title: Monomer-to-dimer transition of Brucella suis type IV secretion system component VirB8 induces conformational changes.
abstract: Secretion systems are protein complexes essential for bacterial virulence and potential targets for antivirulence drugs. In the intracellular pathogen Brucella suis, a type IV secretion system mediates the translocation of virulence factors into host cells and it is essential for pathogenicity. VirB8 is a core component of the secretion system and dimerization is important for functionality of the protein complex. We set out to study dimerization and possible conformational changes of VirB8 from B. suis (VirB8s) using nuclear magnetic resonance, X-ray crystallography, and differential scanning fluorimetry. We identified changes of the protein induced by a concentration-dependent monomer-to-dimer transition of the periplasmic domain (VirB8sp). We also show that the presence of the detergent CHAPS alters several signals in the heteronuclear single quantum coherence (HSQC) spectra and some of these chemical shift changes correspond to those observed during monomer-dimer transition. X-ray analysis of a monomeric variant (VirB8spM102R) demonstrates that significant structural changes occur in the protein's α-helical regions (α2 and α4). We localized chemical shift changes of residues at the dimer interface as well as to the α1 helix that links this interface to a surface groove that binds dimerization inhibitors. Fragment-based screening identified small molecules that bind to VirB8sp and two of them have differential binding affinity for wild-type and the VirB8spM102Rvariant underlining their different conformations. The observed chemical shift changes suggest conformational changes of VirB8s during monomer-dimer transition that may play a role during secretion system assembly or function and they provide insights into the mechanism of inhibitor action.
  
  
  
  BMRB accession no. 26852 and PDB 5JBS.
authors.affiliation: Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Canada.
authors.forename: Mahzad
authors.initials: M
authors.lastname: Sharifahmadian
authors.affiliation: Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Canada.
authors.forename: Tarun
authors.initials: T
authors.lastname: Arya
authors.affiliation: Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Canada.
authors.forename: Benoit
authors.initials: B
authors.lastname: Bessette
authors.affiliation: Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Canada.
authors.forename: Lauriane
authors.initials: L
authors.lastname: Lecoq
authors.affiliation: Institut de Recherche en Immunologie et Cancer (IRIC), Université de Montréal, Canada.
authors.forename: Edward
authors.initials: E
authors.lastname: Ruediger
authors.affiliation: Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Canada.
authors.forename: James G
authors.initials: JG
authors.lastname: Omichinski
authors.affiliation: Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Canada.
authors.forename: Christian
authors.identifier: 0000-0002-0132-2111
authors.initials: C
authors.lastname: Baron
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D041021:Brucella suis / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019281:Dimerization; D005470:Fluorometry; D008823:Micelles; D062105:Molecular Docking Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D019897:Periplasm / Q000737:chemistry; D011487:Protein Conformation; D000069319:Type IV Secretion Systems / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D008823:Micelles; D000069319:Type IV Secretion Systems
keywords: NMR assignment; VirB8; antibiotic resistance; crystal structure; type IV secretion system