Conformational Dynamics of the Pili Constructing Sortase C Enzymes

Abstract

Pili are elongated protein structures that enhance the adhesive abilities and virulence of bacteria. Therefore, understanding the pilin assembly process has important ramifications for the development of new antibacterial compounds. In streptococcus pnuenomae, three Sortase C (SrtC) enzymes are responsible for the construction of pili. In contrast to the housekeeping SrtA class of proteins, SrtC enzymes possess a flexible lid that is thought to modulate access to the active site during the pili construction process. In this work, we have used molecular dynamics simulations, including advanced sampling techniques, to study the conformational dynamics of SrtC. Here, we have probed the lid opening mechanism of three SrtC enzymes (SrtC-1, SrtC-3 and SrtC-3) as well as for two experimentally studied mutants of SrtC-1 (K58G/P60G and R202E). The results suggest that the aforementioned mutations lead to an open lid conformation in SrtC-1, in accordance with experimental findings. In contrast, the salt bridge between the conserved aspartate residue in the lid region and arginine in the active site leads to a robust anchoring of the lid that is highly stable during long conventional molecular dynamics simulations of all the SrtC proteins. This finding is confirmed by umbrella sampling free energy calculations. Excursions from this anchored position are very brief and are to a conformation with a bridged hydrogen bond between the two residues that is mediated by a water molecule. Overall, results show that opening of the active site of SrtC to initiate pili assembly likely requires interactions that are provided by the sorting signal that is contained in the pilin subunit building blocks.