VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD

Casu, Bastien; Mary, Charline; Sverzhinsky, Aleksandr; Fouillen, Aurélien; Nanci, Antonio; Baron, Christian

doi:10.1073/pnas.1802501115

Cited by 16 publications

(20 citation statements)

References 43 publications

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“…The structures are reminiscent of the mammalian nuclear transfer factor NTF2, a protein that also presents as a dimer and undergoes multiple protein–protein interactions . Exceptionally, TraE, a homologue of VirB8 from the conjugation plasmid pKM101, shows a similar structure but with an altered dimerization interface of its periplasmic domain , and the full‐length form of TraE forms a hexamer with a central pore . Likewise, DotI, an inner membrane protein from the Dot/Icm type IV secretion system that is known as a counterpart of VirB8, forms octameric rings in a periplasmic domain crystal, which has been proven to be an intrinsic property rather than a crystal packing artefact .…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of CagV, the Helicobacter pylori homologue of the T4SS protein VirB8

Zhao

Sun

et al. 2019

The FEBS Journal

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The VirB/D type IV secretion system (T4SS) plays an essential role in materials transport between host cells and pathogenic Helicobacter pylori and is considered the major pathogenic mediator of H. pylori‐associated gastric disease. VirB8, an inner membrane protein that interacts with many other proteins, is a crucial component for secretory function. Here, we present a crystal structure of the periplasmic domain of CagV, the VirB8 counterpart in the H. pylori Cag‐T4SS. The structure reveals a fold similar to that of other VirB8 members except for the absence of the α5 helix, a discontinuous β1 strand, a larger angle between the α2 and α3 helices, a more hydrophobic surface groove, but exhibits a different dimer interface. Whether the dimerization occurs in solution was proved by mutagenesis, size‐exclusion chromatography and cross‐linking assays. Unlike the classical dimerization mode, the interface of the CagV dimer is principally formed by several hydrogen bonds, which indicates instability of dimerization. The structure here demonstrates the difference in dimerization among VirB8 homologues and indicates the considerable compositional and functional diversity of them in T4SS. Database Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6IQT.

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Section: Introductionmentioning

confidence: 99%

Crystal structure of CagV, the Helicobacter pylori homologue of the T4SS protein VirB8

Zhao

Sun

et al. 2019

The FEBS Journal

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“…Indeed, the stoichiometry of VirB8 was estimated to 12 in Conj-T4SS from E. coli R388 [40]. In line with, the purification of full-length TraE and TraM, a VirB8 homolog from pKM101 and R64, respectively, revealed that TraE and TraM forms hexamers in solution [39, 41]. Dissimilar to what was described in Gram-negative bacteria, VirB8-like proteins in Gram-positive bacteria are proposed to function as trimers.…”

Section: Discussionmentioning

confidence: 92%

Structural and biochemical analysis of OrfG: the VirB8-like component of the integrative and conjugative element ICESt3 from Streptococcus thermophilus

Cappele

Mohamad-Ali

Leblond‐Bourget

et al. 2020

Preprint

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Conjugative transfer is a major threat to global health since it contributes to the spread of antibiotic resistance genes and virulence factors among commensal and pathogenic bacteria. To allow their transfer, mobile genetic elements including Integrative and Conjugative Elements (ICEs) use a specialized conjugative apparatus related to Type IV secretion systems (Conj-T4SS). Therefore, Conj-T4SSs are excellent targets for strategies that aim to limit the spread of antibiotic resistance. In this study, we combined structural, biochemical and biophysical approaches to study OrfG, a protein that belongs to Conj-T4SS of ICESt3 from Streptococcus thermophilus. Structural analysis of OrfG by X-ray crystallography revealed that OrfG central domain is similar to VirB8-like proteins but displays a different quaternary structure in the crystal. To understand, at a structural level, the common and the diverse features between VirB8-like proteins from both Gram-negative and -positive bacteria, we used an in silico structural alignment method that allowed us to identify different structural classes of VirB8-like proteins. Biochemical and biophysical characterizations of purified OrfG soluble domain and its central and C-terminal subdomains indicated that they are mainly monomeric in solution but able to form an unprecedented 6-mer oligomers. Our study provides new insights into the structural and assembly mode of VirB8-like proteins, a component essential for conjugative transfer and improves our understanding on these under-examined bacterial nanomachines.

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“…We have previously used this approach to identify inhibitors of the VirB8 homolog TraE from the plasmid pKM101 conjugation system. We identified molecules that target a known inhibitor binding site on VirB8-like proteins, and we also identified a new binding site showing the potential for the discovery of bioactive molecules and of novel inhibitor target sites 23,24,33 .…”

Section: Resultsmentioning

confidence: 99%

Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system

Arya

Oudouhou

Casu

et al. 2019

Sci Rep

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Type IV secretion systems are multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system encoded by the cag pathogenicity island encodes 27 proteins and most are essential for virulence. We here present the identification and characterization of inhibitors of Cagα, a hexameric ATPase and member of the family of VirB11-like proteins that is essential for translocation of the CagA cytotoxin into mammalian cells. We conducted fragment-based screening using a differential scanning fluorimetry assay and identified 16 molecules that stabilize the protein suggesting that they bind Cagα. Several molecules affect binding of ADP and four of them inhibit the ATPase activity. Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the active site. Cross-linking suggests that the active molecules change protein conformation and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cagα hexamer. Addition of the molecule 1G2 inhibits the induction of interleukin-8 production in gastric cancer cells after co-incubation with H. pylori suggesting that it inhibits Cagα in vivo . Our results reveal a novel mechanism for the inhibition of the ATPase activity of VirB11-like proteins.

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VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD

Cited by 16 publications

References 43 publications

Crystal structure of CagV, the Helicobacter pylori homologue of the T4SS protein VirB8

Crystal structure of CagV, the Helicobacter pylori homologue of the T4SS protein VirB8

Structural and biochemical analysis of OrfG: the VirB8-like component of the integrative and conjugative element ICESt3 from Streptococcus thermophilus

Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system

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