title: Structural Analysis and Inhibition of TraE from the pKM101 Type IV Secretion System.
abstract: Gram-negative bacteria use type IV secretion systems (T4SSs) for a variety of macromolecular transport processes that include the exchange of genetic material. The pKM101 plasmid encodes a T4SS similar to the well-studied model systems from Agrobacterium tumefaciens and Brucella suis Here, we studied the structure and function of TraE, a homolog of VirB8 that is an essential component of all T4SSs. Analysis by X-ray crystallography revealed a structure that is similar to other VirB8 homologs but displayed an altered dimerization interface. The dimerization interface observed in the X-ray structure was corroborated using the bacterial two-hybrid assay, biochemical characterization of the purified protein, and in vivo complementation, demonstrating that there are different modes of dimerization among VirB8 homologs. Analysis of interactions using the bacterial two-hybrid and cross-linking assays showed that TraE and its homologs from Agrobacterium, Brucella, and Helicobacter pylori form heterodimers. They also interact with heterologous VirB10 proteins, indicating a significant degree of plasticity in the protein-protein interactions of VirB8-like proteins. To further assess common features of VirB8-like proteins, we tested a series of small molecules derived from inhibitors of Brucella VirB8 dimerization. These molecules bound to TraE in vitro, docking predicted that they bind to a structurally conserved surface groove of the protein, and some of them inhibited pKM101 plasmid transfer. VirB8-like proteins thus share functionally important sites, and these can be exploited for the design of specific inhibitors of T4SS function.
authors.affiliation: From the Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Quebec H3C 3J7, Canada, and.
authors.forename: Bastien
authors.initials: B
authors.lastname: Casu
authors.affiliation: From the Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Quebec H3C 3J7, Canada, and.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Smart
authors.affiliation: the SPR-MS Facility, Faculty of Medicine, McGill University, Montréal, Quebec H3G 1Y6, Canada.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Hancock
authors.affiliation: From the Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Quebec H3C 3J7, Canada, and.
authors.forename: Mark
authors.initials: M
authors.lastname: Smith
authors.affiliation: From the Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Quebec H3C 3J7, Canada, and.
authors.forename: Jurgen
authors.initials: J
authors.lastname: Sygusch
authors.affiliation: From the Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Quebec H3C 3J7, Canada, and christian.baron@umontreal.ca.
authors.forename: Christian
authors.identifier: http://orcid.org/0000-0002-0132-2111
authors.initials: C
authors.lastname: Baron
mesh_terms: D016960:Agrobacterium tumefaciens / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D041021:Brucella suis / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D006090:Gram-Negative Bacteria / Q000737:chemistry* / Q000378:metabolism; D016480:Helicobacter pylori / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010957:Plasmids / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D060066:Protein Interaction Maps; D055503:Protein Multimerization; D054852:Small Molecule Libraries / Q000494:pharmacology; D000069319:Type IV Secretion Systems / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D054852:Small Molecule Libraries; D000069319:Type IV Secretion Systems
keywords: bacterial conjugation; membrane protein; plasmid; protein secretion; protein-protein interaction; type IV secretion