Folding Mechanism of β-Helical Passenger Domains from a Bacterial Autotransporter

Abstract

Virulence factors, small peptides attached to larger autotransporter proteins, are a common source of virulence in infectious, Gram-negative bacteria and a new focus for antimicrobial drug development. While the transportation of the virulence factor across the inner membrane is well-studied, the mechanism of how these peptides are secreted through the outer membrane remains unclear due to the absence of traditional energy sources, such as ATP or ion gradients, at the outer membrane. Interestingly, many of these proteins come attached to large β-helical passenger domains, which do not appear to be participating in any of their virulence behavior. In addition, folding of the β-helix occurs significantly faster in vivo than in vitro, so the passenger domain likely folds along a faster, vectorial pathway as its being secreted through the outer membrane. Experimental results show that the relative stability at the N- and C-terminus of the passenger domain largely affects the secretion rate of these proteins, suggesting that the folding of the β-helix structure plays an important role in efficient secretion. In this study, we used three different simulation methods to investigate the folding mechanism and kinetics of the passenger domain of Pertactin, an autotransporter from Bordetella pertussis. Multidimensional replica-exchange umbrella sampling simulations reveal how cooperative folding beginning at the C-terminus enhances the kinetics of folding, while steered molecular dynamics simulations show differences in mechanical responses at the N- and C-terminus. Lastly, we use Markov state modelling to find intermediate folding states as well as possible misfolded structures, which may act as kinetic traps.