Structure and Dynamics of Nucleoproteic and Membrane Assemblies

High-Pressure NMR

C. Roumestand, A. Padilla, K. de Guillen, Y.-S. Yang.

 

 

Collaborations: C. Royer, Á. Garcia (Rensselaer Polytechnic Institute) ; B. Garcia-Moreno and D. Barrick (John Hopkins University) ; R. Winter (University of Dortmund)
Structural and dynamic sensitivity to hydrostatic pressure is a specific and unique property of the folded state. In that sense, it is in contrast with denaturation by temperature or chemical agents that act globally and depend on the exposed surface area of the unfolded state. Pressure perturbation coupled with NMR spectroscopy and novel computational approaches will enable an unprecedented exploration of the complexities of protein energy landscapes. Denaturation by pressure originates from the smaller molar volume of the unfolded states of proteins with respect to their folded counterparts. Several hypotheses have been proposed to explain this decrease in volume: the distinct density between hydrating and bulk water, the pressure-dependent changes in bulk water structure, the loss of internal void volume, or some combination of the previous three. We have demonstrated, using variants of model proteins ∆+PHS Staphylococcal nuclease (SNase) (Roche et al., 2012) and NanK (Rouget et al., 2011), that pressure unfolds proteins owing to the presence of cavities in the folded state, thus solving a 100 year-old conundrum.

 

Structural and dynamic sensitivity to hydrostatic pressure is a specific and unique property of the folded state. In that sense, it is in contrast with denaturation by temperature or chemical agents that act globally and depend on the exposed surface area of the unfolded state. Pressure perturbation coupled with NMR spectroscopy and novel computational approaches will enable an unprecedented exploration of the complexities of protein energy landscapes. Denaturation by pressure originates from the smaller molar volume of the unfolded states of proteins with respect to their folded counterparts. Several hypotheses have been proposed to explain this decrease in volume: the distinct density between hydrating and bulk water, the pressure-dependent changes in bulk water structure, the loss of internal void volume, or some combination of the previous three. We have demonstrated, using variants of model proteins ∆+PHS Staphylococcal nuclease (SNase) (Roche et al., 2012) and NanK (Rouget et al., 2011), that pressure unfolds proteins owing to the presence of cavities in the folded state, thus solving a 100 year-old conundrum.

Structural and energetic mapping of the folding landscapes of cavity variants of ∆+PHS staphylococcal Nuclease (SNase).
Left – A) equilibrium free energy profiles for ∆+PHS (top) as a function of increasing pressure from blue to red, and (below) comparing ∆+PHS at 800 bar to L125A at 600 bar, B) representative structures from the coarse grained simulations constrained by the NMR data. Right - A-E) kinetic mapping of TSE volume for the SNase variants under different solution conditions as noted

 

Beyond demonstrating the major mechanism by which pressure unfolds proteins, high-pressure NMR studies provided unprecedented insights into the structural and energetic features of the folding landscapes of SNase and its mutants. We have developed a coarse grained approach constrained by experimental NMR data that allowed us to characterize structurally and energetically the major conformers on the folding route of these proteins (Roche et al., 2012; see figure). Further exploration of the folding free energy landscapes of these SNase variants was carried out by varying the temperature and also by implementing pressure-jump NMR relaxation kinetics. Temperature studies combined with the HP-NMR and theoretical tools that we have developed lead to the characterization of the effects of temperature, not only on volume changes, but also on the relative stabilities of the various conformers available in the energy landscape. An important effort has been put in implementing RDC measurements in combination with high-pressure NMR. Availability of RDCs under pressure will enormously improve the structural and dynamic description of the folding/unfolding events of proteins. In a first step, pressure resistant alignment media have been identified and RDCs on a model protein have been measured in a range 1-2500 bar, in collaboration with Nathalie Sibille (Team 2) (Sibille et al, in press).

Structural Biology of Infectious Diseases

M. Cohen-Gonsaud, C. Roumestand, P. Barthe, B. Murciano, E. Guca, A. de Visch

 

Biologie structurale de Mycobacterium tuberculosis (MCG)

Collaboration with  : G. Mukamolova (University of Leicester), P. Brodin (Institut Pasteur, Lille), V. Molle (DIMNP – Montpellier), A. Blanc-Potard (DIMNP – Montpellier).

For the next 5 years, the working group “Mtb Structural Biology” led by MCG will continue deciphering the mechanisms around the latency, phospho-regulation, persistence and virulence in M. tuberculosis. This work is also done in collaboration with the “Structures and screening of therapeutic and environmental” team (in collaboration with G. Labesse and JF Guichou) for the drug-design related project. To do so we put in place a network of collaborations with top international and national microbiology laboratories (i.e. University of Leicester, Institut Pasteur Lille), but also collaboration with le local Mtb community (University Montpellier II and CPBS).

 

Resuscitation and Phosphoregulation mechanisms in M.tuberculosis

Collaboration with : V. Molle (DIMNP – Montpellier), L. Kremer (DIMNP – Montpellier), G. Mukamolova (University of Leicester)

Regulation via Ser/Thr Protein Kinases (STPK) has emerged as an essential mechanism in controlling key pathways in the bacillus (metabolism, cell division, expression regulation). Despite the fact that the STPKs have been extensively studied, the molecular mechanisms of its phosphoregulation were poorly understood. In collaboration with different laboratories we engaged studies to understand the activity modulation of STPKs substrates to unravel new regulation pathways within the bacteria. We solved the structure of the unphosphorylated and phosphorylated isoforms of the OdhI protein (also named GarA) in solution, a central regulator of the tricarboxylic acid (TCA). We discovered a major conformational changes upon phosphorylation of a disordered region of the protein (Barthe 2009). We studied a FHA modular protein called Rv0020c. We solved the structure of its domains by NMR and studied their interaction with STPK demonstrating a fine tuning of the STPK/Rv0020c interaction via the phosphorylation of an unstructured STPK domain (Roumestand 2011). Furthermore, we solved the structure of a transcriptional regulator in solution, and revealed a DNA binding regulation mediated by STPK phosphorylation (Cohen-Gonsaud 2009). Other studies in M.tuberculosis focussed on the STPK regulation of the essential mycolic acid biosynthesis (Veyron-Churley 2012), the S-Adenosylhomocysteine hydrolase activity (Corrales 2013), and the CcpA regulation in S. aureus (Leiba 2012).

One of the keys of Mycobacterium tuberculosis’ success as a pathogen is its ability to persist in the host organism in a latent state for years after the first phase of infection. We have been interested for long time in the exit mechanism of latency (Cohen-Gonsaud 2005). The STPK PknB has been proposed to be the receptor for a dormancy exit signal. With proven combined experience in the field, our team and Galiana Mukamolova’s group (University of Leicester) initiated a study to isolate and study the potential molecular determinant of resuscitation in M. tuberculosis. We first solved the structure of the external domain of the kinase in solution that revealed an original fold (Barthe 2010). After extensive studies we can now propose a model for the PknB related resuscitation mechanism (manuscript in preparation).

 

Persistence and Virulence

Collaboration with : A. Blanc-Potard (DIMNP – Montpellier ; K. Brodolin (CPBS, Montpellier), L. Brodin (Institut Pasteur, Lille), G. Mukamolova (University of Leicester)

One of the main projects over the last years has been related to the mechanism that has been unravelled by our collaborator Galiana Mukamolova at the University of Leicester. They demonstrated that three of the main TB antibiotics have the ability to enhance the survival of the bacillus during the stationary phase. Combining molecular modelling, biochemical, genetic, transcriptomic, and animal studies we proposed a complete model to explain the observed phenomenon (Turapov submitted). Another important project has been initiated in mid-2012 in collaboration with the group of Pricille Brodin (Institut Pasteur Lille). The ability of the bacillus to arrest phagosome maturation is a major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, they developed a high-throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 M.tuberculosis mutants. Among them two essential protein were isolated.

 

Biologie structurale d’Apicomplexa (CR)

Collaboration with : S. Delbeq (University of Montpellier), T. Schetters (Intervet International B.V. – The Netherlands), H.Vial (University of Montpellier 2)
The structural study of surface antigens from the merozoite membrane of Babesia is still in progress and constitutes a fruitful collaboration with the team of S. Delbecq (Faculty of Pharmacy, Montpellier) and Intervet® (MSD), one of the leader companies in veterinary vaccines. A new collaboration has been initiated with the group of H. Vial (CNRS UMR 5235, Montpellier) to tackle the malaria parasite Plasmodium falciparum, an Apicomplexa responsible for a toll of one million human deaths per year, with almost half of the human population at risk of contracting the disease. Our objectives are i) to determine the 3D structures of the catalytic domains of PfCCT, an enzyme involved in the metabolism of phospholipids in the parasite, and ii) to study the interaction of the catalytic domains with their own substrates and with compounds inhibiting PC biosynthesis These compounds are mono- and bis-thiazoliums developed in Dr. H. Vial laboratory and are currently under clinical development (Sanofi-Aventis).

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