Multidisciplinary Interrogation of a Crucial Protein Interface in the Type II Secretion System

Abstract

The type IV filament superfamily comprises widespread membrane-associated polymers in prokaryotes. The Type II secretion system (T2SS), a significant virulence pathway in many pathogens, belongs to this superfamily. A knowledge gap in the understanding of the T2SS is the molecular role of a small pseudopilin protein. Using multiple biophysical techniques, we have deciphered how this missing component of the Xcp T2SS architecture is structurally integrated, and thereby also unlocked its function. We demonstrate that the low abundance XcpH is the adapter that bridges a trimeric initiating tip complex XcpIJK with a periplasmic filament of XcpG subunits. Our model reveals that each pseudopilin protein caps an XcpG protofilament in an overall pseudopilus compatible with the dimensions of the periplasm and the outer membrane-spanning secretin through which substrates of the T2SS pass. Unexpectedly, to fulfill its adapter function, the XcpH N-terminal helix must be unwound, a property shared with the XcpG subunits. We provide the first complete structural model of a type IV filament, a result immediately transferable to understanding of other T2SS and the type IV pili.