Structures of two core subunits of the bacterial type IV secretion system, VirB8 from Brucella suis and ComB10 from Helicobacter pylori

Abstract

Type IV secretion systems (T4SSs) are commonly used secretion machineries in Gram-negative bacteria. They are used in the infection of human, animal, or plant cells and the propagation of antibiotic resistance. The T4SS apparatus spans both membranes of the bacterium and generally is composed of 12 proteins, named VirB1-11 and VirD4 after proteins of the canonical Agrobacterium tumefaciens T4SS. The periplasmic core complex of VirB8/VirB10 structurally and functionally links the cytoplasmic NTPases of the system with its outer membrane and pilus components. Here we present crystal structures of VirB8 of Brucella suis, the causative agent of brucellosis, and ComB10, a VirB10 homolog of Helicobacter pylori, the causative agent of gastric ulcers. The structures of VirB8 and ComB10 resemble known folds, albeit with novel secondary-structure modifications unique to and conserved within their respective families. Both proteins crystallized as dimers, providing detailed predictions about their self associations. These structures make a substantial contribution to the repertoire of T4SS component structures and will serve as springboards for future functional and protein-protein interaction studies by using knowledge-based site-directed and deletion mutagenesis.

Fig. 1.

Crystal structure of B. suis pVirB8. (A) Sequence alignment of VirB8 proteins and secondary structure assignment. Amino acids of four representative homologs were aligned, from B. suis (B.sVirB8), A. tumefaciens (A.tVirB8, 21% identical to B.sVirB8), H. pylori Cag [H.pCag10 (HP0530, 14% identity], and ComB [H.pComB8 (HP0030), 18% identity] systems (38). Strictly conserved, strongly conserved, and conserved residues are marked in red, magenta, and light pink, respectively. The modeled region (residues 97–188 and 191–234) is shown as a gray line above the sequence for nonregular structure or as cyan boxes and yellow arrows for α-helices and β-strands, respectively. Green dots mark residues involved in VirB8 self association. Purple stars indicate residues mutated in previous functional studies. (B) Overall fold of pVirB8. Secondary structure representation and labels are as in A. (C) Structure of NTF2, most similar fold to pVirB8. Boxes mark the two major points of difference between the NTF2 and pVirB8 fold, the addition of α4 (blue box), and the loss of two β strands (red box). (D) Surface representation of VirB8 coloring side chains by degree of conservation, as shown in A. Orientation is as in B. (E) pVirB8 dimer. Both monomers are shown in ribbon representation with the monomer on the left shown as in B but turned ≈90° clockwise. The other monomer is in gray. Ile-112 and Tyr-120 are shown in stick representation and colored in magenta and green, respectively. (F) Top-down view of pVirB8 dimer showing side chains involved in interface as marked in A. For clarity, one pVirB8 chain is colored gray, and the other is colored as in B. Residues at the interface are in stick representation, color-coded in green, and labeled. The figure was produced by using pymol, http://pymol.sourceforge.net.

Fig. 2.

Crystal structure of H. pylori pComB10. (A) Sequence alignment of the conserved C-terminal region of VirB10 proteins and secondary structure assignment. Amino acids of five representative homologs were aligned, from H. pylori ComB [H.pComB10, (HP0041/0042)] and Cag [H.pCag7 (HP0527), 25% identical to H.pComB10 over region shown] systems, from A. tumefaciens VirB (A.tVirB10, 21% identity) and Trb (A.tTrbI, 24% identity) systems, and from B. suis VirB (B.sVirB10, 24% identity) system. Strictly conserved, strongly conserved, and conserved residues are marked in red, magenta, and light pink, respectively. The modeled region (amino acids 166–253, 261–294, and 311–376) is shown as a gray line above the sequence for nonregular structure or as cyan boxes for α-helices and yellow, orange, red, or green arrows for β-strands. The “bulge” regions that lie between the central (orange) and platform (red) β-sheets are marked with black arrows. Black and gray dots mark residues in the crystal packing interface. (B) Stereo diagram showing overall fold of pComB10. Representation, color-coding, and labeling of β-strands and α-helices are as in A. The four regions referred to in the text are labeled I, II, III, and IV. (C) View of pComB10 rotated through 90° in the vertical axis. The figure was produced by using pymol.

Fig. 3.

Dimer interface and flexible helical region of pComB10. (A) Crystallographic dimer of pComB10. One monomer (in same orientation as Fig. 2B) is shown as a surface representation of charge potential. The second monomer is shown as a ribbon. α1, β4, β6a, and β6b are shown. (B) Superposition of three representative chains shows conformational flexibility in the protruding helical region. B was produced by using pymol, and A was produced by using grasp (39).