Highlights d All-atom MD simulations extensively characterize the dynamics of EHEC type IV pili d Simulations and mutagenesis reveal H-bond and salt bridge network stabilizing T4P d NMR and MD studies reveal a novel calcium-binding pocket at the pilin interface d Calcium and disulfide bonds stabilize EHEC pili in vitro and in silico

Staphylococcus epidermidisandS. aureusare highly problematic bacteria in hospital settings. This stems, at least in part, from strong abilities to form biofilms on abiotic or biotic surfaces. Biofilms are well-organized multicellular aggregates of bacteria, which, when formed on indwelling medical devices, lead to infections that are difficult to treat. Cell wall-anchored (CWA) proteins are known to be important players in biofilm formation and infection. Many of these proteins have putative stalk-like regions or regions of low complexity near the cell wall-anchoring motif. Recent work demonstrated the strong propensity of the stalk region of theS. epidermidisaccumulation-associated protein (Aap) to remain highly extended under solution conditions that typically induce compaction or other significant conformational changes. This behavior is consistent with the expected function of a stalk-like region that is covalently attached to the cell wall peptidoglycan and projects the adhesive domains of Aap away from the cell surface. In this study, we evaluate whether the ability to resist compaction is a common theme among stalk regions from various staphylococcal CWA proteins. Circular dichroism spectroscopy was used to examine secondary structure changes as a function of temperature and cosolvents along with sedimentation velocity analytical ultracentrifugation and SAXS to characterize structural characteristics in solution. All stalk regions tested are intrinsically disordered, lacking secondary structure beyond random coil and polyproline type II helix, and they all sample highly extended conformations. Remarkably, the Ser-Asp dipeptide repeat region of SdrC exhibited nearly identical behavior in solution when compared to the Aap Pro/Gly-rich region, despite highly divergent sequence patterns, indicating conservation of function by various distinct staphylococcal CWA protein stalk regions.

Type IV pili (T4P) are distinctive dynamic filaments at the surface of many bacteria that can rapidly extend, 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 108 μs 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 was complemented by targeted mutagenesis and biochemical analyses. Our simulations and NMR experiments revealed a conserved set of residues defining a novel calcium-binding site at the interface between three pilin subunits. Calcium binding enhanced T4P stability ex vivo and in vitro, supporting the role of this binding site as a potential pocket for drug design.

Staphylococcus epidermidis and Staphylococcus aureus are highly problematic bacteria in hospital settings. A major challenge is their ability to form biofilms on abiotic or biotic surfaces. Biofilms are well‐organized, multicellular bacterial aggregates that resist antibiotic treatment and often lead to recurrent infections. Bacterial cell wall‐anchored (CWA) proteins are important players in biofilm formation and infection. Many have putative stalk‐like regions or regions of low complexity near the cell wall‐anchoring motif. Recent work demonstrated the strong propensity of the stalk region of S. epidermidis accumulation‐associated protein (Aap) to remain highly extended under solution conditions that typically induce compaction. This behavior is consistent with the expected function of a stalk‐like region that is covalently attached to the cell wall peptidoglycan and projects the adhesive domains of Aap away from the cell surface. In this study, we evaluate whether the ability to resist compaction is a common theme among stalk regions from various staphylococcal CWA proteins. Circular dichroism spectroscopy was used to examine secondary structure changes as a function of temperature and cosolvents along with sedimentation velocity analytical ultracentrifugation, size‐exclusion chromatography, and SAXS to characterize structural characteristics in solution. All stalk regions tested are intrinsically disordered, lacking secondary structure beyond random coil and polyproline type II helix, and they all sample highly extended conformations. Remarkably, the Ser‐Asp dipeptide repeat region of SdrC exhibited nearly identical behavior in solution when compared to the Aap Pro/Gly‐rich region, despite highly divergent sequence patterns, indicating conservation of function by various distinct staphylococcal CWA protein stalk regions.