Freel Meyers, C. L. | bioRxiv Science

bioRxiv2026

MInt-HDX: Leveraging Hydrogen-Deuterium Exchange Mass Spectrometry and Machine-Learning to Improve Protein-Ligand Docking.

Understanding protein structural dynamics is central to elucidating biological function and guiding therapeutic discovery. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) typically offers peptide-level, and sometimes residue-level, time-dependent insights into protein structure, conformational dynamics and/or ligand binding. Yet, translating HDX-MS data into atomic-resolution insights and deriving mechanistic understanding remains a key challenge. Integrative strategies which utilize HDX-MS to inform computational modeling or simulations, traditionally leverage HDX-MS data with physics-based approaches through the calculation of protection factors models. Here, we developed MInt-HDX, a hybrid physics-based, machine- learning framework trained on differential HDX-MS signatures across 11 protein-ligand systems or 1032 individual peptides, using eXtreme Gradient Boosting (XGBoost) to guide small-molecule ligand docking and pose selection. By leveraging XGBoost-predicted interacting residues with three-dimensional clustering and convex-hull geometric algorithms, MInt-HDX first generates HDX-guided candidate docking sites in 3D for physics-based molecular docking and then, following docking, employs HDX-MS-informed XGBoost filtering and scoring functions for ligand- pose ranking. MInt-HDX was validated across 3 protein-ligand systems, consistently resulting in Ligand-RMSD within 3 [A] of the crystallographic ligand conformation, individual steps of MInt- HDX were optimized and its overall performance was assessed against HDX-MS data quality factors and benchmarked against common physics-based and machine learning based docking approaches. Together, this work highlights how machine learning, informed by HDX-MS and aided by physics-based approaches, can bridge the gap between solution-phase HDX-MS data and structural modeling to accelerate protein-ligand discovery pipelines. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/738285v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1bf4574org.highwire.dtl.DTLVardef@68f3c8org.highwire.dtl.DTLVardef@5cd2c1org.highwire.dtl.DTLVardef@10a82c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Aromatic residues in mobile regions distal to the active site support the closed conformation of E. coli DXPS

The essential bacterial enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) is absent in humans, making the enzyme an attractive antimicrobial target. Its product DXP sits at a metabolic branchpoint between the biosynthesis of pyridoxal phosphate (PLP), thiamin diphosphate (ThDP), and isoprenoids. DXP is formed via decarboxylation of pyruvate and subsequent carboligation with D-glyceraldehyde-3-phosphate (D-GAP) in a ThDP-dependent manner. In the current mechanistic model, DXPS follows a ligand-gated mechanism. Pyruvate reacts with ThDP to form C2-lactylThDP (LThDP) which coincides with a shift to a closed conformation. The flexible "spoon" and "fork" motifs become ordered, situating the catalytic residue H299 within the active site which supports LThDP persistence and the closed conformation of the E-LThDP complex until binding of D-GAP. Our goal is to understand the molecular basis for stabilization of the E-LThDP complex in its closed conformation in the absence of D-GAP. We propose the conserved aromatic residues Y288, F298, and F304 in the E. coli DXPS spoon and fork motifs form a cluster upon transition from the open to closed form to position H299 within the active site, necessary for LThDP persistence. Here, we conducted mutagenesis studies to elucidate the roles of Y288, F298, and F304 in conformational cycling and catalysis. On each variant, the conformational equilibrium favored an open state, hindered intermediate formation and persistence, and promoted intermediate release from the active site. Our results support a model in which conserved aromatic residues within the mobile, sequence-diverse spoon and fork motifs promote the closed conformation and support catalysis.

biochemistry↗