Insights into dynamics and gating properties of T2SS secretins.

Secretins are outer membrane (OM) channels found in various bacterial nanomachines that secrete or assemble large extracellular structures. High-resolution 3D structures of type 2 secretion system (T2SS) secretins revealed bimodular channels with a C-module, holding a conserved central gate and an optional top gate, followed by an N-module for which multiple structural organizations have been proposed. Here, we perform a structure-driven in vivo study of the XcpD secretin, which validates one of the organizations of the N-module whose flexibility enables alternative conformations. We also show the existence of the central gate in vivo and its required flexibility, which is key for substrate passage and watertightness control. Last, functional, genomic, and phylogenetic analyses indicate that the optional top gate provides a gain of watertightness. Our data illustrate how the gating properties of T2SS secretins allow these large channels to overcome the duality between the necessity of preserving the OM impermeability while simultaneously promoting the secretion of large, folded effectors.

Refining the Dynamic Network of T2SS Endopilus Tip Heterocomplex Combining cw-EPR and Nitroxide-GdIII Distance Measurements.

The type 2 secretion system (T2SS) is a bacterial nanomachine composed of an inner membrane assembly platform, an outer membrane pore and a dynamic endopilus. T2SS endopili are organized into a homo-multimeric body formed by the major pilin capped by a heterocomplex of four minor pilins. The first model of the T2SS endopilus was recently released, even if structural dynamics insights are still required to decipher the role of each protein in the full tetrameric complex. Here, we applied continuous-wave and pulse EPR spectroscopy using nitroxide-gadolinium orthogonal labelling strategies to investigate the hetero-oligomeric assembly of the minor pilins. Overall, our data are in line with the endopilus model even if they evidenced conformational flexibility and alternative orientations at local scale of specific regions of minor pilins. The integration of different labelling strategies and EPR experiments demonstrates the pertinence of this approach to investigate protein-protein interactions in such multiprotein heterocomplexes.

Structural lessons on bacterial secretins.

To exchange and communicate with their surroundings, bacteria have evolved multiple active and passive mechanisms for trans-envelope transport. Among the pore-forming complexes found in the outer membrane of Gram-negative bacteria, secretins are distinctive homo-oligomeric channels dedicated to the active translocation of voluminous structures such as folded proteins, assembled fibers, virus particles or DNA. Members of the bacterial secretin family share a common cylinder-shaped structure with a gated pore-forming part inserted in the outer membrane, and a periplasmic channel connected to the inner membrane components of the corresponding nanomachine. In this mini-review, we will present what recently determined 3D structures have told us about the mechanisms of translocation through secretins of large substrates to the bacterial surface or in the extracellular milieu.

Structural interactions define assembly adapter function of a type II secretion system pseudopilin.

The type IV filament superfamily comprises widespread membrane-associated polymers in prokaryotes. The type II secretion system (T2SS), a virulence pathway in many pathogens, belongs to this superfamily. A knowledge gap in 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 unlocked its function. We demonstrate that low-abundance XcpH is the adapter that bridges a trimeric initiating tip complex, XcpIJK, with a periplasmic filament of XcpG subunits. Each pseudopilin protein caps an XcpG protofilament in an overall pseudopilus compatible with dimensions of the periplasm and the outer membrane-spanning secretin through which substrates pass. Unexpectedly, to fulfill its adapter function, the XcpH N-terminal helix must be unwound, a property shared with XcpG subunits. We provide an experimentally validated three-dimensional structural model of a complete type IV filament.