Dimerization and interactions of Brucella suis VirB8 with VirB4 and VirB10 are required for its biological activity.
VirB8-like proteins are essential components of type IV secretion systems, bacterial virulence factors that mediate the translocation of effector molecules from many bacterial pathogens into eukaryotic cells. Based on cell biological, genetic, and x-ray crystallographic data, VirB8 was proposed to undergo multiple protein-protein interactions to mediate assembly of the translocation machinery. Here we report the results of a structure-function analysis of the periplasmic domain of VirB8 from the mammalian pathogen Brucella suis, which identifies amino acid residues required for three protein-protein interactions. VirB8 variants changed at residues proposed to be involved in dimerization, and protein-protein interactions were purified and characterized in vitro and in vivo. Changes at M102, Y105, and E214 affected the self-association as measured by analytical ultracentrifugation and gel filtration. The interaction with B. suis VirB10 was reduced by changes at T201, and change at R230 inhibited the interaction with VirB4 in vitro. The in vivo functionality of VirB8 variants was determined by complementation of growth in macrophages by a B. suis virB8 mutant and by using a heterologous assay of type IV secretion system assembly in Agrobacterium tumefaciens. Changes at Y105, T201, R230, and at several other residues impaired the in vivo function of VirB8, suggesting that we have identified interaction sites of relevance in the natural biological context.
Paschos
Athanasios
A
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1.
Patey
Gilles
G
Sivanesan
Durga
D
Gao
Chan
C
Bayliss
Richard
R
Waksman
Gabriel
G
O'callaghan
David
D
Baron
Christian
C
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D041021:Brucella suis / Q000737:chemistry* / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D018360:Crystallography, X-Ray; D019281:Dimerization; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012997:Solvents
D001426:Bacterial Proteins; D012997:Solvents