Deciphering The Conformational Dynamics Of Escherichia Coli Type Iv Pilus

Type IV pili (T4P) are important surface fibers of many bacteria, including the human pathogen Enterohemorrhagic Escherichia coli (EHEC). They are long flexible filaments, capable of rapid extension and retraction. T4P promote biofilm formation, host cell adherence and invasion, motility, DNA uptake and virulence. The structure of the periplasmic domain of EHEC T4P subunit PpdD has been recently determined by Nuclear Magnetic Resonance (NMR) spectroscopy in our group. This structure, combined with cryo-EM density map of the EHEC pilus at 8Å resolution, resulted in an atomistic model of the T4P filament1. We performed extensive all-atom molecular dynamics (MD) simulations to study the wild-type and mutant T4P, in order to characterize their dynamic properties. The MD simulations revealed a set of key residues involved in the interactions between individual subunits, whose functional role was confirmed by the analysis of mutants. Moreover, our NMR analysis revealed putative calcium (Ca2+) binding residues in pilin monomer. Therefore, to test its binding to assembled pili, we performed additional all-atom MD simulations in the presence of Ca2+ and identified a non-canonical calcium binding pocket formed at the interface between three pilin subunits. We characterized the residues interacting with calcium as potential pocket for drug design, and showed their importance for T4P assembly and stability. Finally, we studied the effects of other ions by performing MD simulations of T4P in the presence of sodium (Na+), manganese (Mn2+) or magnesium (Mg2+). Despite stable binding of ions with a positive charge of two during MD simulations, only calcium and manganese increased the overall stability of pili in vitro. This study will allow us to identify the molecular mechanisms behind the conformational dynamics of T4P, and to develop strategies for interfering with its function. 1Bardiaux et al., Structure 27.7 (2019).