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.
Highlights
Plasmids of the IncN incompatibility group have a narrow host range mediating horizontal gene transfer among Enterobacteria
The Bacterial Two-hybrid Assay Shows Heterologous Interactions between Distantly Related VirB8 Homologs—To assess the capacity of VirB8 proteins to interact with homologs from other T4SSs, we expressed full-length VirB8 homologs from A. tumefaciens (VirB8a), VirB8b, TraE, and the VirB8 homolog CagV from Helicobacter pylori as fusions to the T25 and the T18 domains of Bordetella CyaA cytotoxin
The interactions were analyzed in the bacterial twohybrid (BTH) assay using Escherichia coli cyaA deletion strain BTH101 and -galactosidase activity as a readout fused to the cytoplasmic N terminus of the full-length proteins
Summary
T4SS, type IV secretion system; AC, adenylate cyclase; BTH, bacterial two-hybrid; RU, response units; SPR, surface plasmon resonance; TEV, tobacco etch virus; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PDB, Protein Data Bank; DSS, disuccinimidyl suberate. Dimerization and Inhibition of TraE genetic and biochemical analyses suggest that VirB8-like proteins are assembly factors that undergo a series of mostly transient interactions with other T4SS components [15,16,17,18,19,20,21]. Analysis of the TraM protein from the plasmid pIP501 conjugation system from Gram-positive Enterococci and of the TcpC protein from Clostridium perfringens showed that despite the absence of apparent sequence similarity, these proteins had a very similar fold [24, 25] These proteins form trimers, suggesting that VirB8-like proteins may be able to interact via different interfaces of their core structure. We conclude that despite their divergent sequences and the transient nature of their interactions, VirB8like proteins have common features that can be exploited for structure-based design of T4SS inhibitors
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