PMC:4504005 / 7349-22981 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4504005","sourcedb":"PMC","sourceid":"4504005","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4504005","text":"Results\n\nEngineering CLR:RAMP ECD Complexes for Crystallization\nWe previously reported a tethered fusion protein approach to engineer the CLR:RAMP1 and CLR:RAMP2 ECD complexes for crystallization (Moad and Pioszak, 2013), inspired by previous successes using maltose binding protein (MBP) as a “crystallization module” for class B GPCR ECDs (Kumar et al., 2011; Pal et al., 2010; Pioszak et al., 2008, 2009, 2010; Pioszak and Xu, 2008). MBP-RAMP1 or MBP-RAMP2 ECD-CLR ECD fusion proteins in which the two ECDs were covalently tethered with a flexible (Gly-Ser)5 linker were designed to ensure complex stability and enforce 1:1 CLR:RAMP stoichiometry. The tethered RAMP1-CLR ECD fusion was a monomer, whereas the tethered RAMP2-CLR ECD fusion purified as a dimer, but the physiological relevance of oligomerization is unknown. Both proteins selectively bound their respective peptides but failed to yield crystals in the presence of peptides.\nWe reasoned that tether flexibility and oligomerization of the AM1 receptor ECD complex hindered the crystallization efforts. We produced new constructs with a (Gly-Ser-Ala)3 tether designed to decrease flexibility and we identified a single amino acid substitution in the RAMP2 ECD, L106R, which prevented dimerization of the tethered RAMP2-CLR fusion protein (Figure S1A) by disrupting a putative oligomerization interface identified by examining crystal packing in the ligand-free CLR:RAMP2 ECD structure (Kusano et al., 2012). The monomeric RAMP2 L106R-tethered construct retained selectivity for AM over CGRP and bound AM(22-52)NH2 essentially identical to the wild-type tethered fusion in an AlphaScreen competition binding assay (IC50 ∼5–15 μM) (Figures S1B, S1C, and S1H). In a cell-based cAMP signaling assay the full-length AM1 receptor with RAMP2 [L106R] exhibited wild-type response to AM (Figure S1D; Table S4). High-quality crystals of MBP-RAMP2 ECD [L106R]-(GSA)3-CLR ECD grown in the presence of AM(25-52)NH2 were readily obtained (Figure S1E). Crystals of MBP-RAMP1 ECD-(GSA)3-CLR ECD grown in the presence of CGRP(20-37)NH2 diffracted poorly (data not shown); fortunately, high-quality crystals were obtained in the presence of a high-affinity CGRP analog CGRP(27-37)NH2 [D31, P34, F35] (Rist et al., 1998) (Figure S1F). In the competition assay, the CGRP receptor crystallization construct was selective for CGRP over AM and bound the CGRP analog with higher affinity (IC50 ∼0.46 μM) than CGRP(8-37)NH2 (IC50 ∼2 μM) (Figure S1G). The CGRP analog also bound the AM1 receptor crystallization construct with higher affinity than wild-type CGRP but was still lower affinity than AM (Figure S1H). The crystallized proteins thus exhibited peptide selectivity consistent with the intact receptors. The peptides in both crystal forms are antagonist fragments that lack the N-terminal 7TM domain-activating region (Figure S1I). The CGRP analog will hereafter be referred to as CGRPmut.\n\nStructures of the CGRPmut-Bound CLR:RAMP1 and AM-Bound CLR:RAMP2 ECD Heterodimers\nDiffraction data for the CGRPmut- and AM-bound receptor complexes were collected to resolutions of 2.5 and 1.8 Å, respectively (Table 1). The structures were solved by molecular replacement (MR) and refined to good Rwork and Rfree values (Table 1). Three copies of the tethered CGRP receptor fusion and one copy of the tethered AM1 receptor fusion were present in the asymmetric units. Molecule A (Mol A) of the CGRPmut-bound structure had the best electron density and lowest B-factors (Table 1); unless otherwise noted the figures use Mol A. The peptide-bound structures are shown in Figures 1A and 1B. The mFo-DFc electron density maps for the rebuilt MR models showed clear, unambiguous density for CGRPmut and AM (Figures S2A and S2C). MBP sits over the bound peptides, but it does not appear to alter their binding (Figures S2B and S2D). The final models include CGRPmut residues 27–37 and AM residues 35–52 (residues 25–34 were disordered) and the majority of the tethered fusion proteins other than the tethers and ∼20 residues at the C terminus, which were disordered (Figures S2B and S2D). The tethers appeared to be longer than necessary, making it unlikely that they altered RAMP-CLR interactions.\nCGRPmut and AM occupy similar positions near CLR loops 2, 3, and 4; only their C termini are in proximity to the RAMPs (Figures 1A and 1B). Strikingly, CGRPmut adopts a receptor-bound conformation devoid of secondary structure. Receptor-bound AM lacks secondary structure other than one α-helical turn. Shared turn structures near the peptide C termini similarly position the C-terminal residues adjacent to α2 and the α2-α3 loop of the RAMPs. CGRPmut and AM occupy the same face of the CLR ECD as observed for other class B GPCRs with their positions more similar to that of CRF than PTH (Figure 1C). Helix-breaking Pro residues are prevalent in the CGRP, AM, and AM2 sequences and the four residue segment prior to the C-terminal residue contains turn-favoring Pro or Gly residues, consistent with the observed peptide conformations (Figure 1D). The structures are consistent with our knowledge of the architectures of the intact receptor complexes. The ECDs are oriented such that their C termini could continue toward the membrane with a similar number of residues between the termini visible in the structures and the predicted start of the TM segments (∼17 residues for CLR and ∼8 for the RAMPs). The peptides are oriented such that their N termini containing the receptor-activating regions would be directed toward the 7TM domain.\nCGRPmut and AM primarily contact CLR, but key RAMP contacts are also formed (Tables S1 and S2). Two key features of the peptide-binding sites are a hydrophobic patch extending from the base of CLR loop 4 to loop 3 and a pocket extending from the base of CLR loop 4 to loop 2 and the RAMPs (Figure 2). The CLR W72 bulge, previously called the “Trp shelf” (ter Haar et al., 2010), demarcates patch and pocket. The patch comprises the Trp shelf, F92, F95, and Y124. The pockets comprise the Trp shelf, D70, G71, W121, T122, Y124, and RAMP1 W84 and P85 in the CGRP receptor (Figures 2A and 2B) or RAMP2 R97, E101, E105, and P112 in the AM1 receptor (Figures 2C and 2D). CGRPmut G33-A36 and AM S48-G51 form type II β-turns that contact CLR loop 4 in part via hydrogen bonds between the turn main chain and CLR S117, R119, and W121 side chains (Figures 2B and 2D). The β-turns enable the peptide C termini to occupy their respective pockets where their amide groups hydrogen bond with the CLR T122 main chain and their C-terminal residues pack against the Trp shelf, CLR G71, and RAMP1 W84 from the α2-α3 loop, which makes hydrophobic contact with CGRPmut F37 (Figures 2A and 2B), or RAMP2 R97, E101, and E105 from α2, which hydrogen bond with AM Y52 and K46 (Figures 2C and 2D). Prior to the β-turns CGRPmut V32 and AM I47 similarly contact the patch, but moving backward thence the peptides diverge in their interactions. CGRPmut T30 forms main chain- and side chain-mediated hydrogen bonds with CLR D94 on loop 3 and contacts the patch via its side chain methyl group (Figures 2A and 2B). The single helical turn in AM enables K46 to contact the Trp shelf and pack against AM Y52 and AM P43 and A42 contact the patch (Figures 2C and 2D). AM K38-A42 form a series of main chain-mediated hydrogen bonds with the main chain of CLR loop 3 and the side chains of D94 in loop 3 and T37 on α1 (Figure 2D). For the CGRPmut-bound structure, 94% of the solvent accessible surface area (ASA) of the ECD complex buried at the interface with the peptide is from CLR (478 Å2) and only 6% is from RAMP1 (29 Å2). More ASA is buried at the interface with AM, but the majority is still from CLR, 90% (781 Å2), whereas RAMP2 contributes only 10% (85 Å2).\n\nComparisons to Ligand-free and Small Molecule Antagonist-Bound Structures\nSuperpositions of CGRPmut-bound and ligand-free CLR:RAMP1 complexes (ter Haar et al., 2010) revealed clamp-like movement of CLR loops 3 and 4 upon CGRPmut binding, presumably mediated by the CGRPmut T30-CLR D94 interaction and β-turn contacts with CLR loop 4 including the CGRPmut F35-CLR S117 interaction (Figures 3A and 3B). CLR R119 shifts to accommodate the peptide and RAMP1 F83 rotates away from CLR loop 4 (Figure 3B). The RAMP1 position relative to CLR varies in the structures (Figure 3A), but the positions of the α2-α3 loop and W84, which contacts CGRPmut F37, remain relatively similar (Figures 3A and 3B).\nThe CGRPmut-bound structure explains antagonism by the CGRP receptor-selective small molecule drugs olcegepant and telcagepant. Superposition of the CGRPmut-bound and drug-bound structures (ter Haar et al., 2010) indicated that olcegepant and telcagepant block key interactions of the CGRP C-terminal amide and F37 with the receptor pocket by hydrogen bonding with the CLR T122 main chain at the base of the pocket and packing of their piperidyl moieties against the Trp shelf and G71 (Figures 3C and 3D). Olcegepant also hydrogen bonds with CLR D94, thereby blocking the CGRP T30-CLR D94 interaction (Figure 3C). The position of RAMP1 relative to CLR in the peptide-bound versus drug-bound structures varies such that the drugs appear to favor RAMP1 α2 shifting closer to the small molecule binding sites (Figure 3E), presumably due to drug-RAMP1 interactions including packing against W74 (Figures 3C and 3D).\nSuperposition of the AM-bound and ligand-free CLR:RAMP2 complexes (Kusano et al., 2012) revealed minor CLR conformational differences involving the N-terminal region of α1 moving toward AM in the AM-bound state presumably due to AM-CLR T37 contacts (Figure 3F). CLR loops 3 and 4 do not move as in the CGRPmut-bound structure and there are no significant side-chain conformational differences at the peptide-binding site. The position of RAMP2 relative to CLR varies in the two structures (Figure 3F), but the ligand-free RAMP2 position is probably constrained by formation of the dimer of heterodimers in which the C-terminal end of RAMP2 α2 occupies the peptide-binding site of CLR from the opposing heterodimer (Figure 3G). Dimerization of the CLR:RAMP2 ECD heterodimer thus occludes the AM-binding site, which suggested that the RAMP2 L106R substitution (at the end of RAMP2 α2) was key to obtaining AM-bound crystals.\n\nValidation of the Structures for Intact CGRP and AM1 Receptors in Cells\nTo validate the structures, we constructed several Ala substitution mutants in the CLR and RAMP2 ECDs, and they were analyzed for their effects on peptide-stimulated cAMP formation in COS-7 cells. For the CGRP receptor, Ala substitution of CLR W69, D70, K103, or Y91 significantly reduced CGRP potency, likely due to the structural roles of these residues (Figure 4, Table S3). Ala substitution of CLR W72, F92, D94, F95, H114, R119, W121, T122, or Y124, which are contacted by CGRPmut in the structure, reduced potency of the full-length CGRP \u003e20-fold with the CLR D94A mutant being strikingly defective.\nFor the AM1 receptor, Ala substitution of CLR W72, F92, F95, W121, or Y124, which are contacted by AM in the structure, resulted in \u003e40-fold decreases in AM potency (Figure 5A, Table S4). CLR D94, H114, R119, or T122 mutants were less deleterious with 4- to 9-fold decreases in AM potency. Mutation of RAMP2 E101 yielded 26-fold reduced AM potency (Table S4; Watkins et al., 2014). Surprisingly, Ala substitution of RAMP2 R97 or E105 did not affect AM signaling potency despite their contacts with AM (Figure 5B). RAMP2 R97A/E101A and E101A/E105A double mutants had defects similar to the E101A single mutant. These data emphasize that RAMP2 E101 provides the crucial contacts to AM. The Ala substitutions did not significantly alter receptor cell surface expression levels other than reduced AM1 receptor expression with CLR Y124A (Tables S3 and S4).\n\nPeptide Selectivity Determinants\nRAMPs may confer selectivity by providing distinct contacts to the peptides, altering CLR conformation, or a combination of the two. Superposition of the CGRPmut- and AM-bound structures indicated that the RAMPs augment the peptide-binding site pocket with distinct residues from their α2-α3 loop and α2 (Figure 6A). RAMP1 W84 in the α2-α3 loop makes hydrophobic contact with the CGRPmut F37 phenyl ring. This contact would be lost in RAMP2, which has the smaller F111 at the equivalent position. RAMP2 E101 on α2 hydrogen bonds with AM K46 and Y52. The equivalent RAMP1 W74 cannot make these contacts. Two other peptide-proximal RAMP positions differ: RAMP1 F83/RAMP2 G110 on the α2-α3 loop and RAMP1 A70/RAMP2 R97 on α2 (Figure 6B). The F83/G110 position is close to CLR loop 4 and the R119 side chain that has different conformations in the two structures. RAMP2 R97, which participates in the hydrogen bond network near AM Y52, would sterically clash with a Trp at position 111. The small A70 in RAMP1 avoids a clash with W84.\nThe positions of RAMP1 and RAMP2 relative to CLR differ in the two structures and the RAMPs elicit subtly different CLR conformations (Figure 6A). Equivalent RAMP1/2 Cα positions at the end of α2 differ by ∼3 to 4.5 Å such that RAMP2 α2 is closer to the peptide-binding site than RAMP1 α2. A similar 3 to 4.5 Å displacement of the RAMP1/2 α3 helices is accompanied by shifts in the position of the C-terminal end of CLR α1 (Figures 6A and 6B). The RAMPs and CLR α1 appear to move somewhat as a unit relative to the remainder of CLR, which is also evident in the comparisons of the peptide-bound structures to the ligand-free and small molecule antagonist-bound structures (Figures 3A, 3E, and 3F). The subtly different CLR loop 2 positions in the structures may reflect RAMP-dependent differences at the interface with CLR α1 propagated to loop 2 via CLR W69 (Figures 6A and 6B).\nTo explore the contribution of RAMP binding site augmentation to selectivity, we constructed RAMP “swap” mutants in which the four variable residue positions near the peptide C termini were reciprocally exchanged between RAMP1 and RAMP2 (A70/R97, W74/E101, F83/G110, and W84/F111) and we tested their response to CGRP and AM in the cAMP assay (Figures 6C and 6D). The clearest effect was one of a modest decrease in cognate ligand potency. Accordingly, CGRP potency decreased ∼10-fold at the CGRP receptor that included the RAMP1 mutant with RAMP2 residues and AM potency decreased ∼50-fold in the AM1 receptor with the RAMP2 mutant that contained RAMP1 residues. Thus, swapping these RAMP residues was insufficient to exchange pharmacological profiles, but the differing RAMP1/2 positions probably complicated the experiment.\nWe turned to peptide swap experiments to test whether reciprocal exchanges of the C-terminal residues of minimal ECD complex-binding CGRP and AM peptides (Moad and Pioszak, 2013) could exchange their receptor selectivity. In the competition AlphaScreen assay, CGRP(27-37)NH2 [F37Y] retained the ability to bind the CGRP receptor ECD complex (Figure 6E) and did not gain AM-like affinity for the AM1 receptor ECD complex (Figure 6H). CGRPmut [F37Y] retained CGRP receptor ECD complex binding (Figure 6F) and gained the ability to bind the AM1 receptor ECD complex as strongly as AM (Figure 6I). AM(37-52)NH2 [Y52F] exhibited significantly diminished binding to the AM1 receptor ECD complex (Figure 6J) but did not gain increased affinity for the CGRP receptor ECD complex (Figure 6G). These results suggested that the RAMP2 E101-AM Y52 hydrogen bond is a key contributor to AM1 receptor selectivity, whereas Phe as the peptide C-terminal residue is insufficient to confer CGRP receptor selectivity.","divisions":[{"label":"title","span":{"begin":0,"end":7}},{"label":"sec","span":{"begin":9,"end":2937}},{"label":"title","span":{"begin":9,"end":63}},{"label":"p","span":{"begin":64,"end":941}},{"label":"p","span":{"begin":942,"end":2937}},{"label":"sec","span":{"begin":2939,"end":7802}},{"label":"title","span":{"begin":2939,"end":3020}},{"label":"p","span":{"begin":3021,"end":4230}},{"label":"p","span":{"begin":4231,"end":5569}},{"label":"p","span":{"begin":5570,"end":7802}},{"label":"sec","span":{"begin":7804,"end":10331}},{"label":"title","span":{"begin":7804,"end":7877}},{"label":"p","span":{"begin":7878,"end":8496}},{"label":"p","span":{"begin":8497,"end":9408}},{"label":"p","span":{"begin":9409,"end":10331}},{"label":"sec","span":{"begin":10333,"end":11862}},{"label":"title","span":{"begin":10333,"end":10404}},{"label":"p","span":{"begin":10405,"end":11010}},{"label":"p","span":{"begin":11011,"end":11862}},{"label":"title","span":{"begin":11864,"end":11896}},{"label":"p","span":{"begin":11897,"end":12927}},{"label":"p","span":{"begin":12928,"end":13807}},{"label":"p","span":{"begin":13808,"end":14634}}],"tracks":[{"project":"TEST0","denotations":[{"id":"25982113-151-159-8319010","span":{"begin":215,"end":219},"obj":"[\"24115156\", \"24115156\", \"24115156\"]"},{"id":"25982113-234-242-8319011","span":{"begin":356,"end":360},"obj":"[\"21625560\", \"21625560\", \"21625560\"]"},{"id":"25982113-226-234-8319012","span":{"begin":374,"end":378},"obj":"[\"20966082\", \"20966082\", \"20966082\"]"},{"id":"25982113-234-242-8319013","span":{"begin":396,"end":400},"obj":"[\"18801728\", \"18801728\", \"18801728\"]"},{"id":"25982113-226-234-8319014","span":{"begin":402,"end":406},"obj":"[\"19674967\", \"19674967\", \"19674967\"]"},{"id":"25982113-232-240-8319015","span":{"begin":408,"end":412},"obj":"[\"20172855\", \"20172855\", \"20172855\"]"},{"id":"25982113-234-242-8319016","span":{"begin":430,"end":434},"obj":"[\"18375760\", \"18375760\", \"18375760\"]"},{"id":"25982113-233-241-8319017","span":{"begin":1466,"end":1470},"obj":"[\"22102369\", \"22102369\", \"22102369\"]"},{"id":"25982113-221-229-8319018","span":{"begin":2261,"end":2265},"obj":"[\"9438028\", \"9438028\", \"9438028\"]"},{"id":"25982113-71-79-8319019","span":{"begin":5942,"end":5946},"obj":"[\"20826335\", \"20826335\", \"20826335\"]"},{"id":"25982113-87-95-8319020","span":{"begin":7964,"end":7968},"obj":"[\"20826335\", \"20826335\", \"20826335\"]"},{"id":"25982113-79-87-8319021","span":{"begin":8704,"end":8708},"obj":"[\"20826335\", \"20826335\", \"20826335\"]"},{"id":"25982113-82-90-8319022","span":{"begin":9491,"end":9495},"obj":"[\"22102369\", \"22102369\", \"22102369\"]"},{"id":"25982113-85-93-8319023","span":{"begin":11386,"end":11390},"obj":"[\"24199627\", \"24199627\", \"24199627\"]"},{"id":"25982113-173-181-8319024","span":{"begin":14808,"end":14812},"obj":"[\"24115156\", \"24115156\", \"24115156\"]"}],"attributes":[{"subj":"25982113-151-159-8319010","pred":"source","obj":"TEST0"},{"subj":"25982113-234-242-8319011","pred":"source","obj":"TEST0"},{"subj":"25982113-226-234-8319012","pred":"source","obj":"TEST0"},{"subj":"25982113-234-242-8319013","pred":"source","obj":"TEST0"},{"subj":"25982113-226-234-8319014","pred":"source","obj":"TEST0"},{"subj":"25982113-232-240-8319015","pred":"source","obj":"TEST0"},{"subj":"25982113-234-242-8319016","pred":"source","obj":"TEST0"},{"subj":"25982113-233-241-8319017","pred":"source","obj":"TEST0"},{"subj":"25982113-221-229-8319018","pred":"source","obj":"TEST0"},{"subj":"25982113-71-79-8319019","pred":"source","obj":"TEST0"},{"subj":"25982113-87-95-8319020","pred":"source","obj":"TEST0"},{"subj":"25982113-79-87-8319021","pred":"source","obj":"TEST0"},{"subj":"25982113-82-90-8319022","pred":"source","obj":"TEST0"},{"subj":"25982113-85-93-8319023","pred":"source","obj":"TEST0"},{"subj":"25982113-173-181-8319024","pred":"source","obj":"TEST0"}]},{"project":"2_test","denotations":[{"id":"25982113-24115156-118216112","span":{"begin":215,"end":219},"obj":"24115156"},{"id":"25982113-24115156-118216112","span":{"begin":215,"end":219},"obj":"24115156"},{"id":"25982113-21625560-118216113","span":{"begin":356,"end":360},"obj":"21625560"},{"id":"25982113-21625560-118216113","span":{"begin":356,"end":360},"obj":"21625560"},{"id":"25982113-20966082-118216114","span":{"begin":374,"end":378},"obj":"20966082"},{"id":"25982113-20966082-118216114","span":{"begin":374,"end":378},"obj":"20966082"},{"id":"25982113-18801728-118216115","span":{"begin":396,"end":400},"obj":"18801728"},{"id":"25982113-18801728-118216115","span":{"begin":396,"end":400},"obj":"18801728"},{"id":"25982113-19674967-118216116","span":{"begin":402,"end":406},"obj":"19674967"},{"id":"25982113-19674967-118216116","span":{"begin":402,"end":406},"obj":"19674967"},{"id":"25982113-20172855-118216117","span":{"begin":408,"end":412},"obj":"20172855"},{"id":"25982113-20172855-118216117","span":{"begin":408,"end":412},"obj":"20172855"},{"id":"25982113-18375760-118216118","span":{"begin":430,"end":434},"obj":"18375760"},{"id":"25982113-18375760-118216118","span":{"begin":430,"end":434},"obj":"18375760"},{"id":"25982113-22102369-118216119","span":{"begin":1466,"end":1470},"obj":"22102369"},{"id":"25982113-22102369-118216119","span":{"begin":1466,"end":1470},"obj":"22102369"},{"id":"25982113-9438028-118216120","span":{"begin":2261,"end":2265},"obj":"9438028"},{"id":"25982113-9438028-118216120","span":{"begin":2261,"end":2265},"obj":"9438028"},{"id":"25982113-20826335-118216121","span":{"begin":5942,"end":5946},"obj":"20826335"},{"id":"25982113-20826335-118216121","span":{"begin":5942,"end":5946},"obj":"20826335"},{"id":"25982113-20826335-118216122","span":{"begin":7964,"end":7968},"obj":"20826335"},{"id":"25982113-20826335-118216122","span":{"begin":7964,"end":7968},"obj":"20826335"},{"id":"25982113-20826335-118216123","span":{"begin":8704,"end":8708},"obj":"20826335"},{"id":"25982113-20826335-118216123","span":{"begin":8704,"end":8708},"obj":"20826335"},{"id":"25982113-22102369-118216124","span":{"begin":9491,"end":9495},"obj":"22102369"},{"id":"25982113-22102369-118216124","span":{"begin":9491,"end":9495},"obj":"22102369"},{"id":"25982113-24199627-118216125","span":{"begin":11386,"end":11390},"obj":"24199627"},{"id":"25982113-24199627-118216125","span":{"begin":11386,"end":11390},"obj":"24199627"},{"id":"25982113-24115156-71076121","span":{"begin":14808,"end":14812},"obj":"24115156"},{"id":"25982113-24115156-71076121","span":{"begin":14808,"end":14812},"obj":"24115156"},{"id":"25982113-24115156-71076112","span":{"begin":215,"end":219},"obj":"24115156"},{"id":"25982113-24115156-71076112","span":{"begin":215,"end":219},"obj":"24115156"},{"id":"25982113-21625560-71076113","span":{"begin":430,"end":434},"obj":"21625560"},{"id":"25982113-21625560-71076113","span":{"begin":430,"end":434},"obj":"21625560"},{"id":"25982113-20966082-71076113","span":{"begin":430,"end":434},"obj":"20966082"},{"id":"25982113-20966082-71076113","span":{"begin":430,"end":434},"obj":"20966082"},{"id":"25982113-18801728-71076113","span":{"begin":430,"end":434},"obj":"18801728"},{"id":"25982113-18801728-71076113","span":{"begin":430,"end":434},"obj":"18801728"},{"id":"25982113-19674967-71076113","span":{"begin":430,"end":434},"obj":"19674967"},{"id":"25982113-19674967-71076113","span":{"begin":430,"end":434},"obj":"19674967"},{"id":"25982113-20172855-71076113","span":{"begin":430,"end":434},"obj":"20172855"},{"id":"25982113-20172855-71076113","span":{"begin":430,"end":434},"obj":"20172855"},{"id":"25982113-18375760-71076113","span":{"begin":430,"end":434},"obj":"18375760"},{"id":"25982113-18375760-71076113","span":{"begin":430,"end":434},"obj":"18375760"},{"id":"25982113-22102369-71076114","span":{"begin":1466,"end":1470},"obj":"22102369"},{"id":"25982113-22102369-71076114","span":{"begin":1466,"end":1470},"obj":"22102369"},{"id":"25982113-9438028-71076115","span":{"begin":2261,"end":2265},"obj":"9438028"},{"id":"25982113-9438028-71076115","span":{"begin":2261,"end":2265},"obj":"9438028"},{"id":"25982113-20826335-71076116","span":{"begin":5942,"end":5946},"obj":"20826335"},{"id":"25982113-20826335-71076116","span":{"begin":5942,"end":5946},"obj":"20826335"},{"id":"25982113-20826335-71076117","span":{"begin":7964,"end":7968},"obj":"20826335"},{"id":"25982113-20826335-71076117","span":{"begin":7964,"end":7968},"obj":"20826335"},{"id":"25982113-20826335-71076118","span":{"begin":8704,"end":8708},"obj":"20826335"},{"id":"25982113-20826335-71076118","span":{"begin":8704,"end":8708},"obj":"20826335"},{"id":"25982113-22102369-71076119","span":{"begin":9491,"end":9495},"obj":"22102369"},{"id":"25982113-22102369-71076119","span":{"begin":9491,"end":9495},"obj":"22102369"},{"id":"25982113-24199627-71076120","span":{"begin":11386,"end":11390},"obj":"24199627"},{"id":"25982113-24199627-71076120","span":{"begin":11386,"end":11390},"obj":"24199627"},{"id":"T47714","span":{"begin":14808,"end":14812},"obj":"24115156"},{"id":"T56862","span":{"begin":14808,"end":14812},"obj":"24115156"}],"attributes":[{"subj":"25982113-24115156-118216112","pred":"source","obj":"2_test"},{"subj":"25982113-24115156-118216112","pred":"source","obj":"2_test"},{"subj":"25982113-21625560-118216113","pred":"source","obj":"2_test"},{"subj":"25982113-21625560-118216113","pred":"source","obj":"2_test"},{"subj":"25982113-20966082-118216114","pred":"source","obj":"2_test"},{"subj":"25982113-20966082-118216114","pred":"source","obj":"2_test"},{"subj":"25982113-18801728-118216115","pred":"source","obj":"2_test"},{"subj":"25982113-18801728-118216115","pred":"source","obj":"2_test"},{"subj":"25982113-19674967-118216116","pred":"source","obj":"2_test"},{"subj":"25982113-19674967-118216116","pred":"source","obj":"2_test"},{"subj":"25982113-20172855-118216117","pred":"source","obj":"2_test"},{"subj":"25982113-20172855-118216117","pred":"source","obj":"2_test"},{"subj":"25982113-18375760-118216118","pred":"source","obj":"2_test"},{"subj":"25982113-18375760-118216118","pred":"source","obj":"2_test"},{"subj":"25982113-22102369-118216119","pred":"source","obj":"2_test"},{"subj":"25982113-22102369-118216119","pred":"source","obj":"2_test"},{"subj":"25982113-9438028-118216120","pred":"source","obj":"2_test"},{"subj":"25982113-9438028-118216120","pred":"source","obj":"2_test"},{"subj":"25982113-20826335-118216121","pred":"source","obj":"2_test"},{"subj":"25982113-20826335-118216121","pred":"source","obj":"2_test"},{"subj":"25982113-20826335-118216122","pred":"source","obj":"2_test"},{"subj":"25982113-20826335-118216122","pred":"source","obj":"2_test"},{"subj":"25982113-20826335-118216123","pred":"source","obj":"2_test"},{"subj":"25982113-20826335-118216123","pred":"source","obj":"2_test"},{"subj":"25982113-22102369-118216124","pred":"source","obj":"2_test"},{"subj":"25982113-22102369-118216124","pred":"source","obj":"2_test"},{"subj":"25982113-24199627-118216125","pred":"source","obj":"2_test"},{"subj":"25982113-24199627-118216125","pred":"source","obj":"2_test"},{"subj":"25982113-24115156-71076121","pred":"source","obj":"2_test"},{"subj":"25982113-24115156-71076121","pred":"source","obj":"2_test"},{"subj":"25982113-24115156-71076112","pred":"source","obj":"2_test"},{"subj":"25982113-24115156-71076112","pred":"source","obj":"2_test"},{"subj":"25982113-21625560-71076113","pred":"source","obj":"2_test"},{"subj":"25982113-21625560-71076113","pred":"source","obj":"2_test"},{"subj":"25982113-20966082-71076113","pred":"source","obj":"2_test"},{"subj":"25982113-20966082-71076113","pred":"source","obj":"2_test"},{"subj":"25982113-18801728-71076113","pred":"source","obj":"2_test"},{"subj":"25982113-18801728-71076113","pred":"source","obj":"2_test"},{"subj":"25982113-19674967-71076113","pred":"source","obj":"2_test"},{"subj":"25982113-19674967-71076113","pred":"source","obj":"2_test"},{"subj":"25982113-20172855-71076113","pred":"source","obj":"2_test"},{"subj":"25982113-20172855-71076113","pred":"source","obj":"2_test"},{"subj":"25982113-18375760-71076113","pred":"source","obj":"2_test"},{"subj":"25982113-18375760-71076113","pred":"source","obj":"2_test"},{"subj":"25982113-22102369-71076114","pred":"source","obj":"2_test"},{"subj":"25982113-22102369-71076114","pred":"source","obj":"2_test"},{"subj":"25982113-9438028-71076115","pred":"source","obj":"2_test"},{"subj":"25982113-9438028-71076115","pred":"source","obj":"2_test"},{"subj":"25982113-20826335-71076116","pred":"source","obj":"2_test"},{"subj":"25982113-20826335-71076116","pred":"source","obj":"2_test"},{"subj":"25982113-20826335-71076117","pred":"source","obj":"2_test"},{"subj":"25982113-20826335-71076117","pred":"source","obj":"2_test"},{"subj":"25982113-20826335-71076118","pred":"source","obj":"2_test"},{"subj":"25982113-20826335-71076118","pred":"source","obj":"2_test"},{"subj":"25982113-22102369-71076119","pred":"source","obj":"2_test"},{"subj":"25982113-22102369-71076119","pred":"source","obj":"2_test"},{"subj":"25982113-24199627-71076120","pred":"source","obj":"2_test"},{"subj":"25982113-24199627-71076120","pred":"source","obj":"2_test"},{"subj":"T47714","pred":"source","obj":"2_test"},{"subj":"T56862","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"TEST0","color":"#ecb593","default":true},{"id":"2_test","color":"#9b93ec"}]}]}}