Agrobacterium tumefaciens VirB8 structure reveals potential protein-protein interaction sites

Abstract

Bacterial type IV secretion systems (T4SS) translocate DNA and/or 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-A 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 self-interactions 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.

Fig. 1.

Structural features of pVirB8^AT and comparison with pVirB8^BS. Monomer A is colored magenta and cyan and monomer B is gray. (A) Ribbon representation of monomer A with secondary structure elements labeled. (B) Surface rendered image of pVirB8^AT in same orientation as A showing the deep groove. The surface is colored according to the secondary structure: cyan for helical segments, pink for β-strands, and gray for loops. (C) Cα trace of superimposed pVirB8^AT (green with regions of greatest divergence highlighted in cyan) and pVirB8^BS (blue) monomers. (D) Ribbon diagram of the dimer rotated 90° with respect to A. Boxes indicate the three dimer contact regions. The side chains of Arg-107, Asp-113, Asp-115, Thr-116, Tyr-119, and Arg-157 are shown, and two 2-methyl-2-4-pentanediol molecules (yellow) are shown as stick models. (E–G) correspond to boxes D1, D2, and D3 and show details of the three dimer contact regions viewed down the dimer axis, equivalent to a 90° rotation from the view in D. (E) Residues involved in the major region of dimer contact. (F) Interactions across the central channel between the monomers. (G) Four aspartates at the lowest level of the interface.

Fig. 2.

consurf analysis of pVirB8^AT (residues 92–231 of structural model). (A) Multiple sequence alignment of pVirB8^AT and corresponding peptides of selected homologs: Residues in the three most conserved levels are highlighted. The corresponding secondary structural elements are shown. Blue dots indicate residues involved in dimerization. Gray dots indicate residues genetically identified as essential for activity. (B–D) Space filling models of pVirB8^AT. Surface residues are shaded according to degree of conservation. Residues in gray have been shown to be essential for VirB8 activity. Surface exposed residues in the two most conserved levels are labeled. (B) The protein is presented in approximately the same orientation as Fig. 1A. (C) Rotated by 90°. (D) Rotated by 180° with the dimer interface presented face on. GenInfo Identifier numbers for the multiple sequence alignment are A. tumefaciens VirB8, 17939307; B. suis VirB8, 23463398; E. coli TraG, 10955150; Bartonella quintana VirB8, 21260578; and Bordatella pertussis VirB8, 420953.

Fig. 3.

Stick diagram of the loop containing helix 5. The highly conserved residues R210, N213, P214, G216, and V219 are part of this loop. Dashed lines represent the hydrogen bonds between the side chains of Arg-210 and Asn-213 and main chain atoms.