We will seek seek in this BmrA-NMX project to reveal structure features of the different conformational states of BmrA, an ABC efflux pump, using N for NMR, M for Mass spectrometry, and X for X-ray crystallography methods. BmrA is, other members of ABC transporters family, able to transport a wide variety of drugs. Multidrug resistance due to efflux pumps has strong implication in medicine because it is found from humans to bacteria. We will use state-of-the art biochemical studies to identify and characterize different conformational states, using wild-type and mutant forms, and drug-bound forms. Drug binding and transport abilities of the corresponding states of BmrA will be quantified and transposed to crystallogenesis, mass spectrometry and solid-state NMR experiments. These approaches will be used to gain insight into the structural features of the membrane pump. They will deliver complementary structural parameters, such as the overall 3D structure, detailed information on conformational changes as a function of state, and stability and accessibility of secondary structure elements. Sample preparations of the different forms will be established using feed back from the biochemical studies, and crystalline, as well as membrane-bound, drug-bound and mutant forms will be made for the different studies. The unique combination of structural techniques is expected to obtain a comprehensive picture of the processes involved in drug export. Such knowledge will be an important steppingstone towards deciphering the molecular mechanism underlying drug export.

Bacterial pathogens have evolved dedicated strategies to colonise organs, counter-attack and subvert host immune defence systems. Type IV secretion systems (T4SS) are molecular machineries produced by many bacterial pathogens, including Helicobacter pylori, Legionella pneumophila, Brucella suis, Bordetella pertusis or Bartonella henselae. T4SSs consist of around twelve proteins that form a molecular device that spans both bacterial membranes. It is prolonged in the external milieu by a pilus used to adhere to and/or to translocate effectors into host cells. H. pylori is a Gram-negative bacterium that colonises the human stomach in half of the world population. It is estimated that 20% of individuals infected during their childhood will develop peptic ulcer disease, while gastric neoplasia will develop in 2-3% of infected individuals. For these reasons, H. pylori was defined as a group 1 carcinogen by World Health Organization (WHO). Strains associated with the most severe diseases contain the cytotoxin-associated gene cagA. CagA is a unique protein encoded by a gene located in the Cag pathogenicity island (CagPAI). This CagPAI encodes for 28 proteins that assemble a T4SS (cagT4SS) required to deliver CagA into the host cell. After translocation, CagA is tyrosine phosphorylated by host Src kinases and highjacks the signalling system of the cell. This leads to morphological changes in the cell, uncontrolled proliferation and provokes tumor apparition. CagA is therefore the major determinant of cancer development during H. pylori infection and is considered the paradigm of “bacterial carcinogenesis”. The mechanisms of injection of CagA by the cagT4SS are still poorly understood but it was found that the T4SS pilus proteins and CagA utilise a5ß1 integrin as a host cell receptor prior to substrate translocation. Interestingly, a5ß1 integrin, perhaps the most represented member of the integrin family, appears as a common receptor for numerous pathogens. The objective of the proposed research program is to elucidate the structural and molecular basis of integrin a5ß1 utilisation by the H. pylori T4SS complex. Our research initiative builds upon recent exciting developments in our research groups and connects structural studies (X-ray crystallography, Small Angle X-ray Scattering (SAXS) and Electron Microscopy (EM)) with interaction studies and cellular assays. The proposal is based on the current collaborative work performed by three groups that has led to: 1) established protocols for purification of the proteins and interaction studies, 2) high resolution studies of CagA structure and identification of its a5ß1 integrin binding domain, 3) efficient in vivo assays to monitor the functional relevance of the interactions studied. Our short-term goal is to decipher the molecular and structural details of (1) the interactions between pilus protein components (2) their interactions with the integrin a5ß1 receptor and (3) to determine the sequence of binding events/activation leading to CagA injection. The proposed program is timely given the recent maturation of the methods needed to tackle such complex research problems. Our groups have a history of successful collaboration and our expertise is genuinely complementary, hereby ensuring a more complete, integrated vision of the a5ß1 integrin exploitation by H. pylori. Although a5ß1 appears as a common receptor for many pathogens, no structural information is available concerning the details of these interactions. Because an increasing number of pathogens are identified to use integrins to mediate their infection, we believe that our program will impact the study of many infections. The long-term objectives are to better understand the molecular mechanisms of bacterial infection and to provide new therapeutic strategies, urgently needed to combat these infections.

Little is known today about the structure of full-length prions. We recently established, using solid-state NMR studies of the Ure2p and HET-s proteins, that prions show different architectures, and that the building principles between them can differ considerably. We here aim at the study of the Sup35 prion, which poses a formidable challenge considering its large size. Having demonstrated the important role of the globular domain, we here aim to study assembly in the context of the entire proteins, as opposed to fragments, and investigate on a molecular level the role of the functional C-terminal domain of the protein in fibril assembly. We aim thus to compare the structures of fibrillar Sup35NM and full-length Sup35p by solid state NMR. The fragment Sup35NM is often used as a model for the full-length prion. We will compare the structures of fibrillar Sup35NM and full-length Sup35p by solid state NMR and determine whether the structure of this fragment is indeed preserved in the context of the full-length fibril, or if it is different. This question is particularly relevant in the light of our previous results on the full-length HET-s and Ure2p prions, which show that the structure of the isolated prion and globular domains are not conserved in the context of the full-length prions. We will address this question by solid-state NMR, which is an emerging technique for the structural study of insoluble proteins. Important developments over the last decade have pushed this fast evolving technique to become a serious partner in structural biology. If it has been shown in proof-of-principle experiments that amyloid and prion structures can be determined by solid-state NMR methods, it is still difficult to obtain structures of full-length prions. The main difficulties are caused by the large size of these proteins, a property they also share with other insoluble proteins. However, many elements of the technology needed to tackle these problems are now available in the applicants’ laboratories and we propose to fully develop the NMR methodology and, in parallel, apply it to the yeast prion Sup35p that is made of 685 amino-acid residues. The key NMR methodology developed in the context of this proposal will allow us to establish structural models which will be confronted with the large body of biophysical and biochemical and functional knowledge to work out the relationship between structural and biological features of Sup35p. Besides, the protocols and techniques developed will also be applicable to other large proteins and should allow to open structural studies on other insoluble proteins.