Dissecting Protein Interactions, Ligand Binding and Conformational Regulation using Hydrogen Deuterium Exchange Mass Spectrometry

Abstract

Understanding protein interactions and structural dynamics is fundamental to elucidating biological function, disease mechanisms, and therapeutic design. Proteins rarely act in isolation; their activities are mediated through transient or stable interactions with ligands, other proteins, nucleic acids, and membranes. Capturing these dynamic events requires biophysical tools capable of resolving structural changes at high temporal and spatial resolution. Among these, hydrogen-deuterium exchange mass spectrometry (HDX-MS) has emerged as a powerful technique for probing conformational flexibility and interaction interfaces under near-physiological conditions. In this work, we employed HDX-MS, including millisecond-to-second timescales, as a structural biology tool to investigate conformational dynamics and interaction landscapes across a diverse range of protein systems. HDX-MS enabled detailed characterization of cytochrome c (cyt c) binding to phospholipid membranes, revealing that cardiolipin (CL) and the model lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol)(POPG) both induce similar protection in specific interaction sites, particularly the adjacent “L” and “A” regions, while leaving others unperturbed. Expanding to nuclear receptors, HDX-MS provided mechanistic insights into the ligand-induced conformational plasticity of Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR). In CAR, transcriptional activation correlated with stabilization of helix 11 (H11) and can be independent of helix 12 (H12), and distinct structural responses were observed between full agonist and partial agonist. In the CAR: Retinoid X Receptor alpha (RXRα) heterodimer, we uncovered ligand-driven allosteric communication across the dimer interface, with dual ligands producing cooperative stabilization of the CAR ligand-binding domain. In PXR, cooperative binding of furanodienone (FDN) with estrogenic steroids (E2 and EE2) led to enhanced stabilization of helices H3 and H12, promoting full agonist-like activity. Finally, in the lipid hydrolase (ABHD2), HDX-MS enabled mapping of binding interfaces for inhibitors 191R and 192. Dynamic comparison with the catalytically inactive S207A mutant revealed mutation-induced stabilization and altered ligand responsiveness in the absence of crystallographic data. Together, these findings highlight HDX-MS as an effective and versatile technique for elucidating protein-ligand and protein-lipid interactions and highlighting its growing utility in guiding therapeutic development across diverse biological systems.