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 (K(i)=12, 9, and 7 nM, respectively). Photolabeling experiments revealed that the [Bpa(0)]-VIP probe was in physical contact with VPAC1 Q(135), while [Bpa(0)]-PG97-269 was covalently bound to G(62) residue of N-ted, indicating different binding sites. In contrast, photolabeling with [Bpa(6)]- and [Bpa(24)]-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 K(143), T(144), and T(147) residues located in the first transmembrane domain of VPAC1 induced a loss of receptor affinity (IC(50)=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.

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.

Determining membrane protein structures: still a challenge!

Determination of structures and dynamics events of transmembrane proteins is important for the understanding of their function. Analysis of such events requires high-resolution 3D structures of the different conformations coupled with molecular dynamics analyses describing the conformational pathways. However, the solution of 3D structures of transmembrane proteins at atomic level remains a particular challenge for structural biochemists--the need for purified and functional transmembrane proteins causes a 'bottleneck'. There are various ways to obtain 3D structures: X-ray diffraction, electron microscopy, NMR and modelling; these methods are not used exclusively of each other, and the chosen combination depends on several criteria. Progress in this field will improve knowledge of ligand-induced activation and inhibition of membrane proteins in addition to aiding the design of membrane-protein-targeted drugs.

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 alpha-helix membrane anchors in which the K/RhhhYK/Rh motif, also found in cav-3, may play a significant role.

Slightly modifying pseudoproline dipeptides incorporation strategy enables solid phase synthesis of a 54 AA fragment of caveolin-1 encompassing the intramembrane domain.

This work contributes to highlight the benefits of pseudoproline dipeptides introduction in difficult SPPS. We show how a slight modification in the positioning choice conditioned the synthesis achievement of a 54 amino acid long caveolin-1 peptide encompassing the intramembrane domain. Furthermore, we report a side reaction correlated with the coupling steps and generating truncated fragments with a mass deviation of + 42 Da. Considering the need of structural data for membrane proteins, most of which are considered as prevalent therapeutic targets, chemical synthesis provides an interesting alternative pathway to obtain hydrophobic domains by pushing back the frontiers of conventional RP methods of purification.

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.

The vasoactive intestinal peptide (VIP) alpha-Helix up to C terminus interacts with the N-terminal ectodomain of the human VIP/Pituitary adenylate cyclase-activating peptide 1 receptor: photoaffinity, molecular modeling, and dynamics.

The neuropeptide vasoactive intestinal peptide (VIP) strongly impacts on human pathophysiology and does so through interaction with class II G protein-coupled receptors. We characterized the C terminus-binding site of VIP in the N-terminal ectodomain (N-ted) of the human VPAC1 receptor: 1) The probe [(125)I-Bpa(28)]VIP in which the C-terminal residue (Asn(28)) is substituted by a photoreactive p-benzoyl-l-Phe (Bpa) was used to photolabel the receptor. After receptor cleavage and Edman sequencing, it was shown that Asn(28) of VIP is in contact with Lys(127) in the receptor N-ted. Taking into account previous data, it follows that the C-terminal and central parts of VIP from Asn(28) to Phe(6) lie in the N-ted. 2) A three-dimensional model of the N-ted was constructed, the fold being identified as a Sushi domain with two antiparallel beta-sheets and three disulfide bonds. The nuclear magnetic resonance structure of VIP was then docked into this model by taking into account the constraint provided by photoaffinity experiments with [(125)I-Bpa(28)]VIP. It appeared that VIP runs parallel to the beta3-beta4 antiparallel sheets. 3) We performed molecular dynamic simulations over 14 nsec of the complex between VIP and receptor N-ted and the free N-ted. The structural model of the free N-ted is stable, and VIP tends to further stabilize the N-ted structure more especially in the loops connecting the beta-sheets. These structural studies provide a detailed molecular understanding of the VIP-receptor interaction.

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 V(94)TKYWFYR(101) 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.

The human VPAC1 receptor: identification of the N-terminal ectodomain as a major VIP-binding site by photoaffinity labeling and 3D modeling.

The human VPAC1 receptor for VIP and PACAP is a class II Gprotein-coupled receptor (GPCR). The N-terminal ectodomain of the VPAC1 receptor plays a crucial role in VIP binding. Photoaffinity experiments clearly indicated that the 6-28 part of VIP physically interacts with the N-terminal ectodomain. Construction of a 3D model of the N-terminal ectodomain of VPAC1 receptor based on the NMR structure of the mouse CRF receptor 2 indicated the presence of short consensus repeat/Sushi domain. Docking of VIP in the N-terminal ectodomain structural model was performed taking into account the severe constraints provided by photoaffinity. A VIP-binding site was identified on the side of the structured core of the N-terminal ectodomain of the receptor.

Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor.

We previously defined a cholesterol recognition/interaction amino acid consensus sequence [CRAC: L/V-X (1-5)-Y-X (1-5)-R/K] in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), a high-affinity drug and cholesterol-binding protein present in the outer mitochondrial membrane protein. This protein is involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. Reconstituted wild-type recombinant PBR into proteoliposomes demonstrated high-affinity 2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide and cholesterol binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria.

Dynamical characterization of residual and non-native structures in a partially folded protein by (15)N NMR relaxation using a model based on a distribution of correlation times.

A spectral density model based on a truncated lorentzian distribution of correlation times is used to analyze the nanosecond time-scale dynamics of the partially unfolded domain 2 of annexin I from its (15)N NMR relaxation parameters measured at three magnetic field strengths. The use of a distribution of correlation times enables the characterization of the dynamical features of the NH bonds of the protein in terms of heterogeneity of dynamical states in the nanosecond range. The variation along the sequence of the two dynamical parameters introduced, namely the center and the width of the distribution, points out the different types of residual secondary structures present in the D2 domain. Moreover, it allows a physically sensible interpretation of the dynamical behavior of the different residual helices and of the non-native structures. Also, a striking correspondence is found between the parameters obtained using an extended Lipari and Szabo model and the parameters obtained using the distribution of correlation times. This result led us to propose a specific interpretation of the model-free order parameter for internal motions in the nanosecond range in the case of unfolded states.

Most of the structural elements of the globular domain of murine prion protein form fibrils with predominant beta-sheet structure.

The conversion of the cellular prion protein into the beta-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 beta-sheet structures.

Peptide agonist docking in the N-terminal ectodomain of a class II G protein-coupled receptor, the VPAC1 receptor. Photoaffinity, NMR, and molecular modeling.

The neuropeptide vasoactive intestinal peptide (VIP) strongly impacts on human pathophysiology and does so through interaction with class II G protein-coupled receptors named VIP pituitary adenylate cyclase-activating peptide (PACAP) receptors (VPACs). The molecular nature of VIP binding to receptors remains elusive. In this work, we have docked VIP in the human VPAC1 receptor by the following approach. (i) VIP probes containing photolabile residues in positions 6, 22, and 24 of VIP were used to photolabel the receptor. After receptor cleavage and Edman sequencing of labeled receptor fragments, it was shown that Phe6, Tyr22, and Asn24 of VIP are in contact with Asp107, Gly116, and Cys122 in the N-terminal ectodomain (N-ted) of the receptor, respectively. (ii) The structure of VIP was determined by NMR showing a central alpha helix, a disordered N-terminal His1-Phe6 segment and a 3(10) Ser25-Asn28 helix termination. (iii) A three-dimensional model of the N-ted of hVPAC1 was constructed by using the NMR structure of the N-ted of corticotropin-releasing factor receptor 2beta as a template. As expected, the fold is identified as a short consensus repeat with two antiparallel beta sheets and is stabilized by three disulfide bonds. (iv) Taking into account the constraints provided by photoaffinity, VIP was docked into the hVPAC1 receptor N-ted. The 6-28 fragment of VIP nicely lies in the N-ted C-terminal part, but the N terminus region of VIP is free for interacting with the receptor transmembrane region. The data provide a structural rationale to the proposed two-step activation mechanism of VPAC receptor and more generally of class II G protein-coupled receptors.

Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues.

Membrane protein insertion in the lipid bilayer is determining for their activity and is governed by various factors such as specific sequence motifs or key amino-acids. A detailed fluorescence study of such factors is exemplified with PMP1, a small (38 residues) single-membrane span protein that regulates the plasma membrane H(+)-ATPase in yeast and specifically interacts with phosphatidylserines. Such interactions may stabilize raft domains that have been shown to contain H(+)-ATPase. Previous NMR studies of various fragments have focused on the critical role of interfacial residues in the PMP1 structure and intermolecular interactions. The C-terminal domain contains a terminal Phe (F38), a single Trp (W28) and a single Tyr (Y25) that may act together to anchor the protein in the membrane. In order to describe the location and dynamics of W28 and the influence of Y25 on protein insertion within membrane, we carried out a detailed steady-state and time-resolved fluorescence study of the synthetic G13-F38 fragment and its Tyr-less mutant, Y25L in various membrane mimetic systems. Detergent micelles are conveniently used for this purpose. We used dodecylphosphocholine (DPC) in order to compare with and complement previous NMR results. In addition, dodecylmaltoside (DM) was used so that we could apply our recently described new quenching method by two brominated analogs of DM (de Foresta et al. 2002, Eur. Biophys. J. 31:185-97). In both systems, and in the presence and absence of Y25, W28 was shown to be located below but close to the polar headgroup region, as shown by its maximum emission wavelengths (lambda(max)), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (k(q)) by acrylamide. Results were interpreted by comparison with calibration data obtained with fluorescent model peptides. Time-resolved anisotropy measurements were consistent with PMP1 fragment immobilization within peptide-detergent complexes. We tentatively assigned the two major Trp lifetimes to the Trp (chi(1)=60 degrees and 180 degrees ) rotamers, based on the recent lifetime-rotamer correlation proposed for model cyclic peptides (Pan and Barkley 2004, Biophys J 86:3828-35). We also analyzed the role of the hydrophobic anchor, by comparing the micelle binding of fragments of various lengths including the synthesized full-length protein and detected peculiar differences for protein interaction with the polar headgroups of DM or DPC.

Structural basis for the interaction of Asf1 with histone H3 and its functional implications.

Asf1 is a conserved histone chaperone implicated in nucleosome assembly, transcriptional silencing, and the cellular response to DNA damage. We solved the NMR solution structure of the N-terminal functional domain of the human Asf1a isoform, and we identified by NMR chemical shift mapping a surface of Asf1a that binds the C-terminal helix of histone H3. This binding surface forms a highly conserved hydrophobic groove surrounded by charged residues. Mutations within this binding site decreased the affinity of Asf1a for the histone H3/H4 complex in vitro, and the same mutations in the homologous yeast protein led to transcriptional silencing defects, DNA damage sensitivity, and thermosensitive growth. We have thus obtained direct experimental evidence of the mode of binding between a histone and one of its chaperones and genetic data suggesting that this interaction is important in both the DNA damage response and transcriptional silencing.

Deciphering the role of individual acyl chains in the interaction network between phosphatidylserines and a single-spanning membrane protein.

PMP1 is a small single-spanning membrane protein functioning as a regulatory subunit of the yeast plasma membrane H(+)-ATPase. This protein forms a unique helix and exhibits a positively charged cytoplasmic domain that is able to specifically segregate phosphatidylserines (PSs). A marked groove formed at the helix surface is thought to play a major role in the related lipid-protein interaction network. Mutational analysis and (1)H NMR experiments were therefore performed on a synthetic PMP1 fragment using DPC-d(38) micelles as a membrane-like environment, in the presence of small amounts of POPS. A mutation designed for altering the helix groove was shown to disfavor the POPS binding specificity as much as that affecting the electrostatic interaction network. From POPS titration experiments monitored by a full set of one- and two-dimensional NOESY spectra, the association between the phospholipids and the PMP1 peptide has been followed. Our data reveal that the clustering of POPS molecules is promoted from a stabilized framework obtained by coupling the PMP1 helix groove to a POPS sn-2 chain. To our knowledge, the NOE-based titration plots displayed in this report constitute the first NMR data that directly distinguish the role of the sn-1 and sn-2 acyl chains in a lipid-protein interaction. The results are discussed while taking into account our accurate knowledge of the yeast plasma membrane composition and its ability to form functional lipid rafts.

A new consensus sequence for phosphatidylserine recognition by annexins.

Annexins are abundant and ubiquitous proteins that bind, by their four structurally identical domain cores, to phosphatidylserine-containing membranes in the presence of Ca2+. Using molecular simulation and mutagenesis, we have identified a new phosphatidylserine-binding site in annexin V domain 1 and established its structure. The residues involved in this site constitute a consensus sequence highly conserved in all annexins. Remarkably, this consensus sequence is exclusively found in domains 1 or 2, sometimes in both, but never in domains 3 and 4. Such a pattern actually delineates three classes of annexins, shedding new light on the role played by the four-domain core of annexins that could encode specific information discriminating the different annexins that compete within a given cell for membrane binding. Our findings thus provide new strategies for understanding the regulation of the cellular functions of annexins.