Abstract
Type IV pili (T4P) are distinctive dynamic filaments at the surface of many bacteria that can rapidly extend and retract and withstand strong forces. T4P are important virulence factors in many human pathogens, including Enterohemorrhagic Escherichia coli (EHEC). The structure of the EHEC T4P has been determined by integrating nuclear magnetic resonance (NMR) and cryo-electron microscopy data. To better understand pilus assembly, stability, and function, we performed a total of 108ms all-atom molecular dynamics simulations of wild-type and mutant T4P. Extensive characterization of the conformational landscape of T4P in different conditions of temperature, pH, and ionic strength is complemented with targeted mutagenesis and biochemical analyses. Our simulations and NMR experiments reveal a conserved set of residues defining a calcium-binding site at the interface between three pilin subunits. Calcium binding enhances T4P stability exvivo and invitro, supporting the role of this binding site as a potential pocket for drug design.
Highlights
In bacteria, multiple molecular machineries perform a wide variety of biological functions necessary for their survival, social organization, motility, and capacity of infecting host cells
To elucidate the role of ions in pilus dynamics, we carried out molecular dynamics (MD) simulations of T4P in the absence of ions and in the presence of Ca2+, Na+, Mg2+, and Mn2+ and at different salt concentrations
We investigated the effects of ions and salt concentrations by performing MD simulations of T4P in 100 mM salt concentration by placing sodium (Na+), magnesium (Mg2+), or manganese (Mn2+) ions in the calcium binding site, or by increasing the salt concentration to 150 mM in the presence of calcium (Ca2+)
Summary
Multiple molecular machineries perform a wide variety of biological functions necessary for their survival, social organization, motility, and capacity of infecting host cells. Bacterial interactions with their environment often rely on surface polymers called pili, which are assembled through a regular repetition of one or few protein subunits called pilins. Pilins are irreversibly or covalently linked to each other, building fibers with high stability and mechanical resistance This is the case of type I pili from gram-negative (Hospenthal et al, 2017) and sortase-dependent pili from gram-positive bacteria (Kang and Baker, 2012). Dedicated ATPases at the cytoplasmic base of the complex transmit motions to the IM assembly platform complex to drive fiber extension and retraction
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