PhD projects


Structural Biology of Membrane Proteins

PhD project:

Structure-function of chemokine receptors

Principal Investigator:

F. Fieschi, Inst de Biol. Struct. UMR5075 CEA-CNRS-Univ. J.Fourier, 41, rue Jules Horowitz, F38027 Grenoble cedex 1; franck.fieschi|

In collaboration with:

Jean-Luc Popot

Chemokine receptors belong to the 7 transmembrane G-Protein coupled receptors (GPCRs). Chemokines and their associated receptors play a key role in a large variety of physiological and immunological. Amongst these receptors, CXCR4 and CCR5 are of crucial relevance. Besides their poorly documented physiological roles, CCR5 is involved in lymphocyte homeostasis and CXCR4 in embryo development, they have attracted the scientific community by their role in HIV internalization. HIV entry into its target requires indeed the sequential interaction of its envelope glycoprotein with the cellular CD4 molecule and one of its two co-receptors CXCR4 and CCR5. The discovery of the inhibition activity of CXCR4 (the SDF-1 chemokine) or CCR5 (RANTES or MIP-1α or β) ligands on HIV infection triggered new insights in the comprehension of HIV infection and provided new directions in the research of HIV inhibitory molecules. So far the identification of the latter has been mainly empirical, as their rational design would request the characterization of the HIV co-receptors.

Structural studies of membrane proteins are highly limited by the bottleneck of their production in quantities compatible with crystallization trials. When examining the pdb database, it is striking to find out that out of the 43000 protein structures determined so far no more than 125 concern membrane proteins, only 14 being mammalian. Out of these 14 proteins only one is 1 human and 2 were obtained from recombinant sources. Solely one GPCR structure has been resolved so far and concerned bovine rhodopsine, the only GPCR naturally expressed quantity large enough to enable its purification from natural sources to undergo crystallization. In this context we developed a method for the high level production of GPCRs in E.coli, taking CXCR4 and CCR5 as a working model. E.coli has proved to be an amazing machinery for protein recombinant expression but is not compatible with the functional expression of eukaryotic membrane proteins. This is probably due to the unsuccessful insertion of the protein to the prokaryotic membrane and the correlated toxicity for the bacteria. To overcome the problem we decided to address our proteins to inclusion body. Proteins are thus expressed under an aggregated form and purified under denaturing conditions. We are now able to produce mg of pure and homogeneous CCR5 or CXCR4 proteins from one Liter of E.coli culture. The following step consists in refolding the co-receptors by screening a broad range of renaturing buffers and assessing the refolding rate by a functional binding assay. Preliminary experiments have already comforted the feasibility of this approach enabling promising refolding yields1.

- Molecular biology – protein biochemistry (F. Fieschi, first 18 months)

Based on the recent progresses made on the production of CCR5 from inclusion bodies, the production of both receptors will be refined. The refolding procedure needs to be optimised using either classical detergent solution or the incorporation of non-natural surfactants (synthetic amphiphilic compounds without denaturing properties towards proteins on the contrary to classical detergents).

- Ligand binding characterization (F. Fieschi, during 12 months)

Binding activities of over-produced ligands will be addressed by fluorescence and further characterized by surface plasmon resonance.

- Stabilization of the receptors refolded using new amphipols will be assessed (J.L. Popot, 8 months)
- Crystallization and X-ray analysis (E. Pebay-Peyroula, 12 months)

Once the production protocols will be optimized, crystallization trials will be started. Crystallization screens of purified receptors samples will be set up at different conditions in the presence of ligands that were shown to stabilize the proteins. Standard vapor diffusion, as well as lipidic cubic phases will be explored.

Selected Publications:

  1. L. Arcemisbéhère, T. Sen, L. Boudier, M.N. Balestre, G. Gaibelet, E. Detouillon, R. Seyer, V.P. Ratnala, S. Granier, C. Vivès, F. Fieschi, C. Barberis, D. Mesnier, M. Damian, J.L. Banères, T. Durroux and B. Mouillac.
    A generic strategy for G protein-coupled receptor overexpression and refolding: towards biophysical and structural biology studies. Submitted to Journal of Biological Chemistry.
  2. Pocanschi et al. 2006.
    Amphipathic polymers: tools to fold integral membrane proteins to their active form. Biochemistry 45, 13954.
  3. E. Pebay-Peyroula, C. Dahout-Gonzalez, R. Kahn, V. Trézéguet, G.J.-M. Lauquin and G. Brandolin (2003). Structure of mitochondrial ADP/AP carrier in complex with carboxyatractyloside. Nature, 426, 39-44.