Agrobacterium tumefaciens VirB8 structure reveals potential protein–protein interaction sites

Abstract

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 ...

Dimerization and interactions of Brucella suis VirB8 with VirB4 and VirB10 are required for its biological activity

“…The x-ray structures of the periplasmic domains of both the B. suis and A. tumefaciens homologs VirB8sp and VirB8ap have been solved (25,27). Both studies suggest dimer formation, which is further supported by results obtained with the yeast two-hybrid system.…”

“…VirB8sp crystallizes with five molecules in an asymmetric unit, and each of the five chains was found to dimerize. This was suggestive of dimer formation, and the recently described x-ray structure of the periplasmic domain of the A. tumefaciens VirB8 homolog (named in the following VirB8ap for Agrobacterium periplasmic) is in accord with this notion (27). Both x-ray structures suggest potential interaction hotspots, and functionally important residues have been previously identified by random mutagenesis (22).…”

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…VirB8 subunits display sequence similarities mainly in two regions corresponding to residues 100 to 143 and 190 to 235 of VirB8 At . X-ray structures for the periplasmic fragments of B. suis and A. tumefaciens VirB8 subunits each present as a large extended ␤-sheet with five ␣-helices, giving rise to an overall globular fold (21,261). The VirB8 subunits pack as dimers in the crystal structures, and results of mutational analyses suggest that dimerization is physiologically relevant.…”

Swapping of Periplasmic Domains between Brucella suis VirB8 and a pSB102 VirB8 Homologue Allows Heterologous Complementation

“…The carboxy-terminal moiety of the protein of 172 amino acids is believed to be entirely periplasmic. Recently, the three-dimensional structures of the periplasmic domain of VirB8 from both Brucella suis and A. tumefaciens have been determined (1,18). Using site-directed mutagenesis to change selected residues of the B.…”