Determinants of improved CGRP peptide binding kinetics revealed by enhanced molecular simulations

Peptides are desirable therapeutics due to their inherent potency, safety, and ability to engage complex protein surfaces. Slower kinetics of protein-peptide (un)binding can directly influence their drug efficacy and duration of action, in part by improving plasma stability of the peptide. A better understanding of peptide binding mechanisms would benefit for the development of next-generation peptide-based drugs with optimized kinetic properties. The calcitonin receptor-like receptor:receptor activity-modifying proteins 1 (CLR:RAMP1) complex and its endogenous agonist peptide calcitonin gene-related peptide (CGRP) are of particular interest due to their central role in migraine pathophysiology. In this study, we comparatively analyze C-terminal constructs of native CGRP and "ssCGRP," an engineered variant with 430-fold longer residence time in the CLR:RAMP1 complex. Using our high-dimensional weighted-ensemble algorithm, we thoroughly sampled unbinding pathway ensembles for the two peptides. This elucidates the basis of the engineered residence time enhancement for ssCGRP and provides a detailed view of the intra- and intermolecular stabilizing interactions for both peptides in the bound ensemble and along the unbinding transition pathway. The natural dynamics used in weighted ensemble, combined with Markov state modeling, allows for the first comparative analysis of protein-peptide unbinding transition state ensembles between a native and mutant peptide. We find that ssCGRP(27-37) demonstrates enhanced ligand recapture of intermediate unbinding conformations and samples a more heterogeneous bound-state ensemble that entropically stabilizes the bound basin. This study shows the molecular determinants of the residence time of peptides in CLR:RAMP1 and provides valuable insight for the design of long-acting peptide therapeutics.