Molecular dynamics of outer membrane-embedded polysaccharide secretion porins reveals closed resting-state surface gates targetable by virtual fragment screening for drug hotspot identification

Abstract

Recent advances in iterative neural-network analyses (e.g. AlphaFold2 and RoseTTA fold) have been revolutionary for protein 3D-structure prediction, especially for difficult-to-manipulate α-helical/β-barrel integral membrane proteins. These model structures are calculated based on the co-evolution of amino acids within the protein of interest and similarities to existing protein structures; local effects of the membrane on folding and stability of the calculated model structures are not considered. We recently reported the discovery, 3D modelling, and characterization of 18-β-stranded outer-membrane (OM) WzpX, WzpS, and WzpB β-barrel secretion porins for the exopolysaccharide (EPS), major spore coat polysaccharide (MASC), and biosurfactant polysaccharide (BPS) pathways (respectively) in the Gram-negative social predatory bacteriumMyxococcus xanthusDZ2. However, information was not obtained regarding the dynamic behavior of surface-gating WzpX/S/B loop domains, nor on potential treatments to inactivate these porins. Herein, we developed a molecular dynamics (MD) protocol to study the core stability and loop dynamism of neural network-based integral membrane protein structure models embedded in an asymmetric OM bilayer, using theM. xanthusWzpX, WzpS, and WzpB proteins as test candidates. This was accomplished through integration of the CHARMM-graphical user interface (GUI) and Molecular Operating Environment (MOE) workflows to allow for rapid simulation system setup and facilitate data analysis. In addition to serving as a method of model-structure validation, our molecular dynamics simulations revealed minimal movement of extracellular WzpX/S/B loops in the absence of an external stimulus, as well as druggable cavities between the loops. Virtual screening of a commercial fragment library against these cavities revealed putative fragment-binding hotspots on the cell-surface face of each β-barrel, along with key interacting residues, and identified promising hits for the design of potential binders capable of plugging the β-barrels and inhibiting polysaccharide secretion.