MID49126-454 using the Gibson cloning method to introduce mutations51. All constructs were verified using Sanger sequencing. Protein purification Protein purification was performed as described52. Briefly, plasmids containing the WT DRP1 or MID49126-454 sequence were transformed in the BL21-DE3 (RIPL) strain of E. coli. The colonies were inoculated in LB culture medium and grown overnight. Secondary inoculations were done the next morning in ZY medium for auto-induction53,54. The cultures were grown to an OD600 of 0.8 at 37°C in baffled flasks and were shifted to 19°C to grow for another 12 hours. The cultures were spun down and the bacterial pellets were used for protein purification immediately or stored at −80°C. Full length DRP1 WT and mutant variants were purified as described previously for DRP1 WT with modifications25. Briefly, the bacterial pellets were resuspended in buffer A (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 40 mM imidazole, 1 mM DTT, 0.5 mg DNase (Roche) and protease inhibitors (10 mM pepstatin, 50 mM PMSF, 0.5 mM aprotinin and 2 mM leupeptin), followed by cell disruption with a probe sonicator. Lysates were cleared by centrifugation at 40,000×g in Beckman JA 25.50 rotors for 60 min at 4°C. The supernatant was filtered using a 0.45 μm filter and applied to Ni-NTA Agarose beads pre-equilibrated with buffer B (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 40 mM imidazole, 1 mM DTT). Upon the application of the supernatant, the beads were washed with 20 column volumes each of buffer B and buffer C (50 mM HEPES/NaOH (pH 7.5), 800 mM NaCl, 5 mM MgCl2, 40 mM imidazole, 1 mM DTT, 1 mM ATP, 10 mM KCl) followed by buffer D (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 80 mM imidazole, 1 mM DTT, 0.5% (w/v) CHAPS). A final pre-elution wash was done with 20 column volumes of buffer B. Bound DRP1 was eluted with buffer E (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 300 mM imidazole, 1 mM DTT) and dialyzed overnight at 4°C against buffer B without imidazole in the presence of PreScission protease to cleave the N-terminal 10X-His tag. The protein was re-applied to a Ni-NTA column pre-equilibrated with dialysis buffer and was observed to bind the column without the 10X-His tag as well. Subsequently, the protein was eluted with buffer B containing 80 mM imidazole. Pure protein was concentrated with a 30kDa molecular weight cutoff (MWCO) centrifugal concentration device (Millipore). In a final step, DRP1 was purified by size-exclusion chromatography (SEC) on a Superdex-200 column (GE) in buffer F containing 20 mM HEPES/NaOH (pH 7.5), 300 mM NaCl, 2.5 mM MgCl2 and 1 mM DTT. Fractions containing DRP1 were pooled, concentrated, flash-frozen as single use aliquots in liquid nitrogen and stored at −80°C. Exact masses for purified DRP1 proteins were validated by MALDI-TOF mass spectrometry. MID49126-454 was purified as described with the following modifications37. pGEX6p1-MID49126-454 plasmid DNA (human, UNIPROT identifier: Isoform 1 Q96C03-1, also known as MIEF2) was transformed in BL21 (DE3) RIPL cells. The colonies were grown overnight in LB medium and secondary cultures were grown in ZY medium. Cells were grown to an OD600 of 0.8-1, collected by centrifugation and processed immediately or stored at −80°C as described above. The bacterial pellets were lysed as described above in MID-buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT and 0.1% (v/v) Triton X-100). The lysates were pre-cleared at 40,000×g and filtered using a 0.45 μm filter before applying to 3 ml glutathione sepharose beads (GE). After overnight binding to beads, the unbound protein was removed and the beads were washed using 20 column volumes each of MID-buffer A and MID-buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT). The protein was eluted with MID-buffer C (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT and 20 mM reduced glutathione). The eluate was cleaved overnight with PreScission protease while dialyzing against MID-buffer D (20 mM Tris pH 8.0, 100 mM NaCl, 5% glycerol, 1 mM DTT). Cleaved protein was further purified using ion-exchange chromatography using a Q sepharose (GE) column. The low salt buffer for ion-exchange was the same as MID-buffer D and the high salt buffer was MID-buffer E (20 mM Tris pH 8.0, 1 M NaCl, 5% glycerol, 1 mM DTT). The relevant MID49126-454 fractions were pooled, concentrated and further purified using an SEC column pre-equilibrated with MID-buffer F (20 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol, 1 mM DTT). The fractions containing MID49126-454 were pooled, concentrated, flash-frozen in liquid nitrogen and stored as single use aliquots at −80°C. Filament assembly, EM sample preparation, data acquisition and processing To assemble DRP1-MID49126-454 filaments, the proteins were mixed to a final concentration of 2 μM each and kept for an hour at room temperature. The mixture was dialyzed against assembly buffer- 20 mM HEPES pH 7.5, 50 mM KCl, 3 mM MgCl2, 1 mM DTT and 200 μM GMPPCP with or without 0.2% octyl-glucopyranoside (Anatrace). The filaments were observed using negative stain TEM or cryoEM after vitrification. Under these conditions, the mutant DRP1S611D failed to coassemble with MID49, but upon further lowering the ionic strength to 25mM KCl, DRP1S611D displayed detectable but greatly reduced coassembly compared to WT protein. For vitrification, the sample was applied to Quantifoil holey carbon grids (R2/2) using a Vitrobot Mark III with 3.5 μl sample, 5 seconds blotting time and a 0 mm offset at 19°C and 100% humidity. Images were collected on an FEI T30 Polara operating at 300kV at a magnification of 31000X. Images were recorded on a Gatan K2 summit camera in super resolution mode for a final binned pixel size of 1.22 Å/pixel. The movies were dose fractionated, contained 30-40 frames, had a total exposure time of 6-8 seconds with 0.2 seconds per frame and a per-frame dose of 1.1 to 1.4 electrons/Å2. SerialEM was used to automate data collection55. The defocus range was 0.8-3 μm under focus. The data was motion corrected and dose-weighted using UCSF Motioncor256. CTF parameter estimation on the non-dose-weighted but motion-corrected stacks was done using CTFFIND4 and GCTF57,58. Filaments were boxed using the program e2helixboxer.py from the EMAN2 suite59. Particle coordinates were used to extract discrete particles using RELION 1.3-1.460 and all further processing was done within the RELION suite. Multiple rounds of 2D classification identified the well-ordered segments. 3D autorefine was run using a customized Relion1.2 version with the IHRSR algorithm implemented61,62. The consensus helical structure was used to classify the particles without refining helical symmetry (using RELION 1.4), resulting in 2 major classes that differed slightly in rise and twist (Extended data figure 2c). Particles from each class were selected and independently refined again with helical RELION 1.2 and IHRSR. Analysis of these reconstructions revealed that each structure was comprised of three linear filaments that bundle together to form a structure that resembled a triangle in cross-section (Extended data figures 2–3). The vertices of the triangle are formed through asymmetric interactions between the G-domains in adjacent filaments. The triangular arrangement of the bundled helices is unlikely to correspond to a biologically meaningful architecture, and this structure cannot form if the MID49 receptor is embedded in the outer mitochondrial membrane. To further improve signal-to-noise, each of the three filaments in each independent half-map was segmented, extracted, resampled on a common grid and summed using UCSF Chimera63–67. The respective symmetrized but unfiltered half maps from each class were again aligned to a common grid and summed according to the C2 symmetry axis of the DRP1 dimer. In a last step, relion_postprocess was used to add the resulting and fully symmetrized half maps (Extended data figure 2c). These half maps and the final summed map, with differential B-factor sharpening per region (Extended data figures 2c, 4–5), were used for atomic modeling using Rosetta as described below. For the projection structure of the DRP1G362D rings, 2 μM protein was mixed 1:1 molar ratio with MID49126-454 and was allowed to sit at room temperature for an hour. The mixture was dialyzed against the assembly buffer (without detergent) overnight. The sample was collected after 12 hours and vitrified using ultra-thin 3 nm carbon support films (Ted Pella). For vitrification, a Mark III vitrobot was used with 3.5 μl sample, 0 mm offset, 100% humidity and 3.5 seconds blot time. The images were collected using an FEI TF20 microscope and SerialEM for automated data collection. The data were recorded with a Gatan K2 camera operating in super resolution mode to collect dose fractionated movie stacks with a final binned pixel size of 1.234 Å/pixel. 40 frames were collected per stack (0.2 seconds per frame and 1.42 electrons/Å2). The movie stacks were motion corrected and the parameters of the transfer function were estimated as described above. Approximately 2000 particles were picked manually for initial 2D classification in RELION 1.4 and these averages were used as templates for further particle picking by Gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/). Final 2D averages of the entire rings versus quarter segments of the rings were computed using Relion1.4. Liposome and Nanotube reactions Liposomes were made as described before52. Briefly, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) was purchased from Avanti Polar Lipids. DOPS dissolved in chloroform was dried under a steady stream of nitrogen and dried under vacuum for an hour. The dried lipid film was resuspended in n-hexane and dried again under nitrogen. The resulting lipid film was dried under vacuum for 4 hours and was finally resuspended in 20 mM HEPES pH 7.5 and 150 mM KCl. The same protocol was followed for making nanotubes, where the mixture contained 60% D-galactosyl-ß-1,1′ N-nervonoyl-D-erythro-sphingosine (Galactosyl Ceramide), 30% DOPS and 10% Ni2+-NTA DOGS (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] Nickel Salt). For assembly reactions of DRP1G362D over lipid, 0.5-2μM protein was incubated with liposomes or nanotubes for an hour and dialyzed against the assembly buffer without detergent. Model building The general procedure for atomic model interpretation and validation using Rosetta were performed as described68. To obtain an initial model for DRP1, the crystal structure of nucleotide-free DRP1 (PDB ID: 4BEJ)25 was used for the stalk region and DRP1 G domain-BSE structures bound to GMPPCP (PDB ID: 3W6O) were used for the G-domain and BSE regions. Density-guided model completion for DRP1 was carried out with RosettaCM69 using this hybridization of DRP1 crystal structures. A converged solution appeared from the low-energy ensemble of the complete models generated by RosettaCM. However, among the low-energy ensemble, residues 503-610 were found to be extremely flexible without cryoEM density constraints and therefore were omitted for further coordinate refinement. For MID49, the highly homologous mouse MID49 crystal structure (81.3% identity, PDB ID: 4WOY, Extended Data Fig. 5c-d)37 was used to generate a homology model using RosettaCM and used as the starting model. To enable fragment-based, density-guided model refinement with missing residues (503-610, DRP1), Rosetta iterative local rebuilding tool was customized to disallow backbone rebuilding at breaks within a single chain. Multiple rounds of refinement were done for each component against one half map (training map), and the other half map (validation map) was used to monitor overfitting according to the detailed procedure described in Wang et al.68. With the refined model of DRP1 and MID49, we further refined the model in the context of a full assembly that included 8 identical copies of each protein, Mg2+ and nucleotide which included all possible inter-domain molecular interactions in the filament (Extended data figure 5a-b). Pseudo-symmetry was used70 to enable and facilitate the energy evaluations of all neighboring interactions around the asymmetric unit (Green model, shown in Extended data figure 5a) for final model refinement of the full assembly. To this end, refinement was done against the training map. Finally, the half maps were used to determine a weight for the density map that did not introduce overfitting. Using the weight and with the symmetry imposed, the whole assembly of DRP1 and MID49 was refined in the full map, followed by B-factors refinement71. Finally, quantification of buried surface area and the number and nature of the bonds involved for each DRP1-MID49 interaction interface modeled by Rosetta were performed with the PISA server (http://www.ebi.ac.uk/pdbe/pisa/). Visual evaluations of the model-to-map correspondence was carried out in UCSF Chimera using unfiltered and unsharpened maps, maps uniformly sharpened with a range of ad hoc B-factors, and maps processed with a model-based local sharpening and local low-pass filtering procedure to optimize contrast and the visibility of high-resolution features of the map72. To build a molecular model for the closed 12-dimer DRP1 rings, we used the diameter, thickness, and angles revealed by the 2D cryoEM class averages of the DRP1G362D rings stabilized with GMPPCP. The atomic coordinates determined above using RosettaCM were used to build the ring in sections, first with repeating dimers of the interface-2 “X-shaped” stalk, then the BSE and finally the G-domains and the angles between these sections were iteratively adjusted until calculated projections of the molecular model corresponded with the features of the experimental projection densities. Both the top (Fig 5b-c) and the side view (Extended data Figure 10b) were used as constraints. The complete atomic model of ring was finally refined in Phenix73 to minimize clashes. Extended Data Extended Data Figure 1 DRP1 and MID49 assembly states (a-j) DRP1 assembly states visualized with negative stain electron microscopy in the presence of different guanine nucleotides and MID49126-454. Both proteins incubated at 2μM concentration. Bars = 100nm. (k-m) MID49126-454 and MID51132-463 form indistinguishable assemblies with DRP1. (k) DRP1 plus MID49126-454 and GMPPCP, (l) DRP1 plus MID51132-463 and GMPPCP, (m) DRP1 plus both equimolar MID49 and MID51. Bars = 100nm. Extended Data Figure 2 CryoEM and 3D Reconstruction (a) An electron cryo-micrograph of DRP1-MID49126-454 filaments formed with GMPPCP. Bar =100nm. Inset shows a representative 2D class average. Inset bar =10nm. (b) Cross-section of the 3D reconstruction of the filament and the distribution of views determined during helical reconstruction. The length of the cylinders and the color code correspond with the number of particles for that viewing direction (from few to many, blue to red). The 3D structure has been segmented and colored with DRP1 in grey and MID49 in golden yellow. (c) Particle numbers and workflow for the reconstruction protocol. DRP1 density is shown in grey and MID49 is in golden yellow. Extended Data Figure 3 Intra- and Inter-filament Interactions (a) The triangular structure seen in cross section. ‘Side 1’ of the triangular structure has the atomic model placed in the density. The G-domain-to-G-domain contact between adjacent sides is circled. (b) The sum of the 3 sides with the model fit in density. (c) Ribbon diagram of the same atomic model as in (b). The rotated view shows 8 chains each of DRP1 and MID49. The chains further from the reader are rendered transparent. (d) An isolated tetramer of DRP1 from the filament, rendered to highlight the stalk interfaces 1, 2 and 3 observed for DRP1 and other dynamin-family of GTPases. (e) Expanded view of the circular region in (a) illustrating a salt bridge between adjacent G-domains. (f) DRP1-only wild type polymers with GMPPCP versus (g) DRP1 coassembly with wild type MID49126-454 and GMPPCP and high-magnification inset. (h) DRP1E116R mutant polymers versus (i) DRP1E116R mutant coassembly with MiD49126-454. Shorter, single-sided filaments predominate. Disordered “triangular assemblies” were also observed, but were much shorter and infrequent compared with the wild type proteins. Bars = 50nm. Extended Data Figure 4 Resolution Estimates (a-b) Local resolution estimates computed by Resmap74. (a) Histogram of voxel values, (b) Results in A depicted as a heat map of a cross-section through the reconstruction. (c-d) Fourier Shell Correlation plots for (c) the half-maps with and without symmetry and (d) model-to-map correlations for each sub-region of the structure. Extended Data Figure 5 Rosetta-based Model Refinement (a): Complete assembly used for Rosetta-based model building with the asymmetric unit shown in green. (b) Atomic B-factors for one asymmetric unit, DRP1 versus MID49 models (ribbon) and bound GMPPCP (space filling). (c) Sequence alignment between human and mouse MID49 sequences. (d) Overlay of the homology model of human MID49126-454 (blue, with yellow DRR, ribbon) modeled within the cryoEM density overlaid with the mouse MID49 crystal structure (PDB ID: 4WOY, grey ribbon)37 which was used as a constraint. No density attributable to ADP within the nucleotidyltransferase domain was observed. Extended Data figure 6 Map-to-Model Fits and Role of the L1NS and L2S loops Examples of models fit within B-factor sharpened cryoEM density for (a) a helix from the DRP1 stalk, (b) the backbone of the L1NS loop from the stalk, (c) an elongated helix and turn found in MID49, and (d) GMPPCP and Mg within the G-domain. (e) Roles of L1NS and L2S in linear filament formation. Upper panel: An isolated Drp1 tetramer from the cryoEM model. The circled region is expanded in the lower panel. Lower: interactions that the conserved loop L1Ns makes within the assembly. G362 is highlighted. (f) DRP1 stalk and MID49 at receptor interface-3, (g) Dyn3 stalk plus PH domain from PDB 5A3F40, (h) Overlay of (f) and (g). Extended Data Figure 7 Sequence Conservation and Key Interaction Sites (a) Multiple sequence alignment of the regions near and including the DRP1 residues mutated in this study: D190, D221 and G362, G363. The residue numbers apply to human DRP1, isoform 2 (UNIPROT identifier: O00429-3 which is also known as DLP1a). (b) Sequence alignment of MID49 and MID51 at the region around residue R235 of MID49. R235 of MID49 corresponds to R243 of MID51. (c) Sequence alignment of DRP1 Isoform 1 vs Isoform 2 showing the correspondence of S637 (Isoform 1) and S611 (Isoform 2). Extended Data Figure 8 Biochemical and Structural Characterization of Mutants (a-d) Size exclusion chromatography traces for DRP1 wild-type and mutants used in the study. (a) Comparison between wild type (WT) versus G362D, (b) WT versus D221A, and (c) WT versus D190A. (d) Gel filtration standards with annotated molecular weights from the same column and FPLC system. (e-h) DRP1 assembly and coassembly reactions with GMPPCP for (e) DRP1D190A alone, (f) DRP1D190A+MID49126-454, (g) DRP1D221A alone and (h) DRP1D221A+MID49126-454. Bars = 100nm. (i-l) DRP1G362D assembly and coassembly reactions with GMPPCP or GTP. DRP1G362D forms rings but not linear filaments: i,k) without MID49, j,l) with MID49, with i, j) GMPPCP or k, l) GTP. Bars = 100 nm. Extended Data Figure 9 DRP1G362D Rings on Model Membranes (a) DOPS liposomes used in the study. (b) DOPS liposomes after incubation with DRP1G362D showing ring-like assemblies on the membrane and in the background. (c) Lipid nanotubes incubated with DRP1G362D. (d) Power spectrum of the area shown within the dashed square in (b). (e) Power spectrum of the area shown within the dashed square in (c). In both (d) and (e), layer lines indicative of helical geometry are not detectable. Bars = 50nm. Extended Data Figure 10 DRP1G362D forms 12-dimer closed rings (a) 2D class average of the rings. The red dashed circle indicates density that may be attributable to the variable domain. (b) 2D class average of infrequent, orthogonal or “side” views used as a constraint during model building; (c) “top” and (d) “side” projections of the model; (e) “top” and (f) “side” views of the final model rendered as ribbons. Bars =100Å. Green: G-domain, Red: BSE, Purple: Stalk. Supplementary Material 1 2 Sup Table 1 Video 1 Video 2 Video 3

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Title	Structural Basis of Mitochondrial Receptor Binding and Constriction by DRP1
Abstract	Summary Mitochondrial inheritance, genome maintenance, and metabolic adaptation depend on organelle fission by Dynamin-Related Protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogs Mitochondrial Dynamics 49 and 51 (MID49/MID51) and Mitochondrial Fission Factor (MFF), but the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryoEM structure of human, full-length DRP1 coassembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a remarkable elongation and rotation of the G-domain, Bundle-Signaling Element (BSE) and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes polymerization of a linear DRP1 filament with MID49/MID51. Following coassembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery.
Title	Summary
Body	Introduction Fragmentation of the mitochondrial reticulum disperses units of the organelle during cell division1,2, coordinates morphological adaptation with metabolic demand3,4, and quarantines damaged units for turnover5,6. Recent work also led to the discovery of the role mitochondrial fission plays in regulated cell death pathways7–9, brain development and synaptic function10,11, and how certain pathogens disrupt these processes and hijack mitochondrial resources12,13. Finally, there is a growing understanding of how inter-organelle contacts between the ER and mitochondria initiate mitochondrial fission14,15, and how this process impacts mitochondrial genome duplication and integrity16,17. The master regulator that unites these processes across eukaryotic evolution is the membrane-remodeling GTPase DRP12,18,19. DRP1 is necessary but not sufficient for mitochondrial fission because receptors must recruit the enzyme to the Outer Mitochondrial Membrane (OMM). In mammals, these receptors include the paralogs MItochondrial Dynamics proteins MID49 and MID51 and the Mitochondrial Fission Factor, MFF20–24. Following receptor-dependent recruitment, DRP1 assembles into polymers that encircle mitochondria and, via poorly understood mechanisms, channels energy from GTP binding, hydrolysis, and nucleotide exchange into a mechanochemical constriction8,23,25–28. In addition to DRP1 and its OMM receptors, a recent study revealed that a second member of the dynamin-family of GTPases, dynamin-2/DNM2, may enact the final fission event downstream of DRP1-driven constriction of a mitochondrial tubule29. Thus, mitochondrial division is a stepwise reaction regulated by DRP1 receptor binding, oligomerization and guanine nucleotide-dependent conformational dynamics. We and others have reported that the OMM receptors MFF or MID49/51 are independently sufficient to recruit DRP1 to divide mitochondria20,21,23,30. Previous work has suggested that the mitochondrial receptor in yeast, Mdv1p, can coassemble with the DRP1 homolog Dnm1p31. We observed that MID49/51 coassembled with DRP1 to form a copolymer with altered properties, compared with DRP1-only oligomers23. While these results suggest that organelle receptors could nucleate and alter the architecture of a dynamin polymer, the organization and functions of such a coassembly in organelle constriction remain unclear. Here, we report structural snapshots of DRP1-driven constriction through a MID49/51-dependent reaction. We used cryoEM to observe how nucleotide binding to the G-domain induces conformational changes that allosterically propagate through the BSE to open and elongate DRP1 and expose multiple surfaces required for receptor-binding and polymerization. 3D reconstruction revealed how MID49/51 binding stabilized an arrangement of GTP-bound DRP1 tetramers and nucleated polymerization of a linear co-filament. Then, we reconstituted a path-dependent reaction to observe how GTP hydrolysis and nucleotide exchange lead to conformational constriction by the DRP1 polymer. Specifically, when DRP1 subunits within the MID49/51 cofilament were allowed to exchange and hydrolyze GTP, they released the OMM receptors and the polymers shortened while curling into closed rings. Finally, we designed phosphomimetic, structure-based and disease-causing mutations to validate our structural models and to dissect the allosteric rearrangements that govern curling of linear strings into closed and constricted rings following receptor dissociation. Results and Discussion To date, many structural studies of dynamin-family proteins have relied upon mutated or truncated constructs to facilitate crystallization. We purified wild-type, full-length human DRP1 including the N-terminal GTPase domain (G-domain), Bundle Signaling Element (BSE), and four-helix bundle known as the stalk (Fig. 1a). This construct also contained the lipid binding ~100 amino acid region referred to as the variable domain (VD) that resides between the third and fourth α-helices of the stalk, analogous to the Pleckstrin Homology (PH) domain found in endocytic dynamin proteins. A crystal structure of a nucleotide-free and truncated DRP1 mutant revealed the organization of these domains and an overall similarity with the structure of nucleotide-free endocytic dynamin25,32,33. We also purified soluble truncations of MID49 and MID51 engineered to lack their N-terminal transmembrane anchors but include the cytoplasmic nucleotidyltransferase-like domain and the “Dynamin Recruitment Region” (DRR) required for DRP1 binding (Fig 1a)20,34–37. Structure of the DRP1-MID49 cofilament Incubating equimolar ratios of DRP1 with soluble MID49126-454, MID51132-463, or both proteins together, in the presence of Mg2+, GTP or GTP analogs GMPPCP/GTPγS, but not other nucleotides, resulted in cofilament assembly (Extended Data Fig. 1). We focused on the filaments formed with MID49126-454 in the presence of GMPPCP and determined their structure from cryoEM images to an average resolution of 4.2Å (from ~3.5Å to ~8Å, Extended Data Figs. 2–6 and Supplementary Table 1). 3D reconstruction revealed a polymer comprised of three equivalent faces that meet through defined vertices to form a triangular assembly (Extended Data Figs 2–3). A combination of helical reconstruction, segmentation, and single-particle alignment and averaging resolved the elongated DRP1 subunits bound stoichiometrically to MID49126-454, but without assignable density for the majority of the variable domain (Fig. 1b, Extended Data Fig. 2–6). Surprisingly, each chain of DRP1 bound MID49 via four different surfaces, and each MID49 in turn bound four DRP1 molecules to yield a vast interaction network (Figs. 1b-c, Extended Data Figs. 2, 3a-c). MID49 binding to four DRP1 molecules stabilized a linear arrangement of inter-DRP1 interfaces reminiscent of those observed for other dynamin-family proteins (Fig. 1b, Extended Data Figs. 2c, 3d)25,32,33,38–40. We refer to the four distinct surfaces of DRP1 that contribute to MID49/51 binding as receptor interfaces 1 through 4 (Fig. 1c). Structure-based mutants disrupt DRP1-MID49 assembly MID49’s DRR motif occupied the space between two neighboring G-domains and contacted both via receptor interfaces 1 and 2 (buried surface areas of ~530Å2 and ~200 Å2, respectively, Figs. 1b-c, 2a). The precise spacing required for this bivalent G-domain interaction explains why prior mutagenesis efforts suggested that the size and topology of the β4–α4 loop, rather than its exact sequence (which differs between MID49 and MID51) are critical determinants of binding34,35,37. Nevertheless, the structure indicated that R235 of MID49 within the DRR (analogous with R243 in MID51, Extended data Fig. 7) makes key contacts between two neighboring G-domains (Fig. 2a). Accordingly, the MID49R235E point mutant could not coassemble with DRP1 (Fig. 2d-f). In addition, we mutated conserved DRP1 residues involved in receptor interface 1—the largest interface. Both the D190A mutation, which should neutralize a salt bridge with the receptor, and the D221A mutation, which should alter the conformation of a key loop within receptor interface 1, prevented coassembly with MID49 (Fig. 2a). These substitutions also altered DRP1’s self-assembly properties, suggesting pleiotropic effects on nucleotide handling and receptor binding (Extended Data Fig. 8)41. Specifically, D190 and D221 are also involved in nucleotide-dependent G-domain dimerization (PDB:3W6O), leading to the intriguing possibility that MID49 and MID51 may modulate GTP-dependent conformational dynamics. Unexpectedly, MID49 also made contact with the stalk loops of a third and fourth DRP1 molecule through receptor interfaces 3 and 4 (buried surface areas of ~450Å2 and ~230Å2, respectively, Fig. 1c, 2b-c). The DRP1 loops involved in these receptor interfaces, the L1NS and L2S loops, are key determinants of assembly for other dynamin-family oligomers25,32,33,38,40. Receptor interface 3, in particular, involves the conserved loop L1NS and is the site of multiple disease alleles that correlate with elongated mitochondrial morphologies42–44, including G362D and G363D (Fig. 2b, Extended Data Figs. 6b, 6e, 7a). Prior work established that this loop comprises part of the intra-molecular PH domain binding site for the soluble state of endocytic dynamin tetramers (Extended Data Fig. 6f-h)40, and is a determinant of conformational heterogeneity for these and other dynamin-family proteins38,40. The presence of disease alleles near this interface suggests that these mutations may compromise receptor binding and that defects in the recruitment of DRP1 to mitochondria may contribute to pathogenesis. Accordingly, we found that the disease-associated G362D mutant of DRP1 (Fig. 2b) failed to coassemble with MID49 and displayed altered assembly and conformational properties (Extended Data Figs. 6e, 8a, 8i-l). Receptor interface 4 includes Ser611 (equivalent to Ser637 of isoform 1, Extended data Fig. 7c), an intensively studied phosphorylation site for protein kinase A (PKA) or Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα)45–47. Phosphorylation at this site, as well as phosphomimetic alleles of DRP1, have been reported to inhibit mitochondrial fission, although the mechanism of inhibition has been unclear45,48. Our structure suggests that phosphorylation should inhibit MID49 binding, and accordingly we found that the phosphomimetic mutant, S611D, failed to coassemble with MID49126-454 under the same solution conditions that bound the wild type protein (Fig. 2g-i). Nucleotide-induced conformational changes enable receptor binding Understanding the allosteric coupling between nucleotide binding, hydrolysis and exchange and the conformational repertoire of dynamin-family GTPases remains an unmet challenge. We observed that the GMPPCP-bound G-domains and the BSE of DRP1 adopt strikingly different conformations in the cryoEM density compared to the nucleotide-free crystal structure25. In addition to other nucleotide-induced conformational changes within the G-domain, the most salient are the closing of the G2/switch-1 loop to form a closed “lid” over the nucleotide (Figs. 3a-c). Analogous to the conformational change reported for Dynamin-149, the closure of the switch-1 lid propagates through the adjacent beta sheet to push the α-helices of the BSE into an orthogonal position (Supplementary Video 1). When evaluated in the context of an inter-DRP1 stalk interface, the interface-2 “X-shaped” DRP1 dimer, this conformational change is a 90° rotation of the G-domain and a 40Å translation toward the stalk (Fig. 3d, Supplementary Video 2). Two of the four DRP1 surfaces that engage the DRR of MID49/51 (receptor interfaces 1 and 2, Fig. 2a) are inaccessible in the nucleotide-free state but become available for binding upon nucleotide-driven elongation (Fig. 3d, Supplementary Video 2). GTP-hydrolysis induces filaments to curl into rings We next evaluated the dynamics of the DRP1+MID49 filaments in the presence of hydrolyzable GTP, rather than the non-hydrolyzable analog used for 3D reconstruction. Following copolymerization in the presence of the non-hydrolyzable analog, we exchanged GMPPCP for GTP through dialysis and followed the reaction using negative stain TEM at sequential time points until the GTP was exhausted. We observed that the linear, three-sided DRP1-MID49 cofilaments disassembled into shorter, single-sided filaments before disassembling entirely upon complete hydrolysis to GDP (Fig. 4). The thinner single-sided filaments seen at intermediate time points resembled the single-sided filaments we observed after mutagenizing a salt bridge that appears to hold the triangular structure together (Extended data fig. 3e-i). The single-sided filaments, moreover, exhibited intriguing dynamics at intermediate time points (Fig. 4b-c). Specifically, upon reaching a reproducibly narrow range of lengths, the nearly linear single-sided filaments spontaneously curled into closed rings of remarkably uniform diameter (Fig. 4c). A model for closed DRP1 rings In a separate but related experiment, we also evaluated the assembly properties of the DRP1 mutant G362D—which disrupts receptor interface 3—with and without MID49126-454. As described above, this disease-associated residue sits at the base of the L1NS loop and this loop is a key inter-stalk interaction between adjacent DRP1 molecules in the linear filament (Figs. 1c, 2b, Extended Data Fig. 6b, e). We found that DRP1G362D purified as a nearly monodisperse and stable dimer, rather than a mix of tetramers and higher order species observed for the wild-type, full-length protein (Extended Data Fig. 8a). In addition, DRP1G362D exclusively formed rings, not filaments, with or without MID49126-454 and in the presence of GTP or GMPPCP (Fig. 5a, Extended Data Figs. 8i-l). These rings resembled those observed with wild-type DRP1 in all respects except that the wild-type protein only formed closed rings from the linear MID49 copolymer through the path-dependent reaction described above (Fig. 4c versus Fig. 5a). We also observed that these apparently closed DRP1G362D rings could constrict liposomes into membrane tubules and circumscribe lipid nanotubes (Extended Data Fig. 9). Switching to the non-hydrolyzable GTP analog GMPPCP with the DRP1G362D rings improved structural homogeneity, presumably because the rings remain dynamic and eventually disassembled upon complete hydrolysis to GDP (Fig. 4). We imaged the GMPPCP-bound Drp1G362D rings using cryoEM and used 2D class averages of the predominant 12-dimer closed ring to model the differences between the linear filaments and the closed rings (Fig. 5, Extended Data Fig. 10). To account for the projected ring density, the G-domain and the BSE of DRP1 must move even further down toward the stalks. Stalk interface-2 remains constant, as revealed by the “X-shaped” dimer seen in projection (Fig. 5d, e). The curvature of the ring, however, dictates that stalk interfaces 1 and 3, and the conformations of the L1NS and L2S loops, must be extensively remodeled to allow a ~30 degree bending per dimer in comparison with the linear DRP1-MID49 copolymer (Fig. 5d-e, Extended Data Fig. 10e-f, Supplementary Videos 2–3). We did not observe any density for MID49 in the wild-type rings that form by curling of the MID49-DRP1 cofilament in the presence of GTP, nor in our higher-resolution analysis of the Drp1G362D rings that form with or without MID49 present (Fig. 5b-c, Extended Data Fig. 8i-l, 10). This suggests that MID49 binding is incompatible with the curled state of the ring-shaped oligomer, and that constriction therefore requires receptor dissociation (Supplementary Video 3). Discussion We note that with an inner diameter of ~16nm, the closed ring may be sufficient to sever a double-membrane mitochondrion if both the outer and inner membranes are compressed together. Alternatively, if inner membrane fission is separable and precedes outer membrane fission, a 16nm diameter suggests that a single membrane tubule would be stabilized by these rings. The structures we observe in vitro may therefore correspond with a highly-constricted but pre-fission state observed in vitro50 and in living cells when another dynamin-family protein, dynamin-2, is depleted29. Constriction by DRP1, therefore, may stabilize the high degree of membrane curvature that is suitable for the recruitment and final fission event catalyzed by additional dynamin-family enzymes. Together, these findings establish four advances. First, our cryoEM structure and mutagenesis studies revealed how receptor proteins like MID49/MID51 recruit and stabilize a specific nucleotide-bound conformation of DRP1 and initiate polymerization of a cofilament. We speculate that the nearly linear properties of this polymer have adapted to encircle low-curvature mitochondrial tubules. Second, analysis of the MID49-DRP1 copolymer revealed how guanine nucleotide binding induces a conformational rearrangement to expose an avid network of receptor binding sites. We now understand these nucleotide-driven allosteric transformations in the context of both full-length and oligomeric DRP1. Third, a path-dependent constriction reaction revealed GTP-dependent conformational dynamics. In this reaction, nucleotide exchange and hydrolysis led to MID49/51 receptor dissociation, disassembly from the ends of the linear filament, and concomitant curling of the shortening filaments into closed rings. The requirement for MID49/51 receptor dissociation before constriction may explain how overexpression of the MID receptors inhibits mitochondrial fission21. Fourth and finally, analysis of a disease mutant in the L1NS loop, Drp1G362D, highlights this loop as a cardinal determinant of receptor binding as well as the inter-stalk interactions that govern oligomer geometry. Together, these observations teach us how DRP1 performs mechanical work by curling from linear filaments into closed rings around mitochondria. Methods Construct design Wild type (WT) DRP1 isoform 2 sequence was purchased from DNASU (sequence ID HsCD00043627, UNIPROT identifier: O00429-3, also known as Dlp1a) and was cloned into pET16b plasmid (Novagen) between the Nde1 and BamH1 sites. The vector was kindly provided by the laboratory of Wesley I. Sundquist with a 10X-His tag followed by a PreScission protease site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro). Wild-type MID49126-454 sequence was PCR amplified and cloned into pGEX6p1 vector having an N-terminal GST tag followed by a PreScission protease site. Site directed mutagenesis was performed on pET16b-DRP1 and pGEX6p1-MID49126-454 using the Gibson cloning method to introduce mutations51. All constructs were verified using Sanger sequencing. Protein purification Protein purification was performed as described52. Briefly, plasmids containing the WT DRP1 or MID49126-454 sequence were transformed in the BL21-DE3 (RIPL) strain of E. coli. The colonies were inoculated in LB culture medium and grown overnight. Secondary inoculations were done the next morning in ZY medium for auto-induction53,54. The cultures were grown to an OD600 of 0.8 at 37°C in baffled flasks and were shifted to 19°C to grow for another 12 hours. The cultures were spun down and the bacterial pellets were used for protein purification immediately or stored at −80°C. Full length DRP1 WT and mutant variants were purified as described previously for DRP1 WT with modifications25. Briefly, the bacterial pellets were resuspended in buffer A (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 40 mM imidazole, 1 mM DTT, 0.5 mg DNase (Roche) and protease inhibitors (10 mM pepstatin, 50 mM PMSF, 0.5 mM aprotinin and 2 mM leupeptin), followed by cell disruption with a probe sonicator. Lysates were cleared by centrifugation at 40,000×g in Beckman JA 25.50 rotors for 60 min at 4°C. The supernatant was filtered using a 0.45 μm filter and applied to Ni-NTA Agarose beads pre-equilibrated with buffer B (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 40 mM imidazole, 1 mM DTT). Upon the application of the supernatant, the beads were washed with 20 column volumes each of buffer B and buffer C (50 mM HEPES/NaOH (pH 7.5), 800 mM NaCl, 5 mM MgCl2, 40 mM imidazole, 1 mM DTT, 1 mM ATP, 10 mM KCl) followed by buffer D (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 80 mM imidazole, 1 mM DTT, 0.5% (w/v) CHAPS). A final pre-elution wash was done with 20 column volumes of buffer B. Bound DRP1 was eluted with buffer E (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 300 mM imidazole, 1 mM DTT) and dialyzed overnight at 4°C against buffer B without imidazole in the presence of PreScission protease to cleave the N-terminal 10X-His tag. The protein was re-applied to a Ni-NTA column pre-equilibrated with dialysis buffer and was observed to bind the column without the 10X-His tag as well. Subsequently, the protein was eluted with buffer B containing 80 mM imidazole. Pure protein was concentrated with a 30kDa molecular weight cutoff (MWCO) centrifugal concentration device (Millipore). In a final step, DRP1 was purified by size-exclusion chromatography (SEC) on a Superdex-200 column (GE) in buffer F containing 20 mM HEPES/NaOH (pH 7.5), 300 mM NaCl, 2.5 mM MgCl2 and 1 mM DTT. Fractions containing DRP1 were pooled, concentrated, flash-frozen as single use aliquots in liquid nitrogen and stored at −80°C. Exact masses for purified DRP1 proteins were validated by MALDI-TOF mass spectrometry. MID49126-454 was purified as described with the following modifications37. pGEX6p1-MID49126-454 plasmid DNA (human, UNIPROT identifier: Isoform 1 Q96C03-1, also known as MIEF2) was transformed in BL21 (DE3) RIPL cells. The colonies were grown overnight in LB medium and secondary cultures were grown in ZY medium. Cells were grown to an OD600 of 0.8-1, collected by centrifugation and processed immediately or stored at −80°C as described above. The bacterial pellets were lysed as described above in MID-buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT and 0.1% (v/v) Triton X-100). The lysates were pre-cleared at 40,000×g and filtered using a 0.45 μm filter before applying to 3 ml glutathione sepharose beads (GE). After overnight binding to beads, the unbound protein was removed and the beads were washed using 20 column volumes each of MID-buffer A and MID-buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT). The protein was eluted with MID-buffer C (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT and 20 mM reduced glutathione). The eluate was cleaved overnight with PreScission protease while dialyzing against MID-buffer D (20 mM Tris pH 8.0, 100 mM NaCl, 5% glycerol, 1 mM DTT). Cleaved protein was further purified using ion-exchange chromatography using a Q sepharose (GE) column. The low salt buffer for ion-exchange was the same as MID-buffer D and the high salt buffer was MID-buffer E (20 mM Tris pH 8.0, 1 M NaCl, 5% glycerol, 1 mM DTT). The relevant MID49126-454 fractions were pooled, concentrated and further purified using an SEC column pre-equilibrated with MID-buffer F (20 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol, 1 mM DTT). The fractions containing MID49126-454 were pooled, concentrated, flash-frozen in liquid nitrogen and stored as single use aliquots at −80°C. Filament assembly, EM sample preparation, data acquisition and processing To assemble DRP1-MID49126-454 filaments, the proteins were mixed to a final concentration of 2 μM each and kept for an hour at room temperature. The mixture was dialyzed against assembly buffer- 20 mM HEPES pH 7.5, 50 mM KCl, 3 mM MgCl2, 1 mM DTT and 200 μM GMPPCP with or without 0.2% octyl-glucopyranoside (Anatrace). The filaments were observed using negative stain TEM or cryoEM after vitrification. Under these conditions, the mutant DRP1S611D failed to coassemble with MID49, but upon further lowering the ionic strength to 25mM KCl, DRP1S611D displayed detectable but greatly reduced coassembly compared to WT protein. For vitrification, the sample was applied to Quantifoil holey carbon grids (R2/2) using a Vitrobot Mark III with 3.5 μl sample, 5 seconds blotting time and a 0 mm offset at 19°C and 100% humidity. Images were collected on an FEI T30 Polara operating at 300kV at a magnification of 31000X. Images were recorded on a Gatan K2 summit camera in super resolution mode for a final binned pixel size of 1.22 Å/pixel. The movies were dose fractionated, contained 30-40 frames, had a total exposure time of 6-8 seconds with 0.2 seconds per frame and a per-frame dose of 1.1 to 1.4 electrons/Å2. SerialEM was used to automate data collection55. The defocus range was 0.8-3 μm under focus. The data was motion corrected and dose-weighted using UCSF Motioncor256. CTF parameter estimation on the non-dose-weighted but motion-corrected stacks was done using CTFFIND4 and GCTF57,58. Filaments were boxed using the program e2helixboxer.py from the EMAN2 suite59. Particle coordinates were used to extract discrete particles using RELION 1.3-1.460 and all further processing was done within the RELION suite. Multiple rounds of 2D classification identified the well-ordered segments. 3D autorefine was run using a customized Relion1.2 version with the IHRSR algorithm implemented61,62. The consensus helical structure was used to classify the particles without refining helical symmetry (using RELION 1.4), resulting in 2 major classes that differed slightly in rise and twist (Extended data figure 2c). Particles from each class were selected and independently refined again with helical RELION 1.2 and IHRSR. Analysis of these reconstructions revealed that each structure was comprised of three linear filaments that bundle together to form a structure that resembled a triangle in cross-section (Extended data figures 2–3). The vertices of the triangle are formed through asymmetric interactions between the G-domains in adjacent filaments. The triangular arrangement of the bundled helices is unlikely to correspond to a biologically meaningful architecture, and this structure cannot form if the MID49 receptor is embedded in the outer mitochondrial membrane. To further improve signal-to-noise, each of the three filaments in each independent half-map was segmented, extracted, resampled on a common grid and summed using UCSF Chimera63–67. The respective symmetrized but unfiltered half maps from each class were again aligned to a common grid and summed according to the C2 symmetry axis of the DRP1 dimer. In a last step, relion_postprocess was used to add the resulting and fully symmetrized half maps (Extended data figure 2c). These half maps and the final summed map, with differential B-factor sharpening per region (Extended data figures 2c, 4–5), were used for atomic modeling using Rosetta as described below. For the projection structure of the DRP1G362D rings, 2 μM protein was mixed 1:1 molar ratio with MID49126-454 and was allowed to sit at room temperature for an hour. The mixture was dialyzed against the assembly buffer (without detergent) overnight. The sample was collected after 12 hours and vitrified using ultra-thin 3 nm carbon support films (Ted Pella). For vitrification, a Mark III vitrobot was used with 3.5 μl sample, 0 mm offset, 100% humidity and 3.5 seconds blot time. The images were collected using an FEI TF20 microscope and SerialEM for automated data collection. The data were recorded with a Gatan K2 camera operating in super resolution mode to collect dose fractionated movie stacks with a final binned pixel size of 1.234 Å/pixel. 40 frames were collected per stack (0.2 seconds per frame and 1.42 electrons/Å2). The movie stacks were motion corrected and the parameters of the transfer function were estimated as described above. Approximately 2000 particles were picked manually for initial 2D classification in RELION 1.4 and these averages were used as templates for further particle picking by Gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/). Final 2D averages of the entire rings versus quarter segments of the rings were computed using Relion1.4. Liposome and Nanotube reactions Liposomes were made as described before52. Briefly, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) was purchased from Avanti Polar Lipids. DOPS dissolved in chloroform was dried under a steady stream of nitrogen and dried under vacuum for an hour. The dried lipid film was resuspended in n-hexane and dried again under nitrogen. The resulting lipid film was dried under vacuum for 4 hours and was finally resuspended in 20 mM HEPES pH 7.5 and 150 mM KCl. The same protocol was followed for making nanotubes, where the mixture contained 60% D-galactosyl-ß-1,1′ N-nervonoyl-D-erythro-sphingosine (Galactosyl Ceramide), 30% DOPS and 10% Ni2+-NTA DOGS (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] Nickel Salt). For assembly reactions of DRP1G362D over lipid, 0.5-2μM protein was incubated with liposomes or nanotubes for an hour and dialyzed against the assembly buffer without detergent. Model building The general procedure for atomic model interpretation and validation using Rosetta were performed as described68. To obtain an initial model for DRP1, the crystal structure of nucleotide-free DRP1 (PDB ID: 4BEJ)25 was used for the stalk region and DRP1 G domain-BSE structures bound to GMPPCP (PDB ID: 3W6O) were used for the G-domain and BSE regions. Density-guided model completion for DRP1 was carried out with RosettaCM69 using this hybridization of DRP1 crystal structures. A converged solution appeared from the low-energy ensemble of the complete models generated by RosettaCM. However, among the low-energy ensemble, residues 503-610 were found to be extremely flexible without cryoEM density constraints and therefore were omitted for further coordinate refinement. For MID49, the highly homologous mouse MID49 crystal structure (81.3% identity, PDB ID: 4WOY, Extended Data Fig. 5c-d)37 was used to generate a homology model using RosettaCM and used as the starting model. To enable fragment-based, density-guided model refinement with missing residues (503-610, DRP1), Rosetta iterative local rebuilding tool was customized to disallow backbone rebuilding at breaks within a single chain. Multiple rounds of refinement were done for each component against one half map (training map), and the other half map (validation map) was used to monitor overfitting according to the detailed procedure described in Wang et al.68. With the refined model of DRP1 and MID49, we further refined the model in the context of a full assembly that included 8 identical copies of each protein, Mg2+ and nucleotide which included all possible inter-domain molecular interactions in the filament (Extended data figure 5a-b). Pseudo-symmetry was used70 to enable and facilitate the energy evaluations of all neighboring interactions around the asymmetric unit (Green model, shown in Extended data figure 5a) for final model refinement of the full assembly. To this end, refinement was done against the training map. Finally, the half maps were used to determine a weight for the density map that did not introduce overfitting. Using the weight and with the symmetry imposed, the whole assembly of DRP1 and MID49 was refined in the full map, followed by B-factors refinement71. Finally, quantification of buried surface area and the number and nature of the bonds involved for each DRP1-MID49 interaction interface modeled by Rosetta were performed with the PISA server (http://www.ebi.ac.uk/pdbe/pisa/). Visual evaluations of the model-to-map correspondence was carried out in UCSF Chimera using unfiltered and unsharpened maps, maps uniformly sharpened with a range of ad hoc B-factors, and maps processed with a model-based local sharpening and local low-pass filtering procedure to optimize contrast and the visibility of high-resolution features of the map72. To build a molecular model for the closed 12-dimer DRP1 rings, we used the diameter, thickness, and angles revealed by the 2D cryoEM class averages of the DRP1G362D rings stabilized with GMPPCP. The atomic coordinates determined above using RosettaCM were used to build the ring in sections, first with repeating dimers of the interface-2 “X-shaped” stalk, then the BSE and finally the G-domains and the angles between these sections were iteratively adjusted until calculated projections of the molecular model corresponded with the features of the experimental projection densities. Both the top (Fig 5b-c) and the side view (Extended data Figure 10b) were used as constraints. The complete atomic model of ring was finally refined in Phenix73 to minimize clashes. Extended Data Extended Data Figure 1 DRP1 and MID49 assembly states (a-j) DRP1 assembly states visualized with negative stain electron microscopy in the presence of different guanine nucleotides and MID49126-454. Both proteins incubated at 2μM concentration. Bars = 100nm. (k-m) MID49126-454 and MID51132-463 form indistinguishable assemblies with DRP1. (k) DRP1 plus MID49126-454 and GMPPCP, (l) DRP1 plus MID51132-463 and GMPPCP, (m) DRP1 plus both equimolar MID49 and MID51. Bars = 100nm. Extended Data Figure 2 CryoEM and 3D Reconstruction (a) An electron cryo-micrograph of DRP1-MID49126-454 filaments formed with GMPPCP. Bar =100nm. Inset shows a representative 2D class average. Inset bar =10nm. (b) Cross-section of the 3D reconstruction of the filament and the distribution of views determined during helical reconstruction. The length of the cylinders and the color code correspond with the number of particles for that viewing direction (from few to many, blue to red). The 3D structure has been segmented and colored with DRP1 in grey and MID49 in golden yellow. (c) Particle numbers and workflow for the reconstruction protocol. DRP1 density is shown in grey and MID49 is in golden yellow. Extended Data Figure 3 Intra- and Inter-filament Interactions (a) The triangular structure seen in cross section. ‘Side 1’ of the triangular structure has the atomic model placed in the density. The G-domain-to-G-domain contact between adjacent sides is circled. (b) The sum of the 3 sides with the model fit in density. (c) Ribbon diagram of the same atomic model as in (b). The rotated view shows 8 chains each of DRP1 and MID49. The chains further from the reader are rendered transparent. (d) An isolated tetramer of DRP1 from the filament, rendered to highlight the stalk interfaces 1, 2 and 3 observed for DRP1 and other dynamin-family of GTPases. (e) Expanded view of the circular region in (a) illustrating a salt bridge between adjacent G-domains. (f) DRP1-only wild type polymers with GMPPCP versus (g) DRP1 coassembly with wild type MID49126-454 and GMPPCP and high-magnification inset. (h) DRP1E116R mutant polymers versus (i) DRP1E116R mutant coassembly with MiD49126-454. Shorter, single-sided filaments predominate. Disordered “triangular assemblies” were also observed, but were much shorter and infrequent compared with the wild type proteins. Bars = 50nm. Extended Data Figure 4 Resolution Estimates (a-b) Local resolution estimates computed by Resmap74. (a) Histogram of voxel values, (b) Results in A depicted as a heat map of a cross-section through the reconstruction. (c-d) Fourier Shell Correlation plots for (c) the half-maps with and without symmetry and (d) model-to-map correlations for each sub-region of the structure. Extended Data Figure 5 Rosetta-based Model Refinement (a): Complete assembly used for Rosetta-based model building with the asymmetric unit shown in green. (b) Atomic B-factors for one asymmetric unit, DRP1 versus MID49 models (ribbon) and bound GMPPCP (space filling). (c) Sequence alignment between human and mouse MID49 sequences. (d) Overlay of the homology model of human MID49126-454 (blue, with yellow DRR, ribbon) modeled within the cryoEM density overlaid with the mouse MID49 crystal structure (PDB ID: 4WOY, grey ribbon)37 which was used as a constraint. No density attributable to ADP within the nucleotidyltransferase domain was observed. Extended Data figure 6 Map-to-Model Fits and Role of the L1NS and L2S loops Examples of models fit within B-factor sharpened cryoEM density for (a) a helix from the DRP1 stalk, (b) the backbone of the L1NS loop from the stalk, (c) an elongated helix and turn found in MID49, and (d) GMPPCP and Mg within the G-domain. (e) Roles of L1NS and L2S in linear filament formation. Upper panel: An isolated Drp1 tetramer from the cryoEM model. The circled region is expanded in the lower panel. Lower: interactions that the conserved loop L1Ns makes within the assembly. G362 is highlighted. (f) DRP1 stalk and MID49 at receptor interface-3, (g) Dyn3 stalk plus PH domain from PDB 5A3F40, (h) Overlay of (f) and (g). Extended Data Figure 7 Sequence Conservation and Key Interaction Sites (a) Multiple sequence alignment of the regions near and including the DRP1 residues mutated in this study: D190, D221 and G362, G363. The residue numbers apply to human DRP1, isoform 2 (UNIPROT identifier: O00429-3 which is also known as DLP1a). (b) Sequence alignment of MID49 and MID51 at the region around residue R235 of MID49. R235 of MID49 corresponds to R243 of MID51. (c) Sequence alignment of DRP1 Isoform 1 vs Isoform 2 showing the correspondence of S637 (Isoform 1) and S611 (Isoform 2). Extended Data Figure 8 Biochemical and Structural Characterization of Mutants (a-d) Size exclusion chromatography traces for DRP1 wild-type and mutants used in the study. (a) Comparison between wild type (WT) versus G362D, (b) WT versus D221A, and (c) WT versus D190A. (d) Gel filtration standards with annotated molecular weights from the same column and FPLC system. (e-h) DRP1 assembly and coassembly reactions with GMPPCP for (e) DRP1D190A alone, (f) DRP1D190A+MID49126-454, (g) DRP1D221A alone and (h) DRP1D221A+MID49126-454. Bars = 100nm. (i-l) DRP1G362D assembly and coassembly reactions with GMPPCP or GTP. DRP1G362D forms rings but not linear filaments: i,k) without MID49, j,l) with MID49, with i, j) GMPPCP or k, l) GTP. Bars = 100 nm. Extended Data Figure 9 DRP1G362D Rings on Model Membranes (a) DOPS liposomes used in the study. (b) DOPS liposomes after incubation with DRP1G362D showing ring-like assemblies on the membrane and in the background. (c) Lipid nanotubes incubated with DRP1G362D. (d) Power spectrum of the area shown within the dashed square in (b). (e) Power spectrum of the area shown within the dashed square in (c). In both (d) and (e), layer lines indicative of helical geometry are not detectable. Bars = 50nm. Extended Data Figure 10 DRP1G362D forms 12-dimer closed rings (a) 2D class average of the rings. The red dashed circle indicates density that may be attributable to the variable domain. (b) 2D class average of infrequent, orthogonal or “side” views used as a constraint during model building; (c) “top” and (d) “side” projections of the model; (e) “top” and (f) “side” views of the final model rendered as ribbons. Bars =100Å. Green: G-domain, Red: BSE, Purple: Stalk. Supplementary Material 1 2 Sup Table 1 Video 1 Video 2 Video 3
Section	Introduction Fragmentation of the mitochondrial reticulum disperses units of the organelle during cell division1,2, coordinates morphological adaptation with metabolic demand3,4, and quarantines damaged units for turnover5,6. Recent work also led to the discovery of the role mitochondrial fission plays in regulated cell death pathways7–9, brain development and synaptic function10,11, and how certain pathogens disrupt these processes and hijack mitochondrial resources12,13. Finally, there is a growing understanding of how inter-organelle contacts between the ER and mitochondria initiate mitochondrial fission14,15, and how this process impacts mitochondrial genome duplication and integrity16,17. The master regulator that unites these processes across eukaryotic evolution is the membrane-remodeling GTPase DRP12,18,19. DRP1 is necessary but not sufficient for mitochondrial fission because receptors must recruit the enzyme to the Outer Mitochondrial Membrane (OMM). In mammals, these receptors include the paralogs MItochondrial Dynamics proteins MID49 and MID51 and the Mitochondrial Fission Factor, MFF20–24. Following receptor-dependent recruitment, DRP1 assembles into polymers that encircle mitochondria and, via poorly understood mechanisms, channels energy from GTP binding, hydrolysis, and nucleotide exchange into a mechanochemical constriction8,23,25–28. In addition to DRP1 and its OMM receptors, a recent study revealed that a second member of the dynamin-family of GTPases, dynamin-2/DNM2, may enact the final fission event downstream of DRP1-driven constriction of a mitochondrial tubule29. Thus, mitochondrial division is a stepwise reaction regulated by DRP1 receptor binding, oligomerization and guanine nucleotide-dependent conformational dynamics. We and others have reported that the OMM receptors MFF or MID49/51 are independently sufficient to recruit DRP1 to divide mitochondria20,21,23,30. Previous work has suggested that the mitochondrial receptor in yeast, Mdv1p, can coassemble with the DRP1 homolog Dnm1p31. We observed that MID49/51 coassembled with DRP1 to form a copolymer with altered properties, compared with DRP1-only oligomers23. While these results suggest that organelle receptors could nucleate and alter the architecture of a dynamin polymer, the organization and functions of such a coassembly in organelle constriction remain unclear. Here, we report structural snapshots of DRP1-driven constriction through a MID49/51-dependent reaction. We used cryoEM to observe how nucleotide binding to the G-domain induces conformational changes that allosterically propagate through the BSE to open and elongate DRP1 and expose multiple surfaces required for receptor-binding and polymerization. 3D reconstruction revealed how MID49/51 binding stabilized an arrangement of GTP-bound DRP1 tetramers and nucleated polymerization of a linear co-filament. Then, we reconstituted a path-dependent reaction to observe how GTP hydrolysis and nucleotide exchange lead to conformational constriction by the DRP1 polymer. Specifically, when DRP1 subunits within the MID49/51 cofilament were allowed to exchange and hydrolyze GTP, they released the OMM receptors and the polymers shortened while curling into closed rings. Finally, we designed phosphomimetic, structure-based and disease-causing mutations to validate our structural models and to dissect the allosteric rearrangements that govern curling of linear strings into closed and constricted rings following receptor dissociation.
Title	Introduction
Section	Results and Discussion To date, many structural studies of dynamin-family proteins have relied upon mutated or truncated constructs to facilitate crystallization. We purified wild-type, full-length human DRP1 including the N-terminal GTPase domain (G-domain), Bundle Signaling Element (BSE), and four-helix bundle known as the stalk (Fig. 1a). This construct also contained the lipid binding ~100 amino acid region referred to as the variable domain (VD) that resides between the third and fourth α-helices of the stalk, analogous to the Pleckstrin Homology (PH) domain found in endocytic dynamin proteins. A crystal structure of a nucleotide-free and truncated DRP1 mutant revealed the organization of these domains and an overall similarity with the structure of nucleotide-free endocytic dynamin25,32,33. We also purified soluble truncations of MID49 and MID51 engineered to lack their N-terminal transmembrane anchors but include the cytoplasmic nucleotidyltransferase-like domain and the “Dynamin Recruitment Region” (DRR) required for DRP1 binding (Fig 1a)20,34–37. Structure of the DRP1-MID49 cofilament Incubating equimolar ratios of DRP1 with soluble MID49126-454, MID51132-463, or both proteins together, in the presence of Mg2+, GTP or GTP analogs GMPPCP/GTPγS, but not other nucleotides, resulted in cofilament assembly (Extended Data Fig. 1). We focused on the filaments formed with MID49126-454 in the presence of GMPPCP and determined their structure from cryoEM images to an average resolution of 4.2Å (from ~3.5Å to ~8Å, Extended Data Figs. 2–6 and Supplementary Table 1). 3D reconstruction revealed a polymer comprised of three equivalent faces that meet through defined vertices to form a triangular assembly (Extended Data Figs 2–3). A combination of helical reconstruction, segmentation, and single-particle alignment and averaging resolved the elongated DRP1 subunits bound stoichiometrically to MID49126-454, but without assignable density for the majority of the variable domain (Fig. 1b, Extended Data Fig. 2–6). Surprisingly, each chain of DRP1 bound MID49 via four different surfaces, and each MID49 in turn bound four DRP1 molecules to yield a vast interaction network (Figs. 1b-c, Extended Data Figs. 2, 3a-c). MID49 binding to four DRP1 molecules stabilized a linear arrangement of inter-DRP1 interfaces reminiscent of those observed for other dynamin-family proteins (Fig. 1b, Extended Data Figs. 2c, 3d)25,32,33,38–40. We refer to the four distinct surfaces of DRP1 that contribute to MID49/51 binding as receptor interfaces 1 through 4 (Fig. 1c). Structure-based mutants disrupt DRP1-MID49 assembly MID49’s DRR motif occupied the space between two neighboring G-domains and contacted both via receptor interfaces 1 and 2 (buried surface areas of ~530Å2 and ~200 Å2, respectively, Figs. 1b-c, 2a). The precise spacing required for this bivalent G-domain interaction explains why prior mutagenesis efforts suggested that the size and topology of the β4–α4 loop, rather than its exact sequence (which differs between MID49 and MID51) are critical determinants of binding34,35,37. Nevertheless, the structure indicated that R235 of MID49 within the DRR (analogous with R243 in MID51, Extended data Fig. 7) makes key contacts between two neighboring G-domains (Fig. 2a). Accordingly, the MID49R235E point mutant could not coassemble with DRP1 (Fig. 2d-f). In addition, we mutated conserved DRP1 residues involved in receptor interface 1—the largest interface. Both the D190A mutation, which should neutralize a salt bridge with the receptor, and the D221A mutation, which should alter the conformation of a key loop within receptor interface 1, prevented coassembly with MID49 (Fig. 2a). These substitutions also altered DRP1’s self-assembly properties, suggesting pleiotropic effects on nucleotide handling and receptor binding (Extended Data Fig. 8)41. Specifically, D190 and D221 are also involved in nucleotide-dependent G-domain dimerization (PDB:3W6O), leading to the intriguing possibility that MID49 and MID51 may modulate GTP-dependent conformational dynamics. Unexpectedly, MID49 also made contact with the stalk loops of a third and fourth DRP1 molecule through receptor interfaces 3 and 4 (buried surface areas of ~450Å2 and ~230Å2, respectively, Fig. 1c, 2b-c). The DRP1 loops involved in these receptor interfaces, the L1NS and L2S loops, are key determinants of assembly for other dynamin-family oligomers25,32,33,38,40. Receptor interface 3, in particular, involves the conserved loop L1NS and is the site of multiple disease alleles that correlate with elongated mitochondrial morphologies42–44, including G362D and G363D (Fig. 2b, Extended Data Figs. 6b, 6e, 7a). Prior work established that this loop comprises part of the intra-molecular PH domain binding site for the soluble state of endocytic dynamin tetramers (Extended Data Fig. 6f-h)40, and is a determinant of conformational heterogeneity for these and other dynamin-family proteins38,40. The presence of disease alleles near this interface suggests that these mutations may compromise receptor binding and that defects in the recruitment of DRP1 to mitochondria may contribute to pathogenesis. Accordingly, we found that the disease-associated G362D mutant of DRP1 (Fig. 2b) failed to coassemble with MID49 and displayed altered assembly and conformational properties (Extended Data Figs. 6e, 8a, 8i-l). Receptor interface 4 includes Ser611 (equivalent to Ser637 of isoform 1, Extended data Fig. 7c), an intensively studied phosphorylation site for protein kinase A (PKA) or Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα)45–47. Phosphorylation at this site, as well as phosphomimetic alleles of DRP1, have been reported to inhibit mitochondrial fission, although the mechanism of inhibition has been unclear45,48. Our structure suggests that phosphorylation should inhibit MID49 binding, and accordingly we found that the phosphomimetic mutant, S611D, failed to coassemble with MID49126-454 under the same solution conditions that bound the wild type protein (Fig. 2g-i). Nucleotide-induced conformational changes enable receptor binding Understanding the allosteric coupling between nucleotide binding, hydrolysis and exchange and the conformational repertoire of dynamin-family GTPases remains an unmet challenge. We observed that the GMPPCP-bound G-domains and the BSE of DRP1 adopt strikingly different conformations in the cryoEM density compared to the nucleotide-free crystal structure25. In addition to other nucleotide-induced conformational changes within the G-domain, the most salient are the closing of the G2/switch-1 loop to form a closed “lid” over the nucleotide (Figs. 3a-c). Analogous to the conformational change reported for Dynamin-149, the closure of the switch-1 lid propagates through the adjacent beta sheet to push the α-helices of the BSE into an orthogonal position (Supplementary Video 1). When evaluated in the context of an inter-DRP1 stalk interface, the interface-2 “X-shaped” DRP1 dimer, this conformational change is a 90° rotation of the G-domain and a 40Å translation toward the stalk (Fig. 3d, Supplementary Video 2). Two of the four DRP1 surfaces that engage the DRR of MID49/51 (receptor interfaces 1 and 2, Fig. 2a) are inaccessible in the nucleotide-free state but become available for binding upon nucleotide-driven elongation (Fig. 3d, Supplementary Video 2). GTP-hydrolysis induces filaments to curl into rings We next evaluated the dynamics of the DRP1+MID49 filaments in the presence of hydrolyzable GTP, rather than the non-hydrolyzable analog used for 3D reconstruction. Following copolymerization in the presence of the non-hydrolyzable analog, we exchanged GMPPCP for GTP through dialysis and followed the reaction using negative stain TEM at sequential time points until the GTP was exhausted. We observed that the linear, three-sided DRP1-MID49 cofilaments disassembled into shorter, single-sided filaments before disassembling entirely upon complete hydrolysis to GDP (Fig. 4). The thinner single-sided filaments seen at intermediate time points resembled the single-sided filaments we observed after mutagenizing a salt bridge that appears to hold the triangular structure together (Extended data fig. 3e-i). The single-sided filaments, moreover, exhibited intriguing dynamics at intermediate time points (Fig. 4b-c). Specifically, upon reaching a reproducibly narrow range of lengths, the nearly linear single-sided filaments spontaneously curled into closed rings of remarkably uniform diameter (Fig. 4c). A model for closed DRP1 rings In a separate but related experiment, we also evaluated the assembly properties of the DRP1 mutant G362D—which disrupts receptor interface 3—with and without MID49126-454. As described above, this disease-associated residue sits at the base of the L1NS loop and this loop is a key inter-stalk interaction between adjacent DRP1 molecules in the linear filament (Figs. 1c, 2b, Extended Data Fig. 6b, e). We found that DRP1G362D purified as a nearly monodisperse and stable dimer, rather than a mix of tetramers and higher order species observed for the wild-type, full-length protein (Extended Data Fig. 8a). In addition, DRP1G362D exclusively formed rings, not filaments, with or without MID49126-454 and in the presence of GTP or GMPPCP (Fig. 5a, Extended Data Figs. 8i-l). These rings resembled those observed with wild-type DRP1 in all respects except that the wild-type protein only formed closed rings from the linear MID49 copolymer through the path-dependent reaction described above (Fig. 4c versus Fig. 5a). We also observed that these apparently closed DRP1G362D rings could constrict liposomes into membrane tubules and circumscribe lipid nanotubes (Extended Data Fig. 9). Switching to the non-hydrolyzable GTP analog GMPPCP with the DRP1G362D rings improved structural homogeneity, presumably because the rings remain dynamic and eventually disassembled upon complete hydrolysis to GDP (Fig. 4). We imaged the GMPPCP-bound Drp1G362D rings using cryoEM and used 2D class averages of the predominant 12-dimer closed ring to model the differences between the linear filaments and the closed rings (Fig. 5, Extended Data Fig. 10). To account for the projected ring density, the G-domain and the BSE of DRP1 must move even further down toward the stalks. Stalk interface-2 remains constant, as revealed by the “X-shaped” dimer seen in projection (Fig. 5d, e). The curvature of the ring, however, dictates that stalk interfaces 1 and 3, and the conformations of the L1NS and L2S loops, must be extensively remodeled to allow a ~30 degree bending per dimer in comparison with the linear DRP1-MID49 copolymer (Fig. 5d-e, Extended Data Fig. 10e-f, Supplementary Videos 2–3). We did not observe any density for MID49 in the wild-type rings that form by curling of the MID49-DRP1 cofilament in the presence of GTP, nor in our higher-resolution analysis of the Drp1G362D rings that form with or without MID49 present (Fig. 5b-c, Extended Data Fig. 8i-l, 10). This suggests that MID49 binding is incompatible with the curled state of the ring-shaped oligomer, and that constriction therefore requires receptor dissociation (Supplementary Video 3).
Title	Results and Discussion
Section	Structure of the DRP1-MID49 cofilament Incubating equimolar ratios of DRP1 with soluble MID49126-454, MID51132-463, or both proteins together, in the presence of Mg2+, GTP or GTP analogs GMPPCP/GTPγS, but not other nucleotides, resulted in cofilament assembly (Extended Data Fig. 1). We focused on the filaments formed with MID49126-454 in the presence of GMPPCP and determined their structure from cryoEM images to an average resolution of 4.2Å (from ~3.5Å to ~8Å, Extended Data Figs. 2–6 and Supplementary Table 1). 3D reconstruction revealed a polymer comprised of three equivalent faces that meet through defined vertices to form a triangular assembly (Extended Data Figs 2–3). A combination of helical reconstruction, segmentation, and single-particle alignment and averaging resolved the elongated DRP1 subunits bound stoichiometrically to MID49126-454, but without assignable density for the majority of the variable domain (Fig. 1b, Extended Data Fig. 2–6). Surprisingly, each chain of DRP1 bound MID49 via four different surfaces, and each MID49 in turn bound four DRP1 molecules to yield a vast interaction network (Figs. 1b-c, Extended Data Figs. 2, 3a-c). MID49 binding to four DRP1 molecules stabilized a linear arrangement of inter-DRP1 interfaces reminiscent of those observed for other dynamin-family proteins (Fig. 1b, Extended Data Figs. 2c, 3d)25,32,33,38–40. We refer to the four distinct surfaces of DRP1 that contribute to MID49/51 binding as receptor interfaces 1 through 4 (Fig. 1c).
Title	Structure of the DRP1-MID49 cofilament
Section	Structure-based mutants disrupt DRP1-MID49 assembly MID49’s DRR motif occupied the space between two neighboring G-domains and contacted both via receptor interfaces 1 and 2 (buried surface areas of ~530Å2 and ~200 Å2, respectively, Figs. 1b-c, 2a). The precise spacing required for this bivalent G-domain interaction explains why prior mutagenesis efforts suggested that the size and topology of the β4–α4 loop, rather than its exact sequence (which differs between MID49 and MID51) are critical determinants of binding34,35,37. Nevertheless, the structure indicated that R235 of MID49 within the DRR (analogous with R243 in MID51, Extended data Fig. 7) makes key contacts between two neighboring G-domains (Fig. 2a). Accordingly, the MID49R235E point mutant could not coassemble with DRP1 (Fig. 2d-f). In addition, we mutated conserved DRP1 residues involved in receptor interface 1—the largest interface. Both the D190A mutation, which should neutralize a salt bridge with the receptor, and the D221A mutation, which should alter the conformation of a key loop within receptor interface 1, prevented coassembly with MID49 (Fig. 2a). These substitutions also altered DRP1’s self-assembly properties, suggesting pleiotropic effects on nucleotide handling and receptor binding (Extended Data Fig. 8)41. Specifically, D190 and D221 are also involved in nucleotide-dependent G-domain dimerization (PDB:3W6O), leading to the intriguing possibility that MID49 and MID51 may modulate GTP-dependent conformational dynamics. Unexpectedly, MID49 also made contact with the stalk loops of a third and fourth DRP1 molecule through receptor interfaces 3 and 4 (buried surface areas of ~450Å2 and ~230Å2, respectively, Fig. 1c, 2b-c). The DRP1 loops involved in these receptor interfaces, the L1NS and L2S loops, are key determinants of assembly for other dynamin-family oligomers25,32,33,38,40. Receptor interface 3, in particular, involves the conserved loop L1NS and is the site of multiple disease alleles that correlate with elongated mitochondrial morphologies42–44, including G362D and G363D (Fig. 2b, Extended Data Figs. 6b, 6e, 7a). Prior work established that this loop comprises part of the intra-molecular PH domain binding site for the soluble state of endocytic dynamin tetramers (Extended Data Fig. 6f-h)40, and is a determinant of conformational heterogeneity for these and other dynamin-family proteins38,40. The presence of disease alleles near this interface suggests that these mutations may compromise receptor binding and that defects in the recruitment of DRP1 to mitochondria may contribute to pathogenesis. Accordingly, we found that the disease-associated G362D mutant of DRP1 (Fig. 2b) failed to coassemble with MID49 and displayed altered assembly and conformational properties (Extended Data Figs. 6e, 8a, 8i-l). Receptor interface 4 includes Ser611 (equivalent to Ser637 of isoform 1, Extended data Fig. 7c), an intensively studied phosphorylation site for protein kinase A (PKA) or Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα)45–47. Phosphorylation at this site, as well as phosphomimetic alleles of DRP1, have been reported to inhibit mitochondrial fission, although the mechanism of inhibition has been unclear45,48. Our structure suggests that phosphorylation should inhibit MID49 binding, and accordingly we found that the phosphomimetic mutant, S611D, failed to coassemble with MID49126-454 under the same solution conditions that bound the wild type protein (Fig. 2g-i).
Title	Structure-based mutants disrupt DRP1-MID49 assembly
Section	Nucleotide-induced conformational changes enable receptor binding Understanding the allosteric coupling between nucleotide binding, hydrolysis and exchange and the conformational repertoire of dynamin-family GTPases remains an unmet challenge. We observed that the GMPPCP-bound G-domains and the BSE of DRP1 adopt strikingly different conformations in the cryoEM density compared to the nucleotide-free crystal structure25. In addition to other nucleotide-induced conformational changes within the G-domain, the most salient are the closing of the G2/switch-1 loop to form a closed “lid” over the nucleotide (Figs. 3a-c). Analogous to the conformational change reported for Dynamin-149, the closure of the switch-1 lid propagates through the adjacent beta sheet to push the α-helices of the BSE into an orthogonal position (Supplementary Video 1). When evaluated in the context of an inter-DRP1 stalk interface, the interface-2 “X-shaped” DRP1 dimer, this conformational change is a 90° rotation of the G-domain and a 40Å translation toward the stalk (Fig. 3d, Supplementary Video 2). Two of the four DRP1 surfaces that engage the DRR of MID49/51 (receptor interfaces 1 and 2, Fig. 2a) are inaccessible in the nucleotide-free state but become available for binding upon nucleotide-driven elongation (Fig. 3d, Supplementary Video 2).
Title	Nucleotide-induced conformational changes enable receptor binding
Section	GTP-hydrolysis induces filaments to curl into rings We next evaluated the dynamics of the DRP1+MID49 filaments in the presence of hydrolyzable GTP, rather than the non-hydrolyzable analog used for 3D reconstruction. Following copolymerization in the presence of the non-hydrolyzable analog, we exchanged GMPPCP for GTP through dialysis and followed the reaction using negative stain TEM at sequential time points until the GTP was exhausted. We observed that the linear, three-sided DRP1-MID49 cofilaments disassembled into shorter, single-sided filaments before disassembling entirely upon complete hydrolysis to GDP (Fig. 4). The thinner single-sided filaments seen at intermediate time points resembled the single-sided filaments we observed after mutagenizing a salt bridge that appears to hold the triangular structure together (Extended data fig. 3e-i). The single-sided filaments, moreover, exhibited intriguing dynamics at intermediate time points (Fig. 4b-c). Specifically, upon reaching a reproducibly narrow range of lengths, the nearly linear single-sided filaments spontaneously curled into closed rings of remarkably uniform diameter (Fig. 4c).
Title	GTP-hydrolysis induces filaments to curl into rings
Section	A model for closed DRP1 rings In a separate but related experiment, we also evaluated the assembly properties of the DRP1 mutant G362D—which disrupts receptor interface 3—with and without MID49126-454. As described above, this disease-associated residue sits at the base of the L1NS loop and this loop is a key inter-stalk interaction between adjacent DRP1 molecules in the linear filament (Figs. 1c, 2b, Extended Data Fig. 6b, e). We found that DRP1G362D purified as a nearly monodisperse and stable dimer, rather than a mix of tetramers and higher order species observed for the wild-type, full-length protein (Extended Data Fig. 8a). In addition, DRP1G362D exclusively formed rings, not filaments, with or without MID49126-454 and in the presence of GTP or GMPPCP (Fig. 5a, Extended Data Figs. 8i-l). These rings resembled those observed with wild-type DRP1 in all respects except that the wild-type protein only formed closed rings from the linear MID49 copolymer through the path-dependent reaction described above (Fig. 4c versus Fig. 5a). We also observed that these apparently closed DRP1G362D rings could constrict liposomes into membrane tubules and circumscribe lipid nanotubes (Extended Data Fig. 9). Switching to the non-hydrolyzable GTP analog GMPPCP with the DRP1G362D rings improved structural homogeneity, presumably because the rings remain dynamic and eventually disassembled upon complete hydrolysis to GDP (Fig. 4). We imaged the GMPPCP-bound Drp1G362D rings using cryoEM and used 2D class averages of the predominant 12-dimer closed ring to model the differences between the linear filaments and the closed rings (Fig. 5, Extended Data Fig. 10). To account for the projected ring density, the G-domain and the BSE of DRP1 must move even further down toward the stalks. Stalk interface-2 remains constant, as revealed by the “X-shaped” dimer seen in projection (Fig. 5d, e). The curvature of the ring, however, dictates that stalk interfaces 1 and 3, and the conformations of the L1NS and L2S loops, must be extensively remodeled to allow a ~30 degree bending per dimer in comparison with the linear DRP1-MID49 copolymer (Fig. 5d-e, Extended Data Fig. 10e-f, Supplementary Videos 2–3). We did not observe any density for MID49 in the wild-type rings that form by curling of the MID49-DRP1 cofilament in the presence of GTP, nor in our higher-resolution analysis of the Drp1G362D rings that form with or without MID49 present (Fig. 5b-c, Extended Data Fig. 8i-l, 10). This suggests that MID49 binding is incompatible with the curled state of the ring-shaped oligomer, and that constriction therefore requires receptor dissociation (Supplementary Video 3).
Title	A model for closed DRP1 rings
Section	Discussion We note that with an inner diameter of ~16nm, the closed ring may be sufficient to sever a double-membrane mitochondrion if both the outer and inner membranes are compressed together. Alternatively, if inner membrane fission is separable and precedes outer membrane fission, a 16nm diameter suggests that a single membrane tubule would be stabilized by these rings. The structures we observe in vitro may therefore correspond with a highly-constricted but pre-fission state observed in vitro50 and in living cells when another dynamin-family protein, dynamin-2, is depleted29. Constriction by DRP1, therefore, may stabilize the high degree of membrane curvature that is suitable for the recruitment and final fission event catalyzed by additional dynamin-family enzymes. Together, these findings establish four advances. First, our cryoEM structure and mutagenesis studies revealed how receptor proteins like MID49/MID51 recruit and stabilize a specific nucleotide-bound conformation of DRP1 and initiate polymerization of a cofilament. We speculate that the nearly linear properties of this polymer have adapted to encircle low-curvature mitochondrial tubules. Second, analysis of the MID49-DRP1 copolymer revealed how guanine nucleotide binding induces a conformational rearrangement to expose an avid network of receptor binding sites. We now understand these nucleotide-driven allosteric transformations in the context of both full-length and oligomeric DRP1. Third, a path-dependent constriction reaction revealed GTP-dependent conformational dynamics. In this reaction, nucleotide exchange and hydrolysis led to MID49/51 receptor dissociation, disassembly from the ends of the linear filament, and concomitant curling of the shortening filaments into closed rings. The requirement for MID49/51 receptor dissociation before constriction may explain how overexpression of the MID receptors inhibits mitochondrial fission21. Fourth and finally, analysis of a disease mutant in the L1NS loop, Drp1G362D, highlights this loop as a cardinal determinant of receptor binding as well as the inter-stalk interactions that govern oligomer geometry. Together, these observations teach us how DRP1 performs mechanical work by curling from linear filaments into closed rings around mitochondria.
Title	Discussion
Section	Methods Construct design Wild type (WT) DRP1 isoform 2 sequence was purchased from DNASU (sequence ID HsCD00043627, UNIPROT identifier: O00429-3, also known as Dlp1a) and was cloned into pET16b plasmid (Novagen) between the Nde1 and BamH1 sites. The vector was kindly provided by the laboratory of Wesley I. Sundquist with a 10X-His tag followed by a PreScission protease site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro). Wild-type MID49126-454 sequence was PCR amplified and cloned into pGEX6p1 vector having an N-terminal GST tag followed by a PreScission protease site. Site directed mutagenesis was performed on pET16b-DRP1 and pGEX6p1-MID49126-454 using the Gibson cloning method to introduce mutations51. All constructs were verified using Sanger sequencing. Protein purification Protein purification was performed as described52. Briefly, plasmids containing the WT DRP1 or MID49126-454 sequence were transformed in the BL21-DE3 (RIPL) strain of E. coli. The colonies were inoculated in LB culture medium and grown overnight. Secondary inoculations were done the next morning in ZY medium for auto-induction53,54. The cultures were grown to an OD600 of 0.8 at 37°C in baffled flasks and were shifted to 19°C to grow for another 12 hours. The cultures were spun down and the bacterial pellets were used for protein purification immediately or stored at −80°C. Full length DRP1 WT and mutant variants were purified as described previously for DRP1 WT with modifications25. Briefly, the bacterial pellets were resuspended in buffer A (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 40 mM imidazole, 1 mM DTT, 0.5 mg DNase (Roche) and protease inhibitors (10 mM pepstatin, 50 mM PMSF, 0.5 mM aprotinin and 2 mM leupeptin), followed by cell disruption with a probe sonicator. Lysates were cleared by centrifugation at 40,000×g in Beckman JA 25.50 rotors for 60 min at 4°C. The supernatant was filtered using a 0.45 μm filter and applied to Ni-NTA Agarose beads pre-equilibrated with buffer B (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 40 mM imidazole, 1 mM DTT). Upon the application of the supernatant, the beads were washed with 20 column volumes each of buffer B and buffer C (50 mM HEPES/NaOH (pH 7.5), 800 mM NaCl, 5 mM MgCl2, 40 mM imidazole, 1 mM DTT, 1 mM ATP, 10 mM KCl) followed by buffer D (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 80 mM imidazole, 1 mM DTT, 0.5% (w/v) CHAPS). A final pre-elution wash was done with 20 column volumes of buffer B. Bound DRP1 was eluted with buffer E (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 300 mM imidazole, 1 mM DTT) and dialyzed overnight at 4°C against buffer B without imidazole in the presence of PreScission protease to cleave the N-terminal 10X-His tag. The protein was re-applied to a Ni-NTA column pre-equilibrated with dialysis buffer and was observed to bind the column without the 10X-His tag as well. Subsequently, the protein was eluted with buffer B containing 80 mM imidazole. Pure protein was concentrated with a 30kDa molecular weight cutoff (MWCO) centrifugal concentration device (Millipore). In a final step, DRP1 was purified by size-exclusion chromatography (SEC) on a Superdex-200 column (GE) in buffer F containing 20 mM HEPES/NaOH (pH 7.5), 300 mM NaCl, 2.5 mM MgCl2 and 1 mM DTT. Fractions containing DRP1 were pooled, concentrated, flash-frozen as single use aliquots in liquid nitrogen and stored at −80°C. Exact masses for purified DRP1 proteins were validated by MALDI-TOF mass spectrometry. MID49126-454 was purified as described with the following modifications37. pGEX6p1-MID49126-454 plasmid DNA (human, UNIPROT identifier: Isoform 1 Q96C03-1, also known as MIEF2) was transformed in BL21 (DE3) RIPL cells. The colonies were grown overnight in LB medium and secondary cultures were grown in ZY medium. Cells were grown to an OD600 of 0.8-1, collected by centrifugation and processed immediately or stored at −80°C as described above. The bacterial pellets were lysed as described above in MID-buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT and 0.1% (v/v) Triton X-100). The lysates were pre-cleared at 40,000×g and filtered using a 0.45 μm filter before applying to 3 ml glutathione sepharose beads (GE). After overnight binding to beads, the unbound protein was removed and the beads were washed using 20 column volumes each of MID-buffer A and MID-buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT). The protein was eluted with MID-buffer C (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT and 20 mM reduced glutathione). The eluate was cleaved overnight with PreScission protease while dialyzing against MID-buffer D (20 mM Tris pH 8.0, 100 mM NaCl, 5% glycerol, 1 mM DTT). Cleaved protein was further purified using ion-exchange chromatography using a Q sepharose (GE) column. The low salt buffer for ion-exchange was the same as MID-buffer D and the high salt buffer was MID-buffer E (20 mM Tris pH 8.0, 1 M NaCl, 5% glycerol, 1 mM DTT). The relevant MID49126-454 fractions were pooled, concentrated and further purified using an SEC column pre-equilibrated with MID-buffer F (20 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol, 1 mM DTT). The fractions containing MID49126-454 were pooled, concentrated, flash-frozen in liquid nitrogen and stored as single use aliquots at −80°C. Filament assembly, EM sample preparation, data acquisition and processing To assemble DRP1-MID49126-454 filaments, the proteins were mixed to a final concentration of 2 μM each and kept for an hour at room temperature. The mixture was dialyzed against assembly buffer- 20 mM HEPES pH 7.5, 50 mM KCl, 3 mM MgCl2, 1 mM DTT and 200 μM GMPPCP with or without 0.2% octyl-glucopyranoside (Anatrace). The filaments were observed using negative stain TEM or cryoEM after vitrification. Under these conditions, the mutant DRP1S611D failed to coassemble with MID49, but upon further lowering the ionic strength to 25mM KCl, DRP1S611D displayed detectable but greatly reduced coassembly compared to WT protein. For vitrification, the sample was applied to Quantifoil holey carbon grids (R2/2) using a Vitrobot Mark III with 3.5 μl sample, 5 seconds blotting time and a 0 mm offset at 19°C and 100% humidity. Images were collected on an FEI T30 Polara operating at 300kV at a magnification of 31000X. Images were recorded on a Gatan K2 summit camera in super resolution mode for a final binned pixel size of 1.22 Å/pixel. The movies were dose fractionated, contained 30-40 frames, had a total exposure time of 6-8 seconds with 0.2 seconds per frame and a per-frame dose of 1.1 to 1.4 electrons/Å2. SerialEM was used to automate data collection55. The defocus range was 0.8-3 μm under focus. The data was motion corrected and dose-weighted using UCSF Motioncor256. CTF parameter estimation on the non-dose-weighted but motion-corrected stacks was done using CTFFIND4 and GCTF57,58. Filaments were boxed using the program e2helixboxer.py from the EMAN2 suite59. Particle coordinates were used to extract discrete particles using RELION 1.3-1.460 and all further processing was done within the RELION suite. Multiple rounds of 2D classification identified the well-ordered segments. 3D autorefine was run using a customized Relion1.2 version with the IHRSR algorithm implemented61,62. The consensus helical structure was used to classify the particles without refining helical symmetry (using RELION 1.4), resulting in 2 major classes that differed slightly in rise and twist (Extended data figure 2c). Particles from each class were selected and independently refined again with helical RELION 1.2 and IHRSR. Analysis of these reconstructions revealed that each structure was comprised of three linear filaments that bundle together to form a structure that resembled a triangle in cross-section (Extended data figures 2–3). The vertices of the triangle are formed through asymmetric interactions between the G-domains in adjacent filaments. The triangular arrangement of the bundled helices is unlikely to correspond to a biologically meaningful architecture, and this structure cannot form if the MID49 receptor is embedded in the outer mitochondrial membrane. To further improve signal-to-noise, each of the three filaments in each independent half-map was segmented, extracted, resampled on a common grid and summed using UCSF Chimera63–67. The respective symmetrized but unfiltered half maps from each class were again aligned to a common grid and summed according to the C2 symmetry axis of the DRP1 dimer. In a last step, relion_postprocess was used to add the resulting and fully symmetrized half maps (Extended data figure 2c). These half maps and the final summed map, with differential B-factor sharpening per region (Extended data figures 2c, 4–5), were used for atomic modeling using Rosetta as described below. For the projection structure of the DRP1G362D rings, 2 μM protein was mixed 1:1 molar ratio with MID49126-454 and was allowed to sit at room temperature for an hour. The mixture was dialyzed against the assembly buffer (without detergent) overnight. The sample was collected after 12 hours and vitrified using ultra-thin 3 nm carbon support films (Ted Pella). For vitrification, a Mark III vitrobot was used with 3.5 μl sample, 0 mm offset, 100% humidity and 3.5 seconds blot time. The images were collected using an FEI TF20 microscope and SerialEM for automated data collection. The data were recorded with a Gatan K2 camera operating in super resolution mode to collect dose fractionated movie stacks with a final binned pixel size of 1.234 Å/pixel. 40 frames were collected per stack (0.2 seconds per frame and 1.42 electrons/Å2). The movie stacks were motion corrected and the parameters of the transfer function were estimated as described above. Approximately 2000 particles were picked manually for initial 2D classification in RELION 1.4 and these averages were used as templates for further particle picking by Gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/). Final 2D averages of the entire rings versus quarter segments of the rings were computed using Relion1.4. Liposome and Nanotube reactions Liposomes were made as described before52. Briefly, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) was purchased from Avanti Polar Lipids. DOPS dissolved in chloroform was dried under a steady stream of nitrogen and dried under vacuum for an hour. The dried lipid film was resuspended in n-hexane and dried again under nitrogen. The resulting lipid film was dried under vacuum for 4 hours and was finally resuspended in 20 mM HEPES pH 7.5 and 150 mM KCl. The same protocol was followed for making nanotubes, where the mixture contained 60% D-galactosyl-ß-1,1′ N-nervonoyl-D-erythro-sphingosine (Galactosyl Ceramide), 30% DOPS and 10% Ni2+-NTA DOGS (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] Nickel Salt). For assembly reactions of DRP1G362D over lipid, 0.5-2μM protein was incubated with liposomes or nanotubes for an hour and dialyzed against the assembly buffer without detergent. Model building The general procedure for atomic model interpretation and validation using Rosetta were performed as described68. To obtain an initial model for DRP1, the crystal structure of nucleotide-free DRP1 (PDB ID: 4BEJ)25 was used for the stalk region and DRP1 G domain-BSE structures bound to GMPPCP (PDB ID: 3W6O) were used for the G-domain and BSE regions. Density-guided model completion for DRP1 was carried out with RosettaCM69 using this hybridization of DRP1 crystal structures. A converged solution appeared from the low-energy ensemble of the complete models generated by RosettaCM. However, among the low-energy ensemble, residues 503-610 were found to be extremely flexible without cryoEM density constraints and therefore were omitted for further coordinate refinement. For MID49, the highly homologous mouse MID49 crystal structure (81.3% identity, PDB ID: 4WOY, Extended Data Fig. 5c-d)37 was used to generate a homology model using RosettaCM and used as the starting model. To enable fragment-based, density-guided model refinement with missing residues (503-610, DRP1), Rosetta iterative local rebuilding tool was customized to disallow backbone rebuilding at breaks within a single chain. Multiple rounds of refinement were done for each component against one half map (training map), and the other half map (validation map) was used to monitor overfitting according to the detailed procedure described in Wang et al.68. With the refined model of DRP1 and MID49, we further refined the model in the context of a full assembly that included 8 identical copies of each protein, Mg2+ and nucleotide which included all possible inter-domain molecular interactions in the filament (Extended data figure 5a-b). Pseudo-symmetry was used70 to enable and facilitate the energy evaluations of all neighboring interactions around the asymmetric unit (Green model, shown in Extended data figure 5a) for final model refinement of the full assembly. To this end, refinement was done against the training map. Finally, the half maps were used to determine a weight for the density map that did not introduce overfitting. Using the weight and with the symmetry imposed, the whole assembly of DRP1 and MID49 was refined in the full map, followed by B-factors refinement71. Finally, quantification of buried surface area and the number and nature of the bonds involved for each DRP1-MID49 interaction interface modeled by Rosetta were performed with the PISA server (http://www.ebi.ac.uk/pdbe/pisa/). Visual evaluations of the model-to-map correspondence was carried out in UCSF Chimera using unfiltered and unsharpened maps, maps uniformly sharpened with a range of ad hoc B-factors, and maps processed with a model-based local sharpening and local low-pass filtering procedure to optimize contrast and the visibility of high-resolution features of the map72. To build a molecular model for the closed 12-dimer DRP1 rings, we used the diameter, thickness, and angles revealed by the 2D cryoEM class averages of the DRP1G362D rings stabilized with GMPPCP. The atomic coordinates determined above using RosettaCM were used to build the ring in sections, first with repeating dimers of the interface-2 “X-shaped” stalk, then the BSE and finally the G-domains and the angles between these sections were iteratively adjusted until calculated projections of the molecular model corresponded with the features of the experimental projection densities. Both the top (Fig 5b-c) and the side view (Extended data Figure 10b) were used as constraints. The complete atomic model of ring was finally refined in Phenix73 to minimize clashes.
Title	Methods
Section	Construct design Wild type (WT) DRP1 isoform 2 sequence was purchased from DNASU (sequence ID HsCD00043627, UNIPROT identifier: O00429-3, also known as Dlp1a) and was cloned into pET16b plasmid (Novagen) between the Nde1 and BamH1 sites. The vector was kindly provided by the laboratory of Wesley I. Sundquist with a 10X-His tag followed by a PreScission protease site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro). Wild-type MID49126-454 sequence was PCR amplified and cloned into pGEX6p1 vector having an N-terminal GST tag followed by a PreScission protease site. Site directed mutagenesis was performed on pET16b-DRP1 and pGEX6p1-MID49126-454 using the Gibson cloning method to introduce mutations51. All constructs were verified using Sanger sequencing.
Title	Construct design
Section	Protein purification Protein purification was performed as described52. Briefly, plasmids containing the WT DRP1 or MID49126-454 sequence were transformed in the BL21-DE3 (RIPL) strain of E. coli. The colonies were inoculated in LB culture medium and grown overnight. Secondary inoculations were done the next morning in ZY medium for auto-induction53,54. The cultures were grown to an OD600 of 0.8 at 37°C in baffled flasks and were shifted to 19°C to grow for another 12 hours. The cultures were spun down and the bacterial pellets were used for protein purification immediately or stored at −80°C. Full length DRP1 WT and mutant variants were purified as described previously for DRP1 WT with modifications25. Briefly, the bacterial pellets were resuspended in buffer A (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 40 mM imidazole, 1 mM DTT, 0.5 mg DNase (Roche) and protease inhibitors (10 mM pepstatin, 50 mM PMSF, 0.5 mM aprotinin and 2 mM leupeptin), followed by cell disruption with a probe sonicator. Lysates were cleared by centrifugation at 40,000×g in Beckman JA 25.50 rotors for 60 min at 4°C. The supernatant was filtered using a 0.45 μm filter and applied to Ni-NTA Agarose beads pre-equilibrated with buffer B (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 40 mM imidazole, 1 mM DTT). Upon the application of the supernatant, the beads were washed with 20 column volumes each of buffer B and buffer C (50 mM HEPES/NaOH (pH 7.5), 800 mM NaCl, 5 mM MgCl2, 40 mM imidazole, 1 mM DTT, 1 mM ATP, 10 mM KCl) followed by buffer D (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 80 mM imidazole, 1 mM DTT, 0.5% (w/v) CHAPS). A final pre-elution wash was done with 20 column volumes of buffer B. Bound DRP1 was eluted with buffer E (50 mM HEPES/NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 300 mM imidazole, 1 mM DTT) and dialyzed overnight at 4°C against buffer B without imidazole in the presence of PreScission protease to cleave the N-terminal 10X-His tag. The protein was re-applied to a Ni-NTA column pre-equilibrated with dialysis buffer and was observed to bind the column without the 10X-His tag as well. Subsequently, the protein was eluted with buffer B containing 80 mM imidazole. Pure protein was concentrated with a 30kDa molecular weight cutoff (MWCO) centrifugal concentration device (Millipore). In a final step, DRP1 was purified by size-exclusion chromatography (SEC) on a Superdex-200 column (GE) in buffer F containing 20 mM HEPES/NaOH (pH 7.5), 300 mM NaCl, 2.5 mM MgCl2 and 1 mM DTT. Fractions containing DRP1 were pooled, concentrated, flash-frozen as single use aliquots in liquid nitrogen and stored at −80°C. Exact masses for purified DRP1 proteins were validated by MALDI-TOF mass spectrometry. MID49126-454 was purified as described with the following modifications37. pGEX6p1-MID49126-454 plasmid DNA (human, UNIPROT identifier: Isoform 1 Q96C03-1, also known as MIEF2) was transformed in BL21 (DE3) RIPL cells. The colonies were grown overnight in LB medium and secondary cultures were grown in ZY medium. Cells were grown to an OD600 of 0.8-1, collected by centrifugation and processed immediately or stored at −80°C as described above. The bacterial pellets were lysed as described above in MID-buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT and 0.1% (v/v) Triton X-100). The lysates were pre-cleared at 40,000×g and filtered using a 0.45 μm filter before applying to 3 ml glutathione sepharose beads (GE). After overnight binding to beads, the unbound protein was removed and the beads were washed using 20 column volumes each of MID-buffer A and MID-buffer B (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT). The protein was eluted with MID-buffer C (50 mM Tris pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM DTT and 20 mM reduced glutathione). The eluate was cleaved overnight with PreScission protease while dialyzing against MID-buffer D (20 mM Tris pH 8.0, 100 mM NaCl, 5% glycerol, 1 mM DTT). Cleaved protein was further purified using ion-exchange chromatography using a Q sepharose (GE) column. The low salt buffer for ion-exchange was the same as MID-buffer D and the high salt buffer was MID-buffer E (20 mM Tris pH 8.0, 1 M NaCl, 5% glycerol, 1 mM DTT). The relevant MID49126-454 fractions were pooled, concentrated and further purified using an SEC column pre-equilibrated with MID-buffer F (20 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol, 1 mM DTT). The fractions containing MID49126-454 were pooled, concentrated, flash-frozen in liquid nitrogen and stored as single use aliquots at −80°C.
Title	Protein purification
Section	Filament assembly, EM sample preparation, data acquisition and processing To assemble DRP1-MID49126-454 filaments, the proteins were mixed to a final concentration of 2 μM each and kept for an hour at room temperature. The mixture was dialyzed against assembly buffer- 20 mM HEPES pH 7.5, 50 mM KCl, 3 mM MgCl2, 1 mM DTT and 200 μM GMPPCP with or without 0.2% octyl-glucopyranoside (Anatrace). The filaments were observed using negative stain TEM or cryoEM after vitrification. Under these conditions, the mutant DRP1S611D failed to coassemble with MID49, but upon further lowering the ionic strength to 25mM KCl, DRP1S611D displayed detectable but greatly reduced coassembly compared to WT protein. For vitrification, the sample was applied to Quantifoil holey carbon grids (R2/2) using a Vitrobot Mark III with 3.5 μl sample, 5 seconds blotting time and a 0 mm offset at 19°C and 100% humidity. Images were collected on an FEI T30 Polara operating at 300kV at a magnification of 31000X. Images were recorded on a Gatan K2 summit camera in super resolution mode for a final binned pixel size of 1.22 Å/pixel. The movies were dose fractionated, contained 30-40 frames, had a total exposure time of 6-8 seconds with 0.2 seconds per frame and a per-frame dose of 1.1 to 1.4 electrons/Å2. SerialEM was used to automate data collection55. The defocus range was 0.8-3 μm under focus. The data was motion corrected and dose-weighted using UCSF Motioncor256. CTF parameter estimation on the non-dose-weighted but motion-corrected stacks was done using CTFFIND4 and GCTF57,58. Filaments were boxed using the program e2helixboxer.py from the EMAN2 suite59. Particle coordinates were used to extract discrete particles using RELION 1.3-1.460 and all further processing was done within the RELION suite. Multiple rounds of 2D classification identified the well-ordered segments. 3D autorefine was run using a customized Relion1.2 version with the IHRSR algorithm implemented61,62. The consensus helical structure was used to classify the particles without refining helical symmetry (using RELION 1.4), resulting in 2 major classes that differed slightly in rise and twist (Extended data figure 2c). Particles from each class were selected and independently refined again with helical RELION 1.2 and IHRSR. Analysis of these reconstructions revealed that each structure was comprised of three linear filaments that bundle together to form a structure that resembled a triangle in cross-section (Extended data figures 2–3). The vertices of the triangle are formed through asymmetric interactions between the G-domains in adjacent filaments. The triangular arrangement of the bundled helices is unlikely to correspond to a biologically meaningful architecture, and this structure cannot form if the MID49 receptor is embedded in the outer mitochondrial membrane. To further improve signal-to-noise, each of the three filaments in each independent half-map was segmented, extracted, resampled on a common grid and summed using UCSF Chimera63–67. The respective symmetrized but unfiltered half maps from each class were again aligned to a common grid and summed according to the C2 symmetry axis of the DRP1 dimer. In a last step, relion_postprocess was used to add the resulting and fully symmetrized half maps (Extended data figure 2c). These half maps and the final summed map, with differential B-factor sharpening per region (Extended data figures 2c, 4–5), were used for atomic modeling using Rosetta as described below. For the projection structure of the DRP1G362D rings, 2 μM protein was mixed 1:1 molar ratio with MID49126-454 and was allowed to sit at room temperature for an hour. The mixture was dialyzed against the assembly buffer (without detergent) overnight. The sample was collected after 12 hours and vitrified using ultra-thin 3 nm carbon support films (Ted Pella). For vitrification, a Mark III vitrobot was used with 3.5 μl sample, 0 mm offset, 100% humidity and 3.5 seconds blot time. The images were collected using an FEI TF20 microscope and SerialEM for automated data collection. The data were recorded with a Gatan K2 camera operating in super resolution mode to collect dose fractionated movie stacks with a final binned pixel size of 1.234 Å/pixel. 40 frames were collected per stack (0.2 seconds per frame and 1.42 electrons/Å2). The movie stacks were motion corrected and the parameters of the transfer function were estimated as described above. Approximately 2000 particles were picked manually for initial 2D classification in RELION 1.4 and these averages were used as templates for further particle picking by Gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/). Final 2D averages of the entire rings versus quarter segments of the rings were computed using Relion1.4.
Title	Filament assembly, EM sample preparation, data acquisition and processing
Section	Liposome and Nanotube reactions Liposomes were made as described before52. Briefly, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) was purchased from Avanti Polar Lipids. DOPS dissolved in chloroform was dried under a steady stream of nitrogen and dried under vacuum for an hour. The dried lipid film was resuspended in n-hexane and dried again under nitrogen. The resulting lipid film was dried under vacuum for 4 hours and was finally resuspended in 20 mM HEPES pH 7.5 and 150 mM KCl. The same protocol was followed for making nanotubes, where the mixture contained 60% D-galactosyl-ß-1,1′ N-nervonoyl-D-erythro-sphingosine (Galactosyl Ceramide), 30% DOPS and 10% Ni2+-NTA DOGS (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] Nickel Salt). For assembly reactions of DRP1G362D over lipid, 0.5-2μM protein was incubated with liposomes or nanotubes for an hour and dialyzed against the assembly buffer without detergent.
Title	Liposome and Nanotube reactions
Section	Model building The general procedure for atomic model interpretation and validation using Rosetta were performed as described68. To obtain an initial model for DRP1, the crystal structure of nucleotide-free DRP1 (PDB ID: 4BEJ)25 was used for the stalk region and DRP1 G domain-BSE structures bound to GMPPCP (PDB ID: 3W6O) were used for the G-domain and BSE regions. Density-guided model completion for DRP1 was carried out with RosettaCM69 using this hybridization of DRP1 crystal structures. A converged solution appeared from the low-energy ensemble of the complete models generated by RosettaCM. However, among the low-energy ensemble, residues 503-610 were found to be extremely flexible without cryoEM density constraints and therefore were omitted for further coordinate refinement. For MID49, the highly homologous mouse MID49 crystal structure (81.3% identity, PDB ID: 4WOY, Extended Data Fig. 5c-d)37 was used to generate a homology model using RosettaCM and used as the starting model. To enable fragment-based, density-guided model refinement with missing residues (503-610, DRP1), Rosetta iterative local rebuilding tool was customized to disallow backbone rebuilding at breaks within a single chain. Multiple rounds of refinement were done for each component against one half map (training map), and the other half map (validation map) was used to monitor overfitting according to the detailed procedure described in Wang et al.68. With the refined model of DRP1 and MID49, we further refined the model in the context of a full assembly that included 8 identical copies of each protein, Mg2+ and nucleotide which included all possible inter-domain molecular interactions in the filament (Extended data figure 5a-b). Pseudo-symmetry was used70 to enable and facilitate the energy evaluations of all neighboring interactions around the asymmetric unit (Green model, shown in Extended data figure 5a) for final model refinement of the full assembly. To this end, refinement was done against the training map. Finally, the half maps were used to determine a weight for the density map that did not introduce overfitting. Using the weight and with the symmetry imposed, the whole assembly of DRP1 and MID49 was refined in the full map, followed by B-factors refinement71. Finally, quantification of buried surface area and the number and nature of the bonds involved for each DRP1-MID49 interaction interface modeled by Rosetta were performed with the PISA server (http://www.ebi.ac.uk/pdbe/pisa/). Visual evaluations of the model-to-map correspondence was carried out in UCSF Chimera using unfiltered and unsharpened maps, maps uniformly sharpened with a range of ad hoc B-factors, and maps processed with a model-based local sharpening and local low-pass filtering procedure to optimize contrast and the visibility of high-resolution features of the map72. To build a molecular model for the closed 12-dimer DRP1 rings, we used the diameter, thickness, and angles revealed by the 2D cryoEM class averages of the DRP1G362D rings stabilized with GMPPCP. The atomic coordinates determined above using RosettaCM were used to build the ring in sections, first with repeating dimers of the interface-2 “X-shaped” stalk, then the BSE and finally the G-domains and the angles between these sections were iteratively adjusted until calculated projections of the molecular model corresponded with the features of the experimental projection densities. Both the top (Fig 5b-c) and the side view (Extended data Figure 10b) were used as constraints. The complete atomic model of ring was finally refined in Phenix73 to minimize clashes.
Title	Model building
Section	Extended Data Extended Data Figure 1 DRP1 and MID49 assembly states (a-j) DRP1 assembly states visualized with negative stain electron microscopy in the presence of different guanine nucleotides and MID49126-454. Both proteins incubated at 2μM concentration. Bars = 100nm. (k-m) MID49126-454 and MID51132-463 form indistinguishable assemblies with DRP1. (k) DRP1 plus MID49126-454 and GMPPCP, (l) DRP1 plus MID51132-463 and GMPPCP, (m) DRP1 plus both equimolar MID49 and MID51. Bars = 100nm. Extended Data Figure 2 CryoEM and 3D Reconstruction (a) An electron cryo-micrograph of DRP1-MID49126-454 filaments formed with GMPPCP. Bar =100nm. Inset shows a representative 2D class average. Inset bar =10nm. (b) Cross-section of the 3D reconstruction of the filament and the distribution of views determined during helical reconstruction. The length of the cylinders and the color code correspond with the number of particles for that viewing direction (from few to many, blue to red). The 3D structure has been segmented and colored with DRP1 in grey and MID49 in golden yellow. (c) Particle numbers and workflow for the reconstruction protocol. DRP1 density is shown in grey and MID49 is in golden yellow. Extended Data Figure 3 Intra- and Inter-filament Interactions (a) The triangular structure seen in cross section. ‘Side 1’ of the triangular structure has the atomic model placed in the density. The G-domain-to-G-domain contact between adjacent sides is circled. (b) The sum of the 3 sides with the model fit in density. (c) Ribbon diagram of the same atomic model as in (b). The rotated view shows 8 chains each of DRP1 and MID49. The chains further from the reader are rendered transparent. (d) An isolated tetramer of DRP1 from the filament, rendered to highlight the stalk interfaces 1, 2 and 3 observed for DRP1 and other dynamin-family of GTPases. (e) Expanded view of the circular region in (a) illustrating a salt bridge between adjacent G-domains. (f) DRP1-only wild type polymers with GMPPCP versus (g) DRP1 coassembly with wild type MID49126-454 and GMPPCP and high-magnification inset. (h) DRP1E116R mutant polymers versus (i) DRP1E116R mutant coassembly with MiD49126-454. Shorter, single-sided filaments predominate. Disordered “triangular assemblies” were also observed, but were much shorter and infrequent compared with the wild type proteins. Bars = 50nm. Extended Data Figure 4 Resolution Estimates (a-b) Local resolution estimates computed by Resmap74. (a) Histogram of voxel values, (b) Results in A depicted as a heat map of a cross-section through the reconstruction. (c-d) Fourier Shell Correlation plots for (c) the half-maps with and without symmetry and (d) model-to-map correlations for each sub-region of the structure. Extended Data Figure 5 Rosetta-based Model Refinement (a): Complete assembly used for Rosetta-based model building with the asymmetric unit shown in green. (b) Atomic B-factors for one asymmetric unit, DRP1 versus MID49 models (ribbon) and bound GMPPCP (space filling). (c) Sequence alignment between human and mouse MID49 sequences. (d) Overlay of the homology model of human MID49126-454 (blue, with yellow DRR, ribbon) modeled within the cryoEM density overlaid with the mouse MID49 crystal structure (PDB ID: 4WOY, grey ribbon)37 which was used as a constraint. No density attributable to ADP within the nucleotidyltransferase domain was observed. Extended Data figure 6 Map-to-Model Fits and Role of the L1NS and L2S loops Examples of models fit within B-factor sharpened cryoEM density for (a) a helix from the DRP1 stalk, (b) the backbone of the L1NS loop from the stalk, (c) an elongated helix and turn found in MID49, and (d) GMPPCP and Mg within the G-domain. (e) Roles of L1NS and L2S in linear filament formation. Upper panel: An isolated Drp1 tetramer from the cryoEM model. The circled region is expanded in the lower panel. Lower: interactions that the conserved loop L1Ns makes within the assembly. G362 is highlighted. (f) DRP1 stalk and MID49 at receptor interface-3, (g) Dyn3 stalk plus PH domain from PDB 5A3F40, (h) Overlay of (f) and (g). Extended Data Figure 7 Sequence Conservation and Key Interaction Sites (a) Multiple sequence alignment of the regions near and including the DRP1 residues mutated in this study: D190, D221 and G362, G363. The residue numbers apply to human DRP1, isoform 2 (UNIPROT identifier: O00429-3 which is also known as DLP1a). (b) Sequence alignment of MID49 and MID51 at the region around residue R235 of MID49. R235 of MID49 corresponds to R243 of MID51. (c) Sequence alignment of DRP1 Isoform 1 vs Isoform 2 showing the correspondence of S637 (Isoform 1) and S611 (Isoform 2). Extended Data Figure 8 Biochemical and Structural Characterization of Mutants (a-d) Size exclusion chromatography traces for DRP1 wild-type and mutants used in the study. (a) Comparison between wild type (WT) versus G362D, (b) WT versus D221A, and (c) WT versus D190A. (d) Gel filtration standards with annotated molecular weights from the same column and FPLC system. (e-h) DRP1 assembly and coassembly reactions with GMPPCP for (e) DRP1D190A alone, (f) DRP1D190A+MID49126-454, (g) DRP1D221A alone and (h) DRP1D221A+MID49126-454. Bars = 100nm. (i-l) DRP1G362D assembly and coassembly reactions with GMPPCP or GTP. DRP1G362D forms rings but not linear filaments: i,k) without MID49, j,l) with MID49, with i, j) GMPPCP or k, l) GTP. Bars = 100 nm. Extended Data Figure 9 DRP1G362D Rings on Model Membranes (a) DOPS liposomes used in the study. (b) DOPS liposomes after incubation with DRP1G362D showing ring-like assemblies on the membrane and in the background. (c) Lipid nanotubes incubated with DRP1G362D. (d) Power spectrum of the area shown within the dashed square in (b). (e) Power spectrum of the area shown within the dashed square in (c). In both (d) and (e), layer lines indicative of helical geometry are not detectable. Bars = 50nm. Extended Data Figure 10 DRP1G362D forms 12-dimer closed rings (a) 2D class average of the rings. The red dashed circle indicates density that may be attributable to the variable domain. (b) 2D class average of infrequent, orthogonal or “side” views used as a constraint during model building; (c) “top” and (d) “side” projections of the model; (e) “top” and (f) “side” views of the final model rendered as ribbons. Bars =100Å. Green: G-domain, Red: BSE, Purple: Stalk. S
Title	Extended Data
Figure caption	Extended Data Figure 1 DRP1 and MID49 assembly states (a-j) DRP1 assembly states visualized with negative stain electron microscopy in the presence of different guanine nucleotides and MID49126-454. Both proteins incubated at 2μM concentration. Bars = 100nm. (k-m) MID49126-454 and MID51132-463 form indistinguishable assemblies with DRP1. (k) DRP1 plus MID49126-454 and GMPPCP, (l) DRP1 plus MID51132-463 and GMPPCP, (m) DRP1 plus both equimolar MID49 and MID51. Bars = 100nm. E
Title	DRP1 and MID49 assembly states
Figure caption	Extended Data Figure 2 CryoEM and 3D Reconstruction (a) An electron cryo-micrograph of DRP1-MID49126-454 filaments formed with GMPPCP. Bar =100nm. Inset shows a representative 2D class average. Inset bar =10nm. (b) Cross-section of the 3D reconstruction of the filament and the distribution of views determined during helical reconstruction. The length of the cylinders and the color code correspond with the number of particles for that viewing direction (from few to many, blue to red). The 3D structure has been segmented and colored with DRP1 in grey and MID49 in golden yellow. (c) Particle numbers and workflow for the reconstruction protocol. DRP1 density is shown in grey and MID49 is in golden yellow. E
Title	CryoEM and 3D Reconstruction
Figure caption	Extended Data Figure 3 Intra- and Inter-filament Interactions (a) The triangular structure seen in cross section. ‘Side 1’ of the triangular structure has the atomic model placed in the density. The G-domain-to-G-domain contact between adjacent sides is circled. (b) The sum of the 3 sides with the model fit in density. (c) Ribbon diagram of the same atomic model as in (b). The rotated view shows 8 chains each of DRP1 and MID49. The chains further from the reader are rendered transparent. (d) An isolated tetramer of DRP1 from the filament, rendered to highlight the stalk interfaces 1, 2 and 3 observed for DRP1 and other dynamin-family of GTPases. (e) Expanded view of the circular region in (a) illustrating a salt bridge between adjacent G-domains. (f) DRP1-only wild type polymers with GMPPCP versus (g) DRP1 coassembly with wild type MID49126-454 and GMPPCP and high-magnification inset. (h) DRP1E116R mutant polymers versus (i) DRP1E116R mutant coassembly with MiD49126-454. Shorter, single-sided filaments predominate. Disordered “triangular assemblies” were also observed, but were much shorter and infrequent compared with the wild type proteins. Bars = 50nm. E
Title	Intra- and Inter-filament Interactions
Figure caption	Extended Data Figure 4 Resolution Estimates (a-b) Local resolution estimates computed by Resmap74. (a) Histogram of voxel values, (b) Results in A depicted as a heat map of a cross-section through the reconstruction. (c-d) Fourier Shell Correlation plots for (c) the half-maps with and without symmetry and (d) model-to-map correlations for each sub-region of the structure. E
Title	Resolution Estimates
Figure caption	Extended Data Figure 5 Rosetta-based Model Refinement (a): Complete assembly used for Rosetta-based model building with the asymmetric unit shown in green. (b) Atomic B-factors for one asymmetric unit, DRP1 versus MID49 models (ribbon) and bound GMPPCP (space filling). (c) Sequence alignment between human and mouse MID49 sequences. (d) Overlay of the homology model of human MID49126-454 (blue, with yellow DRR, ribbon) modeled within the cryoEM density overlaid with the mouse MID49 crystal structure (PDB ID: 4WOY, grey ribbon)37 which was used as a constraint. No density attributable to ADP within the nucleotidyltransferase domain was observed. E
Title	Rosetta-based Model Refinement
Figure caption	Extended Data figure 6 Map-to-Model Fits and Role of the L1NS and L2S loops Examples of models fit within B-factor sharpened cryoEM density for (a) a helix from the DRP1 stalk, (b) the backbone of the L1NS loop from the stalk, (c) an elongated helix and turn found in MID49, and (d) GMPPCP and Mg within the G-domain. (e) Roles of L1NS and L2S in linear filament formation. Upper panel: An isolated Drp1 tetramer from the cryoEM model. The circled region is expanded in the lower panel. Lower: interactions that the conserved loop L1Ns makes within the assembly. G362 is highlighted. (f) DRP1 stalk and MID49 at receptor interface-3, (g) Dyn3 stalk plus PH domain from PDB 5A3F40, (h) Overlay of (f) and (g). E
Title	Map-to-Model Fits and Role of the L1NS and L2S loops
Figure caption	Extended Data Figure 7 Sequence Conservation and Key Interaction Sites (a) Multiple sequence alignment of the regions near and including the DRP1 residues mutated in this study: D190, D221 and G362, G363. The residue numbers apply to human DRP1, isoform 2 (UNIPROT identifier: O00429-3 which is also known as DLP1a). (b) Sequence alignment of MID49 and MID51 at the region around residue R235 of MID49. R235 of MID49 corresponds to R243 of MID51. (c) Sequence alignment of DRP1 Isoform 1 vs Isoform 2 showing the correspondence of S637 (Isoform 1) and S611 (Isoform 2). E
Title	Sequence Conservation and Key Interaction Sites
Figure caption	Extended Data Figure 8 Biochemical and Structural Characterization of Mutants (a-d) Size exclusion chromatography traces for DRP1 wild-type and mutants used in the study. (a) Comparison between wild type (WT) versus G362D, (b) WT versus D221A, and (c) WT versus D190A. (d) Gel filtration standards with annotated molecular weights from the same column and FPLC system. (e-h) DRP1 assembly and coassembly reactions with GMPPCP for (e) DRP1D190A alone, (f) DRP1D190A+MID49126-454, (g) DRP1D221A alone and (h) DRP1D221A+MID49126-454. Bars = 100nm. (i-l) DRP1G362D assembly and coassembly reactions with GMPPCP or GTP. DRP1G362D forms rings but not linear filaments: i,k) without MID49, j,l) with MID49, with i, j) GMPPCP or k, l) GTP. Bars = 100 nm. E
Title	Biochemical and Structural Characterization of Mutants
Figure caption	Extended Data Figure 9 DRP1G362D Rings on Model Membranes (a) DOPS liposomes used in the study. (b) DOPS liposomes after incubation with DRP1G362D showing ring-like assemblies on the membrane and in the background. (c) Lipid nanotubes incubated with DRP1G362D. (d) Power spectrum of the area shown within the dashed square in (b). (e) Power spectrum of the area shown within the dashed square in (c). In both (d) and (e), layer lines indicative of helical geometry are not detectable. Bars = 50nm. E
Title	DRP1G362D Rings on Model Membranes
Figure caption	Extended Data Figure 10 DRP1G362D forms 12-dimer closed rings (a) 2D class average of the rings. The red dashed circle indicates density that may be attributable to the variable domain. (b) 2D class average of infrequent, orthogonal or “side” views used as a constraint during model building; (c) “top” and (d) “side” projections of the model; (e) “top” and (f) “side” views of the final model rendered as ribbons. Bars =100Å. Green: G-domain, Red: BSE, Purple: Stalk.
Title	DRP1G362D forms 12-dimer closed rings
Section	Supplementary Material 1 2 Sup Table 1 Video 1 Video 2 Video 3
Title	Supplementary Material

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