Nadège Jamin

Class-B GPCR activation: is ligand helix-capping the key?

The class B family of G-protein-coupled receptors (GPCRs) regulates essential physiological functions such as exocrine and endocrine secretions, feeding behaviour, metabolism, growth, and neuro- and immuno-modulations. These receptors are activated by endogenous peptide hormones including secretin, glucagon, vasoactive intestinal peptide, corticotropin-releasing factor and parathyroid hormone. We have identified a common structural motif that is encoded in all class B GPCR-ligand N-terminal sequences. We propose that this local structure, a helix N-capping motif, is formed upon receptor binding and constitutes a key element underlying class B GPCR activation. The folded backbone conformation imposed by the capping structure could serve as a template for a rational design of drugs targeting class B GPCRs in several diseases.

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.

Cell uptake of a biosensor detected by hyperpolarized 129Xe NMR: The transferrin case

For detection of biological events in vitro, sensors using hyperpolarized (129)Xe NMR can become a powerful tool, provided the approach can bridge the gap in sensitivity. Here we propose constructs based on the non-selective grafting of cryptophane precursors on holo-transferrin. This biological system was chosen because there are many receptors on the cell surface, and endocytosis further increases this density. The study of these biosensors with K562 cell suspensions via fluorescence microscopy and (129)Xe NMR indicates a strong interaction, as well as interesting features such as the capacity of xenon to enter the cryptophane even when the biosensor is endocytosed, while keeping a high level of polarization. Despite a lack of specificity for transferrin receptors, undoubtedly due to the hydrophobic character of the cryptophane moiety that attracts the biosensor into the cell membrane, these biosensors allow the first in-cell probing of biological events using hyperpolarized xenon.

Combining IR spectroscopy with fluorescence imaging in a single microscope: Biomedical applications using a synchrotron infrared source (invited)

It has become increasingly clear that infrared microspectroscopy (IRMS) can be an extremely valuable analysis tool when determining the chemical composition of biological and biomedical samples. Frequently, fluorescence illumination is required for sample characterization, and is usually achieved on a separate and dedicated optical microscope. We report the development and use of a single microscope for concomitant fluorescence and synchrotron IRMS. This unique combination has been used to identify changes in the composition of newly remodeled bone after the onset of osteoporosis, misfolded protein structure in Alzheimer’s disease, and cellular changes in apoptosis.It has become increasingly clear that infrared microspectroscopy (IRMS) can be an extremely valuable analysis tool when determining the chemical composition of biological and biomedical samples. Frequently, fluorescence illumination is required for sample characterization, and is usually achieved on a separate and dedicated optical microscope. We report the development and use of a single microscope for concomitant fluorescence and synchrotron IRMS. This unique combination has been used to identify changes in the composition of newly remodeled bone after the onset of osteoporosis, misfolded protein structure in Alzheimer’s disease, and cellular changes in apoptosis.

Hyperpolarized 129Xe NMR signature of living biological cells

We show that the differentiation between internal and external compartments of various biological cells in suspension can be made via simple NMR spectra of hyperpolarized 129Xe. The spectral separation between the signals of 129Xe in these two compartments is already known for red blood cells, because of the strong interaction of the noble gas with hemoglobin. The observation of two separate peaks in the 200‐ppm region can be seen with both eukaryotic and prokaryotic cells, some of which are not known to contain paramagnetic proteins in large quantities. Using different experiments in which the cells are lysed, swell or are blocked in G2 phase, we demonstrate that the low‐field‐shifted peak observed corresponds to xenon in the aqueous pool inside the cells and not in the membranes. The presence of this additional peak is a clear indication of cell integrity, and its integration allows the quantification of the total cell volume. The relaxation time of intracellular xenon is sufficiently long to open up promising perspectives for cell characterization. The exchange time between the inner and outer cell compartments (on the order of 30 ms) renders possible the targeting of intracellular receptors, whereas the observation of chemical shift variations represents a method of revealing the presence of toxic species in the cells. Copyright

Most of the structural elements of the globular domain of murine prion protein form fibrils with predominant β‐sheet structure

The conversion of the cellular prion protein into the β‐sheet‐rich scrapie prion protein is thought to be the key step in the pathogenesis of prion diseases. To gain insight into this structural conversion, we analyzed the intrinsic structural propensity of the amino acid sequence of the murine prion C‐terminal domain. For that purpose, this globular domain was dissected into its secondary structural elements and the structural propensity of the protein fragments was determined. Our results show that all these fragments, excepted that strictly encompassing helix 1, have a very high propensity to form structured aggregates with a dominant content of β‐sheet structures.

Structural properties of POPC monolayers under lateral compression: computer simulations analysis.

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a lipid comprising a saturated and an unsaturated acyl chain, belongs to the class of glycerophosphatidylcholines, major lipids in eukaryotic cell membranes. To get insight into the structural properties of this lipid within monolayers as membrane models, we performed molecular dynamics (MD) simulations of POPC monolayers under compression at the air/water interface. MD simulations were carried out at 300 K and at different surface pressures using the all-atom general Amber force field (GAFF). A good agreement was found between the simulated data and experimental isotherms. At surface pressures greater than 15 mN/m, two orientations of the head groups clearly appear: one nearly parallel to the monolayer interface and another one pointing toward the water. On the basis of the analysis of headgroup dihedral angles, we propose that the conformational variations around the bonds connecting the phosphorus atom to the adjacent oxygens are involved in these two orientations of the headgroup. The glycerol group orientation is characterized by a large distribution centered around 50° with respect to the monolayer normal. The acyl chains are predominantly in trans configuration from 7.5 to 43 mN/m surface pressures. Moreover, the calculated order parameter profiles of both chains suggest an independent behavior of the saturated and unsaturated chains that could be correlated with the formation of chain-type clusters observed along the simulated trajectories.

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

Role of the membrane interface on the conformation of the caveolin scaffolding domain: A CD and NMR study

Circular dichroism (CD) and NMR spectroscopy were used to study the conformational properties of two synthetic peptides, D82–R101 and D82–I109, encompassing the caveolin scaffolding domain (D82–R101), in the presence of dodecylphosphocholine (DPC) micelles. Our data show that a stable helical conformation of the caveolin scaffolding domain in a membrane mimicking system is only obtained for the peptide including the L102–I109 hydrophobic stretch, a part of the caveolin intra‐membrane domain. Through chemical shift variations, an ensemble of six residues of the D82–L109 peptide, mainly located in the V94TKYWFYR101 motif were found to detect the presence of phosphatidylserine solubilized in DPC micelles. Our results constitute a first step for elucidating at a residue level the conformational properties of the central region of the caveolin‐1 protein.

Structural and dynamic properties of juxta-membrane segments of caveolin-1 and caveolin-2 at the membrane interface

Caveolins (cav1–3) are essential membrane proteins found in caveolae. The caveolin scaffolding domain of cav-1 includes a short sequence containing a CRAC motif (V94TKYWFYR101) at its C-terminal end. To investigate the role of this motif in the caveolin–membrane interaction at the atomic level, we performed a detailed structural and dynamics characterization of a cav-1(V94-L102) nonapeptide encompassing this motif and including the first residue of cav-1 hydrophobic domain (L102), in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. Cav-1(V94-L102) partitioned better in DPC and in DM/anionic lipid micelles than in DM micelles, as shown by fluorescence titration and CD. NMR data revealed that this peptide folded as an amphipathic helix located in the polar head group region of DPC micelles. The two tyrosine side-chains, flanked by arginine and lysine residues, are situated on one face of this helix, whereas the phenylalanine and tryptophan side-chains are located on the opposite face. Fluorescence studies showed significant Trp subnanosecond rotations, the presence of several rotamers, and a heterogeneous location within the water/micelle interface. NMR studies of the shorter cav-1(V94-R101) peptide and of the homologous sequence of cav-2(I79SKYVMYKF87) allowed the description of the effect of L102 and of the amino acid variations occurring in cav-2 on the structure and localization in DPC micelles. Based on the topological model of caveolins, our results suggest that the cav-1 and cav-2 nonapeptides studied form interfacial α-helix membrane anchors in which the K/RhhhYK/Rh motif, also found in cav-3, may play a significant role.