Jean-Claude Robert

Secondary and tertiary structures of the transmembrane domains of the translocator protein TSPO determined by NMR. Stabilization of the TSPO tertiary fold upon ligand binding

Numerous biological functions are attributed to the peripheral-type benzodiazepine receptor (PBR) recently renamed translocator protein (TSPO). The best characterized function is the translocation of cholesterol from the outer to inner mitochondrial membrane, which is a rate-determining step in steroid biosynthesis. TSPO drug ligands have been shown to stimulate pregnenolone formation by inducing TSPO-mediated translocation of cholesterol. Until recently, no direct structural data on this membrane protein was available. In a previous paper, we showed that a part of the mouse TSPO (mTSPO) C-terminal region adopts a helical conformation, the side-chain distribution of which provides a groove able to fit a cholesterol molecule. We report here on the overall structural properties of mTSPO. This study was first undertaken by dissecting the protein sequence and studying the conformation of five peptides encompassing the five putative transmembrane domains from (1)H-NMR data. The secondary structure of the recombinant protein in micelles was then studied using CD spectroscopy. In parallel, the stability of its tertiary fold was probed using (1)H-(15)N NMR. This study provides the first experimental evidence for a five-helix fold of mTSPO and shows that the helical conformation of each transmembrane domain is mainly formed through local short-range interactions. Our data show that, in micelles, mTSPO exhibits helix content close to what is expected but an unstable tertiary fold. They reveal that the binding of a drug ligand that stimulates cholesterol translocation is able to stabilize the mTSPO tertiary structure.

Gastric proton pump is expressed in the inner ear and choroid plexus of the rat.

Inner ear fluids and cerebrospinal fluid show remarkably stable ionic concentrations, particularly that of K(+) and H(+), but the mechanisms which control the homeostasis of these media are not well understood. We investigated a possible role of the gastric H, K-ATPase (gH,K-ATPase) pump in this control since this pump is known to be expressed in other tissues than gastric parietal cells. Here, we show by reverse transcription-polymerase chain reaction that the rat gH,K-ATPase alpha- and beta-subunits are expressed in the inner ear (lateral wall, organ of Corti and spiral ganglion cells), while only the alpha-subunit is expressed in the choroid plexus (CP). The presence of the alpha-subunit in the inner ear and CP was confirmed by immunoblotting. Immunohistochemistry localized this protein in the intermediate cells of the stria vascularis, in the spiral ligament and the spiral ganglion. gH,K-ATPase could be involved in the maintenance of H(+) and K(+) equilibria in cerebrospinal and labyrinthine fluids.

Spatial proximity between the VPAC1 receptor and the amino terminus of agonist and antagonist peptides reveals distinct sites of interaction

Vasoactive intestinal peptide (VIP) plays a major role in pathophysiology. Our previous studies demonstrated that the VIP sequence 6‐28 interacts with the N‐terminal ectodomain (N‐ted) of its receptor, VPAC1. Probes for VIP and receptor antagonist PG97‐269 were synthesized with a photolabile residue/Bpa at various positions and used to explore spatial proximity with VPAC1. PG97‐269 probes with Bpa at position 0, 6, and 24 behaved as high‐affinity receptor antagonists (Ki=12, 9, and 7 nM, respectively). Photolabeling experiments revealed that the [Bpa0]‐VIP probe was in physical contact with VPAC1 Q135, while [Bpa0]‐PG97‐269 was covalently bound to G62 residue of N‐ted, indicating different binding sites. In contrast, photolabeling with [Bpa6]‐ and [Bpa24]‐PG97‐269 showed that the distal domains of PG97‐269 interacted with N‐ted, as we previously showed for VIP. Substitution with alanine of the K143, T144, and T147 residues located in the first transmembrane domain of VPAC1 induced a loss of receptor affinity (IC50=1035, 874, and 2070 nM, respectively), and pharmacological studies using VIP2‐28 indicated that these three residues play an important role in VPAC1 interaction with the first histidine residue of VIP. These data demonstrate that VIP and PG97‐269 bind to distinct domains of VPAC1.—Ceraudo, E., Hierso, R., Tan, Y.‐V., Murail, S., Rouyer‐Fessard C., Nicole, P., Robert, J.‐C., Jamin, N., Neumann, J.‐M., Robberecht, P., Laburthe, M., Couvineau, A. Spatial proximity between the VPAC1 receptor and the amino terminus of agonist and antagonist peptides reveals distinct sites of interaction. FASEB J. 26, 2060‐2071 (2012). www.fasebj.org

Interaction between the N-terminal Domain of Gastric H,K-ATPase and the Spectrin Binding Domain of Ankyrin III

We screened a cDNA bank of rabbit gastric fundic mucosa by two-hybrid assays looking for binding partners of the N-terminal domain of the rabbit gastric H,K-ATPase. We extracted five clones sharing more than 90% sequence identity. The longest clone codes for a protein sharing a high identity (96 and 96.8%, respectively) with a fragment of the membrane domain, from Arg-835 to Ser-873, plus the major part of the “spectrin binding domain” going from Glu-874 to Leu-1455 of human and mouse ankyrin III. We conclude that the membrane and spectrin binding domains of the rabbit ankyrin III are candidates for the binding partner of the N-terminal domain of the rabbit gastric H,K-ATPase. To validate the ankyrin-ATPase interaction and to test its specificity, we produced both domains in yeast and bacteria, coimmunoprecipitated them with an anti-ATPase antibody, and copurified them by affinity chromatography. The sequence of rabbit ankyrin III was not known, and this is the first report demonstrating that the ankyrin III and the H,K-ATPase interact with no intermediate. The interaction involves the N-terminal domain of the ATPase on one hand and the spectrin binding domain of the ankyrin on the other.

Characterization of Membrane Protein Preparations: Measurement of Detergent Content and Ligand Binding After Proteoliposomes Reconstitution

The study of membrane proteins is a difficult task due to their natural embedding in hydrophobic environment made by lipids. Solubilization and purification from native membranes or overexpressed system involves the use of detergent to make them soluble while maintaining their structural and functional properties. The choice of detergent is governed not only by their ability to reach these goals, but also by their compatibility with biochemical and structural studies. A different detergent can be used during purification, and characterization of the detergent amounts present in each purification step is crucial. To address this point, we developed a colorimetric method to measure detergent content in different preparations. We analyzed detergent present in the collected fractions from the purification of the recombinant membrane translocator protein (RecTSPO). We followed detergent removal during the reconstitution of RecTSPO in liposomes and observed by electron microscopy the formation of proteoliposomes. We addressed the RecTSPO functionality by testing its ability to bind high affinity drug ligand [(3)H]PK 11195. We described the different parameters that should be controlled in order to optimize the measurement of this ligand binding using a filtration procedure. These protocols are useful to characterize functionality and detergent content of membrane protein, both key factors for further structural studies.

Bacterial Overexpressed Membrane Proteins: An Example: The TSPO

The mitochondrial membrane TranSlocator PrOtein (TSPO) is a 18-kDa transmembrane protein involved in various mitochondrial functions, among which the best characterised is cholesterol transport and steroid formation. Determination of its structure would be an important step to understand the mechanism of transport and its regulation. Purification from native membranes is difficult in respect with amounts of homogeneous purified proteins needed for biophysical, structural, and functional studies. Efficient heterologous overexpression in bacterial system, purification on affinity column, and biochemical characterisation has been successfully developed. Large-scale production of detergent-solubilized TSPO has been obtained with fermentation coupled to fast protein liquid chromatography procedure. Small-scale production at lower cost for isotopically labelled recombinant TSPO and/or detergent is also presented.