Crystal Structure of the VgrG1 Actin Cross-linking Domain of the Vibrio cholerae Type VI Secretion System

• Published in: The Journal of biological chemistry 2012-11, Vol.287 (45), p.38190-38199
• Main Authors: Durand, Eric, Derrez, Estelle, Audoly, Gilles, Spinelli, Silvia, Ortiz-Lombardia, Miguel, Raoult, Didier, Cascales, Eric, Cambillau, Christian
• Format: Article
• Language: English
• Subjects: Actin; Actin Cross-linking; Actins - chemistry; Actins - metabolism; Adenosine Diphosphate - chemistry; Adenosine Diphosphate - metabolism; Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Animals; ATP; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites - genetics; Biochemistry, Molecular Biology; Blotting, Western; Cell Line; Cholera Toxin; Crystal Structure; Crystallography, X-Ray; G-actin; Glutamic Acid - chemistry; Glutamic Acid - genetics; Glutamic Acid - metabolism; Humans; Life Sciences; Magnesium - chemistry; Magnesium - metabolism; Manganese - chemistry; Manganese - metabolism; MARTX; Models, Molecular; Mutation; Protein Binding; Protein Structure and Folding; Protein Structure, Tertiary; Rabbits; Secretion; Type VI Secretion; VgrG; Vibrio cholerae - genetics; Vibrio cholerae - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.390153

Vibrio cholerae is the cause of the diarrheal disease cholera. V. cholerae produces RtxA, a large toxin of the MARTX family, which is targeted to the host cell cytosol, where its actin cross-linking domain (ACD) cross-links G-actin, leading to F-actin depolymerization, cytoskeleton rearrangements, and cell rounding. These effects on the cytoskeleton prevent phagocytosis and bacterial engulfment by macrophages, thus preventing V. cholerae clearance from the gut. The V. cholerae Type VI secretion-associated VgrG1 protein also contains a C-terminal ACD, which shares 61% identity with MARTX ACD and has been shown to covalently cross-link G-actin. Here, we purified the VgrG1 C-terminal domain and determined its crystal structure. The VgrG1 ACD exhibits a V-shaped three-dimensional structure, formed of 12 β-strands and nine α-helices. Its active site comprises five residues that are conserved in MARTX ACD toxin, within a conserved area of ∼10 Å radius. We showed that less than 100 ACD molecules are sufficient to depolymerize the actin filaments of a fibroblast cell in vivo. Mutagenesis studies confirmed that Glu-16 is critical for the F-actin depolymerization function. Co-crystals with divalent cations and ATP reveal the molecular mechanism of the MARTX/VgrG toxins and offer perspectives for their possible inhibition. Background: The toxin VgrG1 actin cross-linking domain (ACD) cross-links G-actin, leading to F-actin depolymerization. It is homologous to the MARTX toxin family. Results: We solved the crystal structure of active VgrG1 ACD. Conclusion: Complexes with ADP/ATP and divalent cations help in understanding ACD active site function. Significance: The structural characterization of ACDs provides opportunities to design compounds targeting their active site.