PMC:4504005 / 19213-22981 JSONTXT

{"project":"TEST0","denotations":[{"id":"25982113-173-181-8319024","span":{"begin":2944,"end":2948},"obj":"[\"24115156\", \"24115156\", \"24115156\"]"}],"text":"Peptide 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."}

{"project":"2_test","denotations":[{"id":"25982113-24115156-71076121","span":{"begin":2944,"end":2948},"obj":"24115156"},{"id":"25982113-24115156-71076121","span":{"begin":2944,"end":2948},"obj":"24115156"},{"id":"T47714","span":{"begin":2944,"end":2948},"obj":"24115156"},{"id":"T56862","span":{"begin":2944,"end":2948},"obj":"24115156"}],"text":"Peptide 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."}