PMC:4504005 / 22983-33312 JSONTXT

{"project":"TEST0","denotations":[{"id":"25982113-233-241-8319025","span":{"begin":855,"end":859},"obj":"[\"20966082\", \"20966082\", \"20966082\"]"},{"id":"25982113-233-241-8319026","span":{"begin":878,"end":882},"obj":"[\"17715056\", \"17715056\", \"17715056\"]"},{"id":"25982113-233-241-8319027","span":{"begin":900,"end":904},"obj":"[\"18375760\", \"18375760\", \"18375760\"]"},{"id":"25982113-149-157-8319028","span":{"begin":1056,"end":1060},"obj":"[\"17785463\", \"17785463\", \"17785463\"]"},{"id":"25982113-173-181-8319029","span":{"begin":1080,"end":1084},"obj":"[\"23247088\", \"23247088\", \"23247088\"]"},{"id":"25982113-194-202-8319030","span":{"begin":1101,"end":1105},"obj":"[\"22102369\", \"22102369\", \"22102369\"]"},{"id":"25982113-218-226-8319031","span":{"begin":1125,"end":1129},"obj":"[\"24115156\", \"24115156\", \"24115156\"]"},{"id":"25982113-230-238-8319033","span":{"begin":1714,"end":1718},"obj":"[\"24115156\", \"24115156\", \"24115156\"]"},{"id":"25982113-15-23-8319034","span":{"begin":1736,"end":1740},"obj":"[\"17785463\", \"17785463\", \"17785463\"]"},{"id":"25982113-204-212-8319035","span":{"begin":2504,"end":2508},"obj":"[\"11444978\", \"11444978\", \"11444978\"]"},{"id":"25982113-127-135-8319037","span":{"begin":2833,"end":2837},"obj":"[\"8527874\", \"8527874\", \"8527874\"]"},{"id":"25982113-148-156-8319038","span":{"begin":2854,"end":2858},"obj":"[\"1988044\", \"1988044\", \"1988044\"]"},{"id":"25982113-177-185-8319039","span":{"begin":2883,"end":2887},"obj":"[\"21830197\", \"21830197\", \"21830197\"]"},{"id":"25982113-117-125-8319041","span":{"begin":3497,"end":3501},"obj":"[\"24115156\", \"24115156\", \"24115156\"]"},{"id":"25982113-167-175-8319042","span":{"begin":3547,"end":3551},"obj":"[\"11444978\", \"11444978\", \"11444978\"]"},{"id":"25982113-186-194-8319043","span":{"begin":3566,"end":3570},"obj":"[\"9438028\", \"9438028\", \"9438028\"]"},{"id":"25982113-104-112-8319045","span":{"begin":3700,"end":3704},"obj":"[\"11444978\", \"11444978\", \"11444978\"]"},{"id":"25982113-235-243-8319046","span":{"begin":4255,"end":4259},"obj":"[\"24115156\", \"24115156\", \"24115156\"]"},{"id":"25982113-122-130-8319048","span":{"begin":5450,"end":5454},"obj":"[\"20188075\", \"20188075\", \"20188075\"]"},{"id":"25982113-144-152-8319049","span":{"begin":5472,"end":5476},"obj":"[\"24199627\", \"24199627\", \"24199627\"]"},{"id":"25982113-235-243-8319050","span":{"begin":7485,"end":7489},"obj":"[\"24199627\", \"24199627\", \"24199627\"]"},{"id":"25982113-99-107-8319051","span":{"begin":8202,"end":8206},"obj":"[\"18593822\", \"18593822\", \"18593822\"]"},{"id":"25982113-105-113-8319052","span":{"begin":8208,"end":8212},"obj":"[\"21402116\", \"21402116\", \"21402116\"]"},{"id":"25982113-115-123-8319053","span":{"begin":8330,"end":8334},"obj":"[\"16959943\", \"16959943\", \"16959943\"]"},{"id":"25982113-132-140-8319054","span":{"begin":8347,"end":8351},"obj":"[\"18593822\", \"18593822\", \"18593822\"]"},{"id":"25982113-138-146-8319055","span":{"begin":8353,"end":8357},"obj":"[\"21402116\", \"21402116\", \"21402116\"]"}],"text":"Discussion\nRAMPs are an important class of accessory membrane proteins that modulate GPCR pharmacology. The CGRPmut-bound CLR:RAMP1 ECD and AM-bound CLR:RAMP2 ECD structures presented here expand our understanding of the mechanisms by which peptides can bind to class B GPCRs and increase our understanding of how RAMPs enable peptide selectivity. The engineered tethered ECD fusion proteins used for crystallization exhibited the same peptide selectivity rank order as the intact receptors, which indicated that they are valid reagents for studying selective peptide binding. The purified proteins bound their respective peptides with apparent affinities in the low μM range (Figures S1C, S1G, and S1H), which are lower than the affinities of the agonist peptides for intact receptors but typical for truncated peptides at class B GPCR ECDs (Pal et al., 2010; Parthier et al., 2007; Pioszak and Xu, 2008).\nThe oligomeric states of CLR:RAMP complexes has been a source of debate with evidence for 1:1, 2:1, and 2:2 CLR:RAMP stoichiometries (Héroux et al., 2007; Hill and Pioszak, 2013; Kusano et al., 2012; Moad and Pioszak, 2013; Watkins et al., 2013a). Dimerization of the purified CLR:RAMP2 ECD heterodimer to form a 2:2 complex may be an artifact because the RAMP2 L106R mutation prevented oligomerization yet did not affect AM1 receptor function (Figure S1). Occlusion of the AM-binding site by dimerization explains our inability to measure AM binding to the tethered RAMP2-CLR ECD fusion protein in an assay using μM receptor concentration, whereas AM binding was readily measured in an assay using nM receptor concentration where the dimeric species was likely not significantly present (Moad and Pioszak, 2013). Héroux et al. (2007) provided evidence for a homo-oligomer of CLR with a single RAMP1 as the functional CGRP receptor in cells. We cannot rule out a role for higher-order oligomerization in the function of the intact receptors, but the structures indicate that the 1:1 heterodimers are sufficient to bind peptides.\nThe CGRPmut and AM peptides adopted receptor-bound conformations different from typical α-helical class B GPCR peptide ligands. CGRPmut and AM are characterized by a shared turn structure that positions their C-terminal residue to occupy the pocket near the RAMP. Previous studies indicated the presence of turns in the C-terminal region of CGRP (and an absence of α-helix in this area), but how this region interacted with the receptor was unclear (Carpenter et al., 2001; Watkins et al., 2013b). Prior to the turns, the peptides diverge in their structure and interactions with CLR, but they contact the same area of CLR with little or no peptide secondary structure. NMR structures of CGRP and AM suggested that α-helix is restricted to residues 8-18/22-34 of these peptides (Boulanger et al., 1995; Breeze et al., 1991; Pérez-Castells et al., 2012; Watkins et al., 2013a) and indeed the C-terminal regions of both contain helix-breaking Pro residues (Figure 1D). Accordingly, the lack of substantial helical content in the bound peptide fragments is consistent with what is known about the structures of the full-length peptides. A turn structure and paucity of α-helicity may be a general feature of the receptor ECD-binding portions of CT family peptides.\nThe CGRPmut and AM binding modes are consistent with peptide mutagenesis studies. CGRP T30, V32, and F37 and the C-terminal amide were important for binding purified CLR:RAMP1 ECD (Moad and Pioszak, 2013) and intact CGRP receptor (Carpenter et al., 2001; Rist et al., 1998; Watkins et al., 2013b). Modified CGRP peptides as short as 30–37 maintained the ability to bind the receptor (Carpenter et al., 2001), consistent with this region providing most of the contacts. Increased affinity of CGRPmut over that of CGRP can be explained by their differences in the turn region (Figure 1D). P34 favors β-turn formation better than S34 and F35 provides better hydrophobic contact to CLR loop 4 than K35. AM P43, K46, I47, G51, Y52, and the C-terminal amide were most critical for binding purified CLR:RAMP2 ECD and intact AM1 receptor, and truncation beyond residue 38 diminished binding even though the K38-V41 side chains were not important (Moad and Pioszak, 2013; Watkins et al., 2013a).\nMapping the receptor mutagenesis data (Figures 4 and 5) onto the surface of the receptor structures (Figures 7A and 7B) suggests that CGRP binds in a similar manner to CGRPmut and that the structures are good models for full-length CGRP and AM binding to intact receptors. Mutation of CLR residues that form the shared binding site diminished CGRP and AM potencies, and the effects of some of the mutations were similar for both peptides (e.g., F92). Noteworthy divergent effects of several mutations support the differences in the structures. CLR D94 was far more important for CGRP action than AM, consistent with the crucial role of D94 in contacting CGRP T30 and its less important role in contacting the AM main chain. CLR R119A diminished the potency of CGRP much more than that of AM, which may reflect an important role for the different R119 conformations observed in the two structures. CLR W72A was more deleterious for AM action than CGRP, which is consistent with the greater number of AM contacts to CLR W72 as compared to CGRP.\nRAMP1 W84 and RAMP2 E101 were previously identified as key residues for CGRP and AM function, respectively (Moore et al., 2010; Watkins et al., 2014). These data are explained by how these residues augment the binding site pocket (Figures 7A and 7B). Apparently, packing of the CGRP F37 and AM Y52 phenyl rings against the CLR Trp shelf and G71 is insufficient for strong binding. RAMP1 W84 or RAMP2 E101 is required to complete the pocket to enable strong “anchoring” of the peptide C termini. RAMP2 R97 and E105 also augment the pocket, but the R97A and E105A mutants did not diminish AM potency. These data along with the peptide swap data indicate that the RAMP2 E101-Y52 hydrogen bond is the crucial AM anchoring contact. Ionic interactions of AM K46 and RAMP2 E101/E105 do not appear to be significant. The main role of AM K46 thus appears to be intramolecular packing against Y52 and contacting the Trp shelf.\nDistinct RAMP binding site augmentation clearly contributes to peptide selectivity (Figure 6). RAMP2 E101 favors AM binding because it can hydrogen bond with Y52 and RAMP2 F111 discourages CGRP binding because it is too small to contact the F37 phenyl ring. Indeed, the F37Y swap in CGRPmut conferred strong affinity for the AM1 receptor ECD complex and the Y52F swap in AM(37-52)NH2 significantly diminished its binding. The lack of Glu at RAMP1 position 74 would disfavor strong AM binding. RAMP1 W84 enables strong CGRP binding by contacting F37, but this contact alone is apparently insufficient for selectivity because AM(37-52)NH2 [Y52F] did not gain affinity for the CGRP receptor ECD complex.\nModeling the AM-bound AM2 receptor ECD complex (Supplemental Experimental Procedures) suggests that RAMP3 augments the binding site as a RAMP1-2 hybrid (Figure 7C). RAMP3 E74, which is equivalent to RAMP2 E101, would favor AM binding by hydrogen bonding with Y52. RAMP3 W84, which is equivalent to RAMP1 W84, could contact the AM Y52 and CGRP F37 phenyl rings, thereby explaining diminished potency of both peptides at the AM2 receptor with RAMP3 W84A and why CGRP is more active at the AM2 receptor than the AM1 receptor (Watkins et al., 2014). The key RAMP residues proposed as selectivity determinants are conserved across species: W84 and a lack of Glu at position 74 in RAMP1, E101 and F or Y at position 111 in RAMP2, and E74 and W84 in RAMP3 (Figure 7D). A small amino acid is conserved at position 70 in RAMP1/3, which would avoid steric clash with W84. Notably, the lack of conservation of RAMP2 R97 and E105 is consistent with the mutagenesis data that indicated that these residues are not critical for AM signaling.\nPrevious RAMP single swap mutant studies supported the importance of Glu at position 74/101 as a determinant for AM selectivity. RAMP1 W74E had no effect on CGRP potency but increased AM potency at the CGRP receptor (Qi et al., 2008, 2011). RAMP3 E74W decreased AM potency at the AM2 receptor, while having a negligible effect on CGRP potency (Hay et al., 2006; Qi et al., 2008, 2011). More extensive quadruple swap mutants in this study failed to exchange the pharmacological profiles, but these experiments are complicated by the different RAMP positions relative to CLR and variable RAMP effects on CLR conformation.\nThe failure of the CGRP and AM peptide C-terminal residue swaps to exchange their receptor preferences (Figure 6) strongly suggests that RAMP binding site augmentation alone is insufficient to account for selectivity. Thus, the subtle differences in CLR conformation in the two structures may also be important for selectivity. RAMP-induced changes in CLR R119 side-chain conformation and/or subtle shifting of loop 2 may sufficiently alter the pocket to favor one peptide over the other. Future studies will be required to determine to what extent such allostery contributes to selectivity. Peptide selectivity determinants may also exist in portions of the receptors that were not addressed in this study.\nIn summary, the structures presented here provide the first structural views of any accessory membrane protein modulating GPCR ligand binding and may provide a basis for understanding modulation of other GPCRs by accessory proteins. Our data indicate that RAMPs determine peptide selectivity of CLR through a combination of binding site augmentation and alteration of CLR conformation. It is striking that relatively minor differences in RAMP-specific peptide contacts and subtle RAMP-induced changes in CLR conformation lead to such profoundly different pharmacological profiles. Of practical value, the structures may inform rational drug design targeting CLR:RAMP complexes with clinical relevance for migraine headache and cardiovascular disorders. Lastly, the MBP-tethered ECD fusion protein approach to crystallization should facilitate structural studies of other CT family peptides bound to their respective receptor ECD complexes, which will enable a more complete understanding of how RAMPs modulate both CLR and CTR."}

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egin":900,"end":904},"obj":"18375760"},{"id":"25982113-24115156-71076124","span":{"begin":1714,"end":1718},"obj":"24115156"},{"id":"25982113-24115156-71076124","span":{"begin":1714,"end":1718},"obj":"24115156"},{"id":"25982113-17785463-71076125","span":{"begin":1736,"end":1740},"obj":"17785463"},{"id":"25982113-17785463-71076125","span":{"begin":1736,"end":1740},"obj":"17785463"},{"id":"25982113-24115156-71076128","span":{"begin":3497,"end":3501},"obj":"24115156"},{"id":"25982113-24115156-71076128","span":{"begin":3497,"end":3501},"obj":"24115156"},{"id":"25982113-11444978-71076130","span":{"begin":3700,"end":3704},"obj":"11444978"},{"id":"25982113-11444978-71076130","span":{"begin":3700,"end":3704},"obj":"11444978"},{"id":"25982113-20188075-71076132","span":{"begin":5472,"end":5476},"obj":"20188075"},{"id":"25982113-20188075-71076132","span":{"begin":5472,"end":5476},"obj":"20188075"},{"id":"25982113-24199627-71076132","span":{"begin":5472,"end":5476},"obj":"24199627"},{"id":"25982113-24199627-71076132","span":{"begin":5472,"end":5476},"obj":"24199627"},{"id":"25982113-24199627-71076133","span":{"begin":7485,"end":7489},"obj":"24199627"},{"id":"25982113-24199627-71076133","span":{"begin":7485,"end":7489},"obj":"24199627"},{"id":"25982113-18593822-71076134","span":{"begin":8208,"end":8212},"obj":"18593822"},{"id":"25982113-18593822-71076134","span":{"begin":8208,"end":8212},"obj":"18593822"},{"id":"25982113-21402116-71076134","span":{"begin":8208,"end":8212},"obj":"21402116"},{"id":"25982113-21402116-71076134","span":{"begin":8208,"end":8212},"obj":"21402116"},{"id":"25982113-16959943-71076135","span":{"begin":8353,"end":8357},"obj":"16959943"},{"id":"25982113-16959943-71076135","span":{"begin":8353,"end":8357},"obj":"16959943"},{"id":"25982113-18593822-71076135","span":{"begin":8353,"end":8357},"obj":"18593822"},{"id":"25982113-18593822-71076135","span":{"begin":8353,"end":8357},"obj":"18593822"},{"id":"25982113-21402116-71076135","span":{"begin":8353,"end":8357},"obj":"21402116"},{"id":"25982113-21402116-71076135","span":{"begin":8353,"end":8357},"obj":"21402116"}],"text":"Discussion\nRAMPs are an important class of accessory membrane proteins that modulate GPCR pharmacology. The CGRPmut-bound CLR:RAMP1 ECD and AM-bound CLR:RAMP2 ECD structures presented here expand our understanding of the mechanisms by which peptides can bind to class B GPCRs and increase our understanding of how RAMPs enable peptide selectivity. The engineered tethered ECD fusion proteins used for crystallization exhibited the same peptide selectivity rank order as the intact receptors, which indicated that they are valid reagents for studying selective peptide binding. The purified proteins bound their respective peptides with apparent affinities in the low μM range (Figures S1C, S1G, and S1H), which are lower than the affinities of the agonist peptides for intact receptors but typical for truncated peptides at class B GPCR ECDs (Pal et al., 2010; Parthier et al., 2007; Pioszak and Xu, 2008).\nThe oligomeric states of CLR:RAMP complexes has been a source of debate with evidence for 1:1, 2:1, and 2:2 CLR:RAMP stoichiometries (Héroux et al., 2007; Hill and Pioszak, 2013; Kusano et al., 2012; Moad and Pioszak, 2013; Watkins et al., 2013a). Dimerization of the purified CLR:RAMP2 ECD heterodimer to form a 2:2 complex may be an artifact because the RAMP2 L106R mutation prevented oligomerization yet did not affect AM1 receptor function (Figure S1). Occlusion of the AM-binding site by dimerization explains our inability to measure AM binding to the tethered RAMP2-CLR ECD fusion protein in an assay using μM receptor concentration, whereas AM binding was readily measured in an assay using nM receptor concentration where the dimeric species was likely not significantly present (Moad and Pioszak, 2013). Héroux et al. (2007) provided evidence for a homo-oligomer of CLR with a single RAMP1 as the functional CGRP receptor in cells. We cannot rule out a role for higher-order oligomerization in the function of the intact receptors, but the structures indicate that the 1:1 heterodimers are sufficient to bind peptides.\nThe CGRPmut and AM peptides adopted receptor-bound conformations different from typical α-helical class B GPCR peptide ligands. CGRPmut and AM are characterized by a shared turn structure that positions their C-terminal residue to occupy the pocket near the RAMP. Previous studies indicated the presence of turns in the C-terminal region of CGRP (and an absence of α-helix in this area), but how this region interacted with the receptor was unclear (Carpenter et al., 2001; Watkins et al., 2013b). Prior to the turns, the peptides diverge in their structure and interactions with CLR, but they contact the same area of CLR with little or no peptide secondary structure. NMR structures of CGRP and AM suggested that α-helix is restricted to residues 8-18/22-34 of these peptides (Boulanger et al., 1995; Breeze et al., 1991; Pérez-Castells et al., 2012; Watkins et al., 2013a) and indeed the C-terminal regions of both contain helix-breaking Pro residues (Figure 1D). Accordingly, the lack of substantial helical content in the bound peptide fragments is consistent with what is known about the structures of the full-length peptides. A turn structure and paucity of α-helicity may be a general feature of the receptor ECD-binding portions of CT family peptides.\nThe CGRPmut and AM binding modes are consistent with peptide mutagenesis studies. CGRP T30, V32, and F37 and the C-terminal amide were important for binding purified CLR:RAMP1 ECD (Moad and Pioszak, 2013) and intact CGRP receptor (Carpenter et al., 2001; Rist et al., 1998; Watkins et al., 2013b). Modified CGRP peptides as short as 30–37 maintained the ability to bind the receptor (Carpenter et al., 2001), consistent with this region providing most of the contacts. Increased affinity of CGRPmut over that of CGRP can be explained by their differences in the turn region (Figure 1D). P34 favors β-turn formation better than S34 and F35 provides better hydrophobic contact to CLR loop 4 than K35. AM P43, K46, I47, G51, Y52, and the C-terminal amide were most critical for binding purified CLR:RAMP2 ECD and intact AM1 receptor, and truncation beyond residue 38 diminished binding even though the K38-V41 side chains were not important (Moad and Pioszak, 2013; Watkins et al., 2013a).\nMapping the receptor mutagenesis data (Figures 4 and 5) onto the surface of the receptor structures (Figures 7A and 7B) suggests that CGRP binds in a similar manner to CGRPmut and that the structures are good models for full-length CGRP and AM binding to intact receptors. Mutation of CLR residues that form the shared binding site diminished CGRP and AM potencies, and the effects of some of the mutations were similar for both peptides (e.g., F92). Noteworthy divergent effects of several mutations support the differences in the structures. CLR D94 was far more important for CGRP action than AM, consistent with the crucial role of D94 in contacting CGRP T30 and its less important role in contacting the AM main chain. CLR R119A diminished the potency of CGRP much more than that of AM, which may reflect an important role for the different R119 conformations observed in the two structures. CLR W72A was more deleterious for AM action than CGRP, which is consistent with the greater number of AM contacts to CLR W72 as compared to CGRP.\nRAMP1 W84 and RAMP2 E101 were previously identified as key residues for CGRP and AM function, respectively (Moore et al., 2010; Watkins et al., 2014). These data are explained by how these residues augment the binding site pocket (Figures 7A and 7B). Apparently, packing of the CGRP F37 and AM Y52 phenyl rings against the CLR Trp shelf and G71 is insufficient for strong binding. RAMP1 W84 or RAMP2 E101 is required to complete the pocket to enable strong “anchoring” of the peptide C termini. RAMP2 R97 and E105 also augment the pocket, but the R97A and E105A mutants did not diminish AM potency. These data along with the peptide swap data indicate that the RAMP2 E101-Y52 hydrogen bond is the crucial AM anchoring contact. Ionic interactions of AM K46 and RAMP2 E101/E105 do not appear to be significant. The main role of AM K46 thus appears to be intramolecular packing against Y52 and contacting the Trp shelf.\nDistinct RAMP binding site augmentation clearly contributes to peptide selectivity (Figure 6). RAMP2 E101 favors AM binding because it can hydrogen bond with Y52 and RAMP2 F111 discourages CGRP binding because it is too small to contact the F37 phenyl ring. Indeed, the F37Y swap in CGRPmut conferred strong affinity for the AM1 receptor ECD complex and the Y52F swap in AM(37-52)NH2 significantly diminished its binding. The lack of Glu at RAMP1 position 74 would disfavor strong AM binding. RAMP1 W84 enables strong CGRP binding by contacting F37, but this contact alone is apparently insufficient for selectivity because AM(37-52)NH2 [Y52F] did not gain affinity for the CGRP receptor ECD complex.\nModeling the AM-bound AM2 receptor ECD complex (Supplemental Experimental Procedures) suggests that RAMP3 augments the binding site as a RAMP1-2 hybrid (Figure 7C). RAMP3 E74, which is equivalent to RAMP2 E101, would favor AM binding by hydrogen bonding with Y52. RAMP3 W84, which is equivalent to RAMP1 W84, could contact the AM Y52 and CGRP F37 phenyl rings, thereby explaining diminished potency of both peptides at the AM2 receptor with RAMP3 W84A and why CGRP is more active at the AM2 receptor than the AM1 receptor (Watkins et al., 2014). The key RAMP residues proposed as selectivity determinants are conserved across species: W84 and a lack of Glu at position 74 in RAMP1, E101 and F or Y at position 111 in RAMP2, and E74 and W84 in RAMP3 (Figure 7D). A small amino acid is conserved at position 70 in RAMP1/3, which would avoid steric clash with W84. Notably, the lack of conservation of RAMP2 R97 and E105 is consistent with the mutagenesis data that indicated that these residues are not critical for AM signaling.\nPrevious RAMP single swap mutant studies supported the importance of Glu at position 74/101 as a determinant for AM selectivity. RAMP1 W74E had no effect on CGRP potency but increased AM potency at the CGRP receptor (Qi et al., 2008, 2011). RAMP3 E74W decreased AM potency at the AM2 receptor, while having a negligible effect on CGRP potency (Hay et al., 2006; Qi et al., 2008, 2011). More extensive quadruple swap mutants in this study failed to exchange the pharmacological profiles, but these experiments are complicated by the different RAMP positions relative to CLR and variable RAMP effects on CLR conformation.\nThe failure of the CGRP and AM peptide C-terminal residue swaps to exchange their receptor preferences (Figure 6) strongly suggests that RAMP binding site augmentation alone is insufficient to account for selectivity. Thus, the subtle differences in CLR conformation in the two structures may also be important for selectivity. RAMP-induced changes in CLR R119 side-chain conformation and/or subtle shifting of loop 2 may sufficiently alter the pocket to favor one peptide over the other. Future studies will be required to determine to what extent such allostery contributes to selectivity. Peptide selectivity determinants may also exist in portions of the receptors that were not addressed in this study.\nIn summary, the structures presented here provide the first structural views of any accessory membrane protein modulating GPCR ligand binding and may provide a basis for understanding modulation of other GPCRs by accessory proteins. Our data indicate that RAMPs determine peptide selectivity of CLR through a combination of binding site augmentation and alteration of CLR conformation. It is striking that relatively minor differences in RAMP-specific peptide contacts and subtle RAMP-induced changes in CLR conformation lead to such profoundly different pharmacological profiles. Of practical value, the structures may inform rational drug design targeting CLR:RAMP complexes with clinical relevance for migraine headache and cardiovascular disorders. Lastly, the MBP-tethered ECD fusion protein approach to crystallization should facilitate structural studies of other CT family peptides bound to their respective receptor ECD complexes, which will enable a more complete understanding of how RAMPs modulate both CLR and CTR."}