Bacterial type IV secretion systems (T4SS) translocate DNA and͞or proteins to recipient cells, thus providing a mechanism for conjugative transfer of genetic material and bacterial pathogenesis. Here we describe the first structure of a core component from the archetypal Agrobacterium tumefaciens T4SS: the 2.2-Å resolution crystal structure of the VirB8 periplasmic domain (pVirB8 AT ). VirB8 forms a dimer in the crystal, and we identify residues likely important for stabilization of the dimer interface. Structural comparison of pVirB8 AT with Brucella suis VirB8 confirms that the monomers have a similar fold. In addition, the pVirB8 AT dimer superimposes very closely on the B. suis VirB8 dimer, supporting the proposal that dimer formation in the crystal reflects selfinteractions that are biologically significant. The evolutionary conservation level for each residue was obtained from a data set of 84 VirB8 homologs and projected onto the protein structure to indicate conserved surface patches that likely contact other T4SS proteins.bacterial protein export ͉ conjugation ͉ type IV secretion T ype IV secretion systems (T4SS) function as conjugation systems, DNA uptake͞release systems, and effector protein translocator systems (1) and are required for virulence of several human pathogens including Bartonella henselae, Bordetella pertussis, Brucella suis, Helicobacter pylori, and Legionella pneumophila, as well as the plant pathogen Agrobacterium tumefaciens (2). The A. tumefaciens T4SS is composed of 12 proteins, VirB1-11 and VirD4. The known properties of these components are well reviewed (1), and each contributes to one or more functional subgroups. The A. tumefaciens system genetically transforms plant cells with a specific DNA element, called the T-DNA. VirB4, VirB11, and VirD4 are NTPases located at the inner membrane that may provide energy for DNA transfer or assembly of the secretion system. VirB6-VirB10 likely form a ''core'' complex that spans the inner and outer membranes. VirB2, VirB5, and VirB7 assemble the pilus. VirB1 is a lytic transglycosidase thought to promote T4SS assembly by lysis of the peptidoglycan layer. Crystal structures of several T4SS components have been published, including homologs of hexameric NTPases VirB11 and VirD4 (3, 4), VirB5 (5), VirB8, and VirB10 (6). A hexameric structure for A. tumefaciens VirB4 has been recently proposed (7).A. tumefaciens VirB8 is a bitopic membrane protein with a short N-terminal cytoplasmically exposed domain, a transmembrane helix, and a large C-terminal periplasmic domain. A T-DNA immunoprecipitation assay has identified contacts between the DNA substrate and six of the T4SS components, including VirB8 (8). It was recently proposed that VirB8 is the scaffold for polar assembly of the A. tumefaciens T4SS (9). Yeast two-hybrid interaction studies suggest that the periplasmic portion of VirB8 contacts VirB1, VirB4, VirB9, VirB10, and VirB11 (10, 11). Biochemical studies confirm that VirB8 interacts with VirB9, VirB10, and itself in vitro (11), and that ...

The Agrobacterium T-DNA transporter belongs to a growing class of evolutionarily conserved transporters, called type IV secretion systems (T4SSs). VirB4, 789 aa, is the largest T4SS component, providing a rich source of possible structural domains. Here, we use a variety of bioinformatics methods to predict that the C-terminal domain of VirB4 (including the Walker A and B nucleotide-binding motifs) is related by divergent evolution to the cytoplasmic domain of TrwB, the coupling protein required for conjugative transfer of plasmid R388 from Escherichia coli. This prediction is supported by detailed sequence and structure analyses showing conservation of functionally and structurally important residues between VirB4 and TrwB. The availability of a solved crystal structure for TrwB enables the construction of a comparative model for VirB4 and the prediction that, like TrwB, VirB4 forms a hexamer. These results lead to a model in which VirB4 acts as a docking site at the entrance of the T4SS channel and acts in concert with VirD4 and VirB11 to transport substrates (T-strand linked to VirD2 or proteins such as VirE2, VirE3, or VirF) through the T4SS. structure prediction ͉ bacterial transport ͉ NTPase ͉ homology modeling

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Gram-negative type IV secretion systems (T4SSs) transfer proteins and DNA to eukaryotic and/or prokaryotic recipients resulting in pathogenesis or conjugative DNA transfer. VirB4, one of the most conserved proteins in these systems, has both energetic and structural roles in substrate translocation. We previously predicted a structural model for the large C-terminal domain (residues 425-789) of VirB4 of Agrobacterium tumefaciens. Here we have defined a homology-based structural model for Agrobacterium VirB11. Both VirB4 and VirB11 models predict hexameric oligomers. Yeast two-hybrid interactions define peptides in the C terminus of VirB4 and the N terminus of VirB11 that interact with each other. These interactions were mapped onto the homology models to predict direct interactions between the hexameric interfaces of VirB4 and VirB11 such that the VirB4 C terminus stacks above VirB11 in the periplasm. In support of this, fractionation and Western blotting show that the VirB4 C terminus is localized to the membrane and periplasm rather than the cytoplasm of cells. Additional high resolution yeast two-hybrid results demonstrate interactions between the C terminus of VirB4 and the periplasmic portions of VirB1, VirB8, and VirB10. Genetic studies reveal dominant negative interactions and thus function of the VirB4 C terminus in vivo. The above data are integrated with the existing body of literature to propose a structural, periplasmic role for the C-terminal half of the Agrobacterium VirB4 protein.