Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma (J.M.B., M.L.W., A.M.R., A.A.P.) and School of Biological Sciences, University of Auckland, Auckland, New Zealand (D.L.H.).
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma (J.M.B., M.L.W., A.M.R., A.A.P.) and School of Biological Sciences, University of Auckland, Auckland, New Zealand (D.L.H.)
Mol Pharmacol. 2018 Apr;93(4):355-367. doi: 10.1124/mol.117.110916. Epub 2018 Jan 23.
Binding of the vasodilator peptides adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) to the class B G protein-coupled receptor calcitonin receptor-like receptor (CLR) is modulated by receptor activity-modifying proteins (RAMPs). RAMP1 favors CGRP, whereas RAMP2 and RAMP3 favor AM. Crystal structures of peptide-bound RAMP1/2-CLR extracellular domain (ECD) heterodimers suggested RAMPs alter ligand preference through direct peptide contacts and allosteric modulation of CLR. Here, we probed this dual mechanism through rational structure-guided design of AM and CGRP antagonist variants. Variants were characterized for binding to purified RAMP1/2-CLR ECD and for antagonism of the full-length CGRP (RAMP1:CLR), AM (RAMP2:CLR), and AM (RAMP3:CLR) receptors. Short nanomolar affinity AM(37-52) and CGRP(27-37) variants were obtained through substitutions including AM S45W/Q50W and CGRP K35W/A36S designed to stabilize their -turn. K46L and Y52F substitutions designed to exploit RAMP allosteric effects and direct peptide contacts, respectively, yielded AM variants with selectivity for the CGRP receptor over the AM receptor. AM(37-52) S45W/K46L/Q50W/Y52F exhibited nanomolar potency at the CGRP receptor and micromolar potency at AM A 2.8-Å resolution crystal structure of this variant bound to the RAMP1-CLR ECD confirmed that it bound as designed. CGRP(27-37) N31D/S34P/K35W/A36S exhibited potency and selectivity comparable to the traditional antagonist CGRP(8-37). Giving this variant the ability to contact RAMP2 through the F37Y substitution increased affinity for AM, but it still preferred the CGRP receptor. These potent peptide antagonists with altered selectivity inform the development of AM/CGRP-based pharmacological tools and support the hypothesis that RAMPs alter CLR ligand selectivity through allosteric effects and direct peptide contacts.
舒血管肽肾上腺髓质素 (AM) 和降钙素基因相关肽 (CGRP) 与 B 类 G 蛋白偶联受体降钙素受体样受体 (CLR) 的结合受受体活性修饰蛋白 (RAMP) 调节。RAMP1 有利于 CGRP，而 RAMP2 和 RAMP3 有利于 AM。肽结合的 RAMP1/2-CLR 细胞外结构域 (ECD) 异二聚体的晶体结构表明，RAMP 通过直接的肽接触和 CLR 的变构调节改变配体的偏好。在这里，我们通过合理的基于结构的 AM 和 CGRP 拮抗剂变体设计来探测这种双重机制。对变体与纯化的 RAMP1/2-CLR ECD 的结合以及对全长 CGRP (RAMP1:CLR)、AM (RAMP2:CLR) 和 AM (RAMP3:CLR) 受体的拮抗作用进行了表征。通过包括 AM S45W/Q50W 和 CGRP K35W/A36S 的取代获得了具有短纳摩尔亲和力的 AM(37-52)和 CGRP(27-37)变体，这些取代旨在稳定它们的β-转角。分别设计 K46L 和 Y52F 取代以利用 RAMP 变构效应和直接肽接触，得到了对 CGRP 受体具有选择性的 AM 变体，而对 AM 受体的选择性较低。AM(37-52)S45W/K46L/Q50W/Y52F 在 CGRP 受体上具有纳摩尔效力，在 AM A 2.8-Å 分辨率的晶体结构中，该变体与 RAMP1-CLR ECD 结合，证实其按设计结合。CGRP(27-37)N31D/S34P/K35W/A36S 表现出与传统拮抗剂 CGRP(8-37)相当的效力和选择性。通过 F37Y 取代赋予该变体与 RAMP2 接触的能力，增加了对 AM 的亲和力，但它仍然优先与 CGRP 受体结合。这些具有改变的选择性的强效肽拮抗剂为 AM/CGRP 为基础的药理学工具的开发提供了信息，并支持 RAMP 通过变构效应和直接肽接触改变 CLR 配体选择性的假设。
