Dynamic association of an assembly platform subcomplex of the bacterial type two secretion system

Type II secretion systems (T2SS) allow diderm bacteria to secrete hydrolytic enzymes, adhesins or toxins important for growth and virulence. T2SS-mediated secretion of folded proteins from the periplasm to the cell surface requires the assembly of periplasmic filaments called pseudopili. Similarly to related type IV pili, pseudopili are polymerized in the inner membrane fostered by the essential assembly platform (AP) complex and the energy provided by the ATPase motor. To understand the role of the AP in the assembly and secretion mechanism, we determined the first structure of the heterodimer complex of two key components from Klebsiella oxytoca T2SS, PulL and PulM, by a combination of biophysical approaches, NMR and X-ray crystallography. We also showed in vivo how the assembly of their periplasmic ferredoxin-like domains is crucial for the secretion process. Cysteine scanning and cross-linking data in the cellular context allowed us to understand the PulL-PulM binding and orientation with respect to the membrane. Together with our data on the relative abundance of PulL and PulM and their partners, our findings support a model of the AP as a dynamic hub that orchestrates pseudopilus polymerization, with PulL being stationary close to the ATPase and PulM recruiting monomer subunits in the inner membrane, guiding them to the AP to be inserted at the base of the filament.