1. Academic Validation
  2. Structural Analysis and Inhibition of TraE from the pKM101 Type IV Secretion System

Structural Analysis and Inhibition of TraE from the pKM101 Type IV Secretion System

  • J Biol Chem. 2016 Nov 4;291(45):23817-23829. doi: 10.1074/jbc.M116.753327.
Bastien Casu 1 Jonathan Smart 1 Mark A Hancock 2 Mark Smith 1 Jurgen Sygusch 1 Christian Baron 3
Affiliations

Affiliations

  • 1 From the Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Quebec H3C 3J7, Canada, and.
  • 2 the SPR-MS Facility, Faculty of Medicine, McGill University, Montréal, Quebec H3G 1Y6, Canada.
  • 3 From the Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, Quebec H3C 3J7, Canada, and christian.baron@umontreal.ca.
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.

Keywords

bacterial conjugation; membrane protein; plasmid; protein secretion; protein-protein interaction; type IV secretion.

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