The type IV secretion system core component VirB8 interacts via the ß1-strand with VirB10.

In this work, we provide evidence for the interactions between VirB8 and VirB10, two core components of the type IV secretion system (T4SS). Using nuclear magnetic resonance experiments, we identified residues on the ß1-strand of Brucella VirB8 that undergo chemical shift changes in the presence of VirB10. Bacterial two-hybrid experiments confirm the importance of the ß1-strand, whereas phage display experiments suggest that the α2-helix of VirB8 may also contribute to the interaction with VirB10. Conjugation assays using the VirB8 homolog TraE as a model show that several residues on the ß1-strand of TraE are important for T4SS function. Together, our results suggest that the ß1-strand of VirB8-like proteins is essential for their interaction with VirB10 in the T4SS complex.

Monomer-to-dimer transition of Brucella suis type IV secretion system component VirB8 induces conformational changes.

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 VirB8spM102R variant 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. DATABASE: BMRB accession no. 26852 and PDB 5JBS.

Type IV Secretion in Agrobacterium tumefaciens and Development of Specific Inhibitors.

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) comprises 12 membrane-bound proteins, and it assembles a surface-exposed T-pilus. It is considered to be the archetypical system that is generally used to orient the nomenclature of other T4SS. Whereas the sequence similarities between T4SSs from different organisms are often limited, the general mechanism of action appears to be conserved, and the evolutionary relationship to bacterial conjugation systems and to T4SSs from animal pathogens is well established. Agrobacterium is a natural genetic engineer that is extensively used for the generation of transgenic plants for research and for agro-biotechnological applications. It also served as an early model for the understanding of pathogen-host interactions and for the transfer of macromolecular virulence factors into host cells. The knowledge on the mechanism of its T4SS inspired the search for small molecules that inhibit the virulence of bacterial pathogens and of bacterial conjugation. Inhibitors of bacterial virulence and of conjugation have interesting potential as alternatives to antibiotics and as inhibitors of antimicrobial resistance gene transfer. Mechanistic work on the Agrobacterium T4SS will continue to inspire the search for inhibitor target sites and drug design.

Role of the N-terminal seven residues of surfactant protein B (SP-B).

Breathing is enabled by lung surfactant, a mixture of proteins and lipids that forms a surface-active layer and reduces surface tension at the air-water interface in lungs. Surfactant protein B (SP-B) is an essential component of lung surfactant. In this study we probe the mechanism underlying the important functional contributions made by the N-terminal 7 residues of SP-B, a region sometimes called the "insertion sequence". These studies employed a construct of SP-B, SP-B (1-25,63-78), also called Super Mini-B, which is a 41-residue peptide with internal disulfide bonds comprising the N-terminal 7-residue insertion sequence and the N- and C-terminal helices of SP-B. Circular dichroism, solution NMR, and solid state (2)H NMR were used to study the structure of SP-B (1-25,63-78) and its interactions with phospholipid bilayers. Comparison of results for SP-B (8-25,63-78) and SP-B (1-25,63-78) demonstrates that the presence of the 7-residue insertion sequence induces substantial disorder near the centre of the lipid bilayer, but without a major disruption of the overall mechanical orientation of the bilayers. This observation suggests the insertion sequence is unlikely to penetrate deeply into the bilayer. The 7-residue insertion sequence substantially increases the solution NMR linewidths, most likely due to an increase in global dynamics.