Résumé
The overproduction and purification of membrane proteins are intrinsically difficult, making their analysis challenging. We purified the TraE membrane protein from a bacterial conjugation system that is involved in plasmid transfer. Our results suggest that this protein forms hexamers with a central pore, and we also show that it binds to the TraD protein. The structure of TraE is completely different from that of the previously characterized periplasmic domain. This has intriguing implications for the role of TraE and of its interaction partner TraD in substrate translocation across the bacterial cell envelope. This work makes an important contribution to understanding of the mechanism of plasmid transfer, contributing to the design of approaches to inhibit the spread of antibiotic resistance genes. Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.