Biological systems

Antibiotic resistance and bacterial cell wall

Contact : J.P.Simorre

Collaborations :

• Centre d’Ingénierie des Protéines, Université Liège, Belgium • Institute for Cell and Molecular Biosciences, Newcastle upon Tyne, England Although antibiotics have drastically reduced illness and death from infectious diseases, bacteria have exhibited a remarkable capacity to quickly become resistant to antibiotics.Peptidoglycan (murein), the major component of the bacterial cell wall, is essential for resistance to the osmotic pressure of the cytoplasm, plays a crucial role in cell division, determines cell shape, and provides a scaffold for anchoring various polymers to the cell surface. Peptidoglycan is also recognized by the innate immune system and its synthesis is inhibited by major classes of antibiotics including the ß-lactams.Since protein recognition motifs are not only defined by the chemical constitution of peptidoglycan subunits but also by their three-dimensional organization in the polymer, the absence of structure at atomic resolution constitutes a stumbling block to a better understanding of the molecular recognition mechanisms and of the catalytic pathways used by peptidoglycan-interacting proteins. During the recent years, we have developed a strategy based on liquid state and solid state NMR to develop the analysis of the interaction of proteins with peptidoglycan and to build the first experimental model of bacterial peptidoglycan 3D-structure. Our results will permit identification of the mode of recognition of peptidoglycan by proteins involved in immunity, virulence, and peptidoglycan morphogenesis.

Figure: EM of the bacterial cell wall and chemical formula of the peptidoglycan polymer

Heavy metal resistance in Cupriavidus metallidurans CH34

Contact : B.Bersch

Collaborations :

• Jacques Covès, Laboratoire des Protéines Membranaires, IBS, Grenoble
• SCK-CEN, Mol, Belgium
• Département de Protéomique et de Biochimie des Protéines, Mons, Belgium

Cupriavidus metallidurans CH34 is a beta-Proteobacterium which thrives in the presence of millimolar concentration of multiple heavy-metals (Cu(II), Zn(II), Cd(II), Co(II), Pb(II), Hg(II), Ni(II), and Cr(VI)) and represents an important model to study hevy metal resistance. The genome of C. metallidurans CH34 contains two large plasmids pMOL28 (164 ORFs, 171 459 bp) and pMOL30 (250 ORFs, 233 720 bp) carrying gene clusters that encode resistance machineries to various heavy metals.

We are studying the structural and metal-binding properties of proteins involved in resistance to different heavy metals (mercury, copper, cobalt, nickel ...) using NMR in combination with different spectroscopies (RPE, UV-Vis, fluorescence ...) and biochemical techniques.

Membrane proteins

Contact : D.Marion

Structure determination of integral membrane proteins is one of the most important challenges of structural biology. Membrane proteins have key roles in many diseases and more than half of all drug targets are membrane proteins. Beside X-ray diffraction, NMR has become an alternative for structural studies of these proteins as a result of recent progress in NMR spectroscopy, isotopic labeling and detergent solubilization. A collaboration has been recently initiated with a research group developing vaccine against cowdriosis or heartwater in the French West Indies. The tick-borne intracellular bacterium Ehrlichia ruminatium is responsible for this disease in ruminants in sub-Saharan Africa and in the Caribbean. Several types of vaccines have been developed but exhibit limited efficiency in the field as a result of the genetic diversity of the various strains. In infection with E. ruminatium, the serological response is directed against outer-membrane proteins of approximately 30 kDa (map1). The whole map1gene cluster contains 16 paralogs that are regulated differently in the host and vector cell environments in vitro. These proteins are predicted to form ß-barrel structures and thus exposing long loops on the extracellular side that are likely to play an important role in the immunological response. According to in silico structure predictions, these loops show also a large diversity among the paralogs and the various strains. In order to understand the genetic diversity of map1 in terms of 3D structure, an NMR study has been initiated and preliminary expression and solubilisation investigations are under way. Structural information obtained from this study may help to design second-generation vaccines with increased efficiency against the various E. ruminatium strains.

Structural Studies of the Molecular Interaction Networks Underlying miRNA Biogenesis

Contact : J.Boisbouvier

Collaborations : IBR Rosario, Argentina

Biosynthesis of micro-RNAs (miRNA) occurs via a conserved processing pathway in which the long primary miRNA transcript undergoes double strand cleavage at two locations. The final product is a short, 21-25 nucleotide single-stranded miRNA that guides the RNA interference machinery. How the processing enzymes and accessory proteins assemble and co-ordinate this process remain unknown. Recent 3D structures of small portions of the processing machinery have revealed interesting mechanistic insights, but considerable areas remain poorly understood, particularly at atomic resolution.

Outline of the human miRNA biogenesis pathway.

A systematic structural characterization of the biomolecular associations that underlie miRNA biogenesis in plants and humans has been undertaken. Domains derived from proteins involved in immature miRNA processing have been produced using a semi-automated cloning and expression platform. A RNAse-free wetlab has been set up for large-scale production and purification of microRNA precursors. The complex network of protein-protein and protein-RNA interactions is being mapped by NMR spectroscopy and other biochemical techniques. In combination with high-resolution protein and RNA structures, these data are allowing a better understanding of the mechanisms that drive substrate recognition and formation of processing complexes in miRNA maturation.

Structural Studies of Viral RNAs and associated complexes

Contact : J.Boisbouvier

Collaborations : UVHCI- Grenoble, IECB (Bordeaux), Univ. Ottawa (Canada)

The genome of viruses such as the Human Immunodeficiency Virus or the Hepatisis C Virus is composed by RNA. After infecting a cell, viral RNA serves as a template for new copies of viral chromosomal RNA but is also used as mRNA in the synthesis of viral proteins that compose the virus envelope. In order to understand the mechanisms of RNA virus replication at an atomic level and to develop or improve drugs to block virus multiplication, we are studying the structure of key viral RNA motifs and their interaction with drugs or host-cell machineries.

Structure of the TAR-TAR*GA complex. 17 structures of the NMR ensemble has been superimposed with a rmsd equal to 0.26 Å

A first target of interest is the TAR stable stem-loop structure of HIV RNA, which plays a crucial role during the life cycle of the virus. The apical loop of TAR acts as a binding site for essential cellular cofactors required for the replication of HIV. High-affinity aptamers directed against the apical loop of TAR have been identified by the SELEX approach. The structure of the TAR/apatamer "kissing complex" was determined using liquid crystal NMR spectroscopy, and represents one of the highest-resolution RNA structures determined in solution to date. This precise model reveals the molecular origins of the high stability of HIV TAR RNA bound to its SELEX RNA aptamer. A non-canonical loop-closing GA base pair was found to be stabilized by a network of intersugar hydrogen bonds, which in turn accounts for the greatly reduced dissociation constant of the complex relative to those without the GA pair. By systematic permutations of the loop closing base pair, we establish that the identified atomic interactions, which form the basis for the high stability of the complex, are maintained in several other kissing complexes. This study will contribute to the development or improvement of drugs against RNA loops of viruses or pathogens as well as the conception of biochemical tools targeting RNA hairpins involved in important biological functions.

A second target studied in our laboratory is the Internal Ribosome Entry Sites of Hepatisis C Viral RNA. HCV IRES is a genomic non-coding region, recognized by host proteins (initiation factors eIFs and ribosomal proteins), which form ribonucleoprotein complexes (RNP) with the IRES. These RNPs are relevant for viral translation initiation and virus amplification. In order to elucidate the initiation mechanism of viral RNA translation into viral proteins, we are studying the structure of IRES domains in interaction with host cells proteins.