HDX-MS guided ensemble reweighting approach reveals cryptic drug binding sites in the cytoplasmic heme binding protein, PhuS.

Abstract

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a valuable approach to probe protein structure and dynamics. However, interpretation of HDX-MS data is often qualitative and typically limited to peptide-level resolution. Ongoing efforts aim to integrate experimental HDX-MS data with molecular dynamics (MD) approaches, to provide a high-resolution interpretation of the HDX-MS data. One such approach is an HDX-MS based maximum-entropy reweighting approach (HDXer) to reweight computationally generated ensembles to HDX-MS data. Here, HDXer is used in conjunction with enhanced MD (eMD) to characterize the structural dynamics of PhuS. PhuS is a cytoplasmic heme binding protein from P. aeruginosa which shuttles exogenous heme to Heme Oxygenase (HemO) for degradation and is essential for virulence. Through the implementation of a workflow that integrates eMD, HDXer, and dimensional reduction, we had previously modeled the native state ensemble of apo-PhuS revealing a locally diverse apo-PhuS ensemble characterized by large rearrangements of helices surrounding the heme binding. Here, the representative structures from the modeled native ensemble were used as starting points for computer aided drug design (CADD). Implementing the Site Identification by Ligand Competitive Saturation (SILCS) CADD method approximately 1.3 million compounds were screened for each representative structure as well as the published apo PhuS crystal structure, yielding a library of 50 chemically representative candidate compounds. The candidate compounds were then screened using a fluorescence quenching assay. From these assays, several hits were confirmed to bind apo PhuS at nanomolar binding affinities. Most significantly, none of the compounds were derived from CADD using the published apo PhuS crystal structure, demonstrating the utility of HDX-MS guiding ensemble reweighting in revealing cryptic drug binding site in CADD approaches.