| T&barbelow;ype IV s&barbelow;ecretion s&barbelow;ystems (T4SSs) are complex protein machineries mediating the transfer of DNA and protein substrates across the cell envelope to bacterial or eukaryotic cells generally by direct cell-to-cell contact. They play a key role in the pathogenicity of several important Gram-negative pathogens. According to the well-studied A. tumefaciens T4SS model, the eleven VirB proteins (VirB 1 through VirB 11) elaborate a trans-envelope apparatus and a T-pilus structure. The T-pilus is an extracellular filament that mediates the attachment between the bacterium and the host recipient. VirB2 is the major component of the T-pilus, but the roles of other VirB proteins in T-pilus assembly remained undefined.; In the present study, we solved the crystal structure of TraC, a homolog of VirB5. The crystal structure shows that TraC/VirB5 is a single-domain protein with a mostly alpha-helical elongated structure. Results from functional studies based on the structural information suggest that VirB5-like proteins bind along the pilus, including the pilus tip and base. In addition, some conserved amino acid residues on the surface of TraC have been identified to promote pilus assembly or to be involved in protein-protein interactions. The work presented here provides insights into the biological function(s) of TraC/VirB5 proteins during the type IV secretion pathway.; VirB4 is an essential component of T4SS and VirB8 is a crucial structural and functional component of T4SS. In this study, we found that VirB4 is essential for the stabilization of VirB3 and VirB8. Analysis of VirB protein complexes extracted from the membranes with mild detergent revealed that VirB2-VirB5 pilus complex formation depended on VirB4; and VirB3 and VirB8 co-fractionated with T-pilus components VirB2, VirB5, and VirB7. Direct interactions between VirB4 and VirB8 as well as between VirB8 and VirB5 have been identified using different biochemical methods. A pathway of T-pilus assembly via VirB4-VirB8-VirB5-VirB2 (VirB3) sequential interactions is suggested. Here, VirB4 localizes at the base of a trans-envelope interaction sequence, and by stabilization of VirB8, it mediates the incorporation of VirB5 and VirB2 into extracellular pili. |
