Biology subjects

Pioszak, A. A.

Publications and source records attributed to Pioszak, A. A..

4 recordsLinked to original sources

bioRxiv2025

Determinants of Improved CGRP Peptide Binding Kinetics Revealed by Enhanced Molecular Simulations

Peptides are desirable therapeutics due to their inherent potency, safety, cost-effectiveness and ability to engage large or more 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. The CLR:RAMP1 complex and its endogenous agonist peptide CGRP are of particularly high interest because of their central role in migraine pathophysiology. A better understanding of peptide binding mechanisms is needed for the development of next-generation peptide-based drugs with optimized kinetic properties. In this study, we comparatively analyze constructs of native CGRP and "ssCGRP", an engineered variant with 430-fold longer residence time on the CLR:RAMP1 complex. Using large-scale computational resources and our high-dimensional weighted-ensemble algorithm, we then thoroughly sample and compare unbinding path 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 path. The bias-free nature of the sampling approach in combination with Markov state modeling allows for the calculation of committor values and the first analysis of protein-peptide binding transition state ensembles. Through analysis of the unbinding committor, we find that ssCGRP(27-37) also 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 peptide residence time at CLR:RAMP1 and provides valuable insight for the design of long-acting peptide therapeutics.

molecular biology↗

bioRxiv2024

Amylin receptor subunit interactions are modulated by agonists and determine signaling

Three amylin receptors (AMYRs) mediate the metabolic actions of the peptide hormone amylin and are drug targets for diabetes and obesity. AMY1R, AMY2R, and AMY3R are heterodimers consisting of the G protein-coupled calcitonin receptor (CTR) paired with a RAMP1, -2, or -3 accessory subunit, respectively, which increases amylin potency. Little is known about AMYR subunit interactions and their role in signaling. Here, we show that the AMYRs have distinct basal subunit equilibriums that are modulated by peptide agonists and determine the cAMP signaling phenotype. Using a novel biochemical assay that resolves the AMYR heterodimers and free subunits, we found that the AMY1/2R subunit equilibriums favored free CTR and RAMP1/2, and rat amylin and CGRP agonists promoted subunit association. A stronger CTR-RAMP3 transmembrane domain interface yielded a more stable AMY3R, and human and salmon calcitonin agonists promoted AMY3R dissociation. Similar changes in subunit association-dissociation were observed in live cell membranes, and G protein coupling and cAMP signaling assays showed how these altered signaling. Our findings reveal regulation of heteromeric GPCR signaling through subunit interaction dynamics.

biochemistry↗

bioRxiv2023

Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of theadrenomedullin2 G protein-coupled receptor

The signaling peptides adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and CGRP have overlapping and distinct functions in the cardiovascular, lymphatic, and nervous systems by activating three shared receptors comprised of the class B GPCR CLR in complex with a RAMP1, -2, or -3 modulatory subunit. Here, we report that AM2/IMD, which is thought to be a non-selective agonist, is kinetically selective for CLR-RAMP3, known as the AM2R. AM2/IMD-AM2R elicited substantially longer duration cAMP signaling than the eight other peptide-receptor combinations due to AM2/IMD slow off-rate binding kinetics. The regions responsible for the slow off-rate were mapped to the AM2/IMD mid-region and the RAMP3 extracellular domain. MD simulations revealed how these bestow enhanced stability to the complex. Our results uncover AM2/IMD-AM2R as a cognate pair with unique temporal features, define the mechanism of kinetic selectivity, and explain how AM2/IMD and RAMP3 collaborate to shape the signaling output of a clinically important GPCR.

biochemistry↗

bioRxiv2020

Picomolar affinity antagonist and sustained signaling agonist peptide ligands for the adrenomedullin and calcitonin gene-related peptide receptors

The calcitonin receptor-like G protein-coupled receptor (CLR) mediates adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) actions including vasodilation, cardioprotection, and nociception. Receptor activity-modifying proteins (RAMP1-3) determine CLR ligand selectivity through an unresolved mechanism. CLR-RAMP complexes are drug targets, but short AM and CGRP plasma half-lives limit their therapeutic utility. We used combinatorial peptide library and rational design approaches to probe selectivity determinants and develop short AM and CGRP variants with [~]1000-fold increased receptor extracellular domain affinities. Binding and structural studies explained the increased affinities and defined roles for AM Lys46 and RAMP modulation of CLR conformation in selectivity. In longer scaffolds that also bind the CLR transmembrane domain the variants generated picomolar affinity antagonists, one with an estimated 12.5 hr CGRP receptor residence time, and sustained signaling agonists ss-AM and ss-CGRP. This work clarifies the RAMP-modulated ligand selectivity mechanism and provides AM and CGRP variants with promise as long-acting therapeutics.

pharmacology and toxicology↗