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

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 -helical/{beta}-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-{beta}-stranded outer-membrane (OM) WzpX, WzpS, and WzpB {beta}-barrel secretion porins for the exopolysaccharide (EPS), major spore coat polysaccharide (MASC), and biosurfactant polysaccharide (BPS) pathways (respectively) in the Gram-negative social predatory bacterium Myxococcus xanthus DZ2. 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 the M. xanthus WzpX, 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 {beta}-barrel, along with key interacting residues, and identified promising hits for the design of potential binders capable of plugging the {beta}-barrels and inhibiting polysaccharide secretion.