External release of entropy by synchronized movements of local secondary structures drives folding of a small, disulfide-bonded protein.

A crucial mechanism to the formation of native, fully functional, 3D structures from local secondary structures is unraveled in this study. Through the introduction of various amino acid substitutions at four canonical ß-turns in a three-fingered protein, Toxin α from Naja nigricollis, we found that the release of internal entropy to the external environment through the globally synchronized movements of local substructures plays a crucial role. Throughout the folding process, the folding species were saturated with internal entropy so that intermediates accumulated at the equilibrium state. Their relief from the equilibrium state was accomplished by the formation of a critical disulfide bridge, which could guide the synchronized movement of one of the peripheral secondary structure. This secondary structure collided with a core central structure, which flanked another peripheral secondary structure. This collision displaced the internal thermal fluctuations from the first peripheral structure to the second peripheral structure, where the displaced thermal fluctuations were ultimately released as entropy. Two protein folding processes that acted in succession were identified as the means to establish the flow of thermal fluctuations. The first process was the time-consuming assembly process, where stochastic combinations of colliding, native-like, secondary structures provided candidate structures for the folded protein. The second process was the activation process to establish the global mutual relationships of the native protein in the selected candidate. This activation process was initiated and propagated by a positive feedback process between efficient entropy release and well-packed local structures, which moved in synchronization. The molecular mechanism suggested by this experiment was assessed with a well-defined 3D structure of erabutoxin b because one of the turns that played a critical role in folding was shared with erabutoxin b.

Bee Venom Phospholipase A2, a Good "Chauffeur" for Delivering Tumor Antigen to the MHC I and MHC II Peptide-Loading Compartments of the Dendritic Cells: The Case of NY-ESO-1.

Bee venom phospholipase A2 (bvPLA2) is a small, 15kDa enzyme which hydrolyses many phospholipids through interfacial binding. The mutated bvPLA2H34Q (bvPLA2m), in which histidine-34 is replaced by glutamine, is not catalytically active. This protein has been shown to be a suitable membrane anchor and has been suggested as a suitable tumor-antigen vector for the development of novel dendritic cell-based vaccines. To confirm this feature, in this study the fusion protein PNY, composed of NY-ESO-1(NY(s)) fused to the C-terminus of bvPLA2m, was engineered. bvPLA2m enhanced the binding of NY(s) to the membrane of human monocyte-derived dendritic cells (DCs) and, once taken up by the cells, the antigen fused to the vector was directed to both MHC I and MHC II peptide-loading compartments. bvPLA2m was shown to increase the cross-presentation of the NY(s)-derived, restricted HLA-A*02 peptide, NY-ESO-1157-165(NY157-165), at the T1 cell surface. DCs loaded with the fusion protein induced cross-priming of NY(s)-specific CD8 + T-cells with greater efficiency than DCs loaded with NY(s). Sixty-five percent of these NY(s)-specific CD8+ T-cell lines could also be activated with the DCs pulsed with the peptide, NY157-165. Of these CD8+ T-cell lines, two were able to recognize the human melanoma cell line, SK-MEL-37, in a context of HLA-A*02. Only a small number of bvPLA2m CD8+ T-cell lines were induced, indicating the low immunogenicity of the protein. It was concluded that bvPLA2m can be used as a membrane-binding vector to promote MHC class II peptide presentation and MHC class I peptide cross-presentation. Such a system can, therefore, be tested for the preparation of cell-based vaccines.

Structural model of ligand-G protein-coupled receptor (GPCR) complex based on experimental double mutant cycle data: MT7 snake toxin bound to dimeric hM1 muscarinic receptor.

The snake toxin MT7 is a potent and specific allosteric modulator of the human M1 muscarinic receptor (hM1). We previously characterized by mutagenesis experiments the functional determinants of the MT7-hM1 receptor interaction (Fruchart-Gaillard, C., Mourier, G., Marquer, C., Stura, E., Birdsall, N. J., and Servent, D. (2008) Mol. Pharmacol. 74, 1554-1563) and more recently collected evidence indicating that MT7 may bind to a dimeric form of hM1 (Marquer, C., Fruchart-Gaillard, C., Mourier, G., Grandjean, O., Girard, E., le Maire, M., Brown, S., and Servent, D. (2010) Biol. Cell 102, 409-420). To structurally characterize the MT7-hM1 complex, we adopted a strategy combining double mutant cycle experiments and molecular modeling calculations. First, thirty-three ligand-receptor proximities were identified from the analysis of sixty-one double mutant binding affinities. Several toxin residues that are more than 25 Å apart still contact the same residues on the receptor. As a consequence, attempts to satisfy all the restraints by docking the toxin onto a single receptor failed. The toxin was then positioned onto two receptors during five independent flexible docking simulations. The different possible ligand and receptor extracellular loop conformations were described by performing simulations in explicit solvent. All the docking calculations converged to the same conformation of the MT7-hM1 dimer complex, satisfying the experimental restraints and in which (i) the toxin interacts with the extracellular side of the receptor, (ii) the tips of MT7 loops II and III contact one hM1 protomer, whereas the tip of loop I binds to the other protomer, and (iii) the hM1 dimeric interface involves the transmembrane helices TM6 and TM7. These results structurally support the high affinity and selectivity of the MT7-hM1 interaction and highlight the atypical mode of interaction of this allosteric ligand on its G protein-coupled receptor target.

Irditoxin, a novel covalently linked heterodimeric three-finger toxin with high taxon-specific neurotoxicity.

A novel heterodimeric three-finger neurotoxin, irditoxin, was isolated from venom of the brown treesnake Boiga irregularis (Colubridae). Irditoxin subunit amino acid sequences were determined by Edman degradation and cDNA sequencing. The crystal structure revealed two subunits with a three-finger protein fold, typical for "nonconventional" toxins such as denmotoxin, bucandin, and candoxin. This is the first colubrid three-finger toxin dimer, covalently connected via an interchain disulfide bond. Irditoxin showed taxon-specific lethality toward birds and lizards and was nontoxic toward mice. It produced a potent neuromuscular blockade at the avian neuromuscular junction (IC(50)=10 nM), comparable to alpha-bungarotoxin, but was three orders of magnitude less effective at the mammalian neuromuscular junction. Covalently linked heterodimeric three-finger toxins found in colubrid venoms constitute a new class of venom peptides, which may be a useful source of new neurobiology probes and therapeutic leads.

Conserved structural determinants in three-fingered protein domains.

The three-dimensional structures of some components of snake venoms forming so-called 'three-fingered protein' domains (TFPDs) are similar to those of the ectodomains of activin, bone morphogenetic protein and transforming growth factor-beta receptors, and to a variety of proteins encoded by the Ly6 and Plaur genes. The analysis of sequences of diverse snake toxins, various ectodomains of the receptors that bind activin and other cytokines, and numerous gene products encoded by the Ly6 and Plaur families of genes has revealed that they differ considerably from each other. The sequences of TFPDs may consist of up to six disulfide bonds, three of which have the same highly conserved topology. These three disulfide bridges and an asparagine residue in the C-terminal part of TFPDs are essential for the TFPD-like fold. Analyses of the three-dimensional structures of diverse TFPDs have revealed that the three highly conserved disulfides impose a major stabilizing contribution to the TFPD-like fold, in both TFPDs contained in some snake venoms and ectodomains of several cellular receptors, whereas the three remaining disulfide bonds impose specific geometrical constraints in the three fingers of some TFPDs.

Increasing the humoral immunogenic properties of the HIV-1 Tat protein using a ligand-stabilizing strategy.

Tat is regarded as an attractive target for the development of an AIDS vaccine. However, works suggest that Tat is a poorly immunogenic protein and therefore we attempted to increase its immunogenic potency. As we observed that Tat is highly sensitive to enzymatic degradation in vitro we tried to make it less susceptible to proteolysis using ligands. We complexed Tat101 with various sulfated sugars and observed that some of these ligands made the protein more resistant to proteolysis and more immunogenic. In a more thorough study, we observed that a low-molecular-weight heparin fragment, called Hep6000, altered both the cell-binding capacity and transactivating activity of Tat101, suggesting that this sulfated polysaccharide can make the protein less toxic. Sera raised against Tat101 and Tat101/Hep6000 similarly bound mainly to the N-terminal region of the protein, indicating that formation of the complex does not alter the B-cell immunodominant region. Anti-Tat101/Hep6000 antisera neutralized the transactivating activity of Tat101 more efficiently than anti-Tat101 antisera. Altogether, these results indicate that stabilization of Tat101 using sulfated sugars increases its immunogenicity and might be of value in increasing its vaccine efficacy.

Structural basis for the NAD-hydrolysis mechanism and the ARTT-loop plasticity of C3 exoenzymes.

C3-like exoenzymes are ADP-ribosyltransferases that specifically modify some Rho GTPase proteins, leading to their sequestration in the cytoplasm, and thus inhibiting their regulatory activity on the actin cytoskeleton. This modification process goes through three sequential steps involving NAD-hydrolysis, Rho recognition, and binding, leading to Rho ADP-ribosylation. Independently, three distinct residues within the ARTT loop of the C3 exoenzymes are critical for each of these steps. Supporting the critical role of the ARTT loop, we have shown previously that it adopts a distinct conformation upon NAD binding. Here, we present seven wild-type and ARTT loop-mutant structures of C3 exoenzyme of Clostridium botulinum free and bound to its true substrate, NAD, and to its NAD-hydrolysis product, nicotinamide. Altogether, these structures expand our understanding of the conformational diversity of the C3 exoenzyme, mainly within the ARTT loop.

Creation of intercellular bonds by anchoring protein ligands to membranes using the diphtheria toxin T domain.

We describe the creation of cell adhesion mediated by cell surface engineering. The Flt3-ligand was fused to a membrane anchor made of the diphtheria toxin translocation domain. The fusion protein was attached to the surface of a cell by an acid pulse. Contact with another cell expressing the receptor Flt3 lead to its activation. This activity involved direct cell-cell contact. A mean force of 20 nN was needed to separate functionalized cells after 5 min of contact. Overall, we showed that it is possible to promote specific cell-cell adhesion by attaching protein ligands at the surface of cells.

Concerted protonation of key histidines triggers membrane interaction of the diphtheria toxin T domain.

The translocation domain (T domain) of the diphtheria toxin contributes to the transfer of the catalytic domain from the cell endosome to the cytosol, where it blocks protein synthesis. Translocation is initiated when endosome acidification induces the interaction of the T domain with the membrane of the compartment. We found that the protonation of histidine side chains triggers the conformational changes required for membrane interaction. All histidines are involved in a concerted manner, but none is indispensable. However, the preponderance of each histidine varies according to the transition observed. The pair His(223)-His(257) and His(251) are the most sensitive triggers for the formation of the molten globule state in solution, whereas His(322)-His(323) and His(251) are the most sensitive triggers for membrane binding. Interestingly, the histidines are located at key positions throughout the structure of the protein, in hinges and at the interface between each of the three layers of helices forming the domain. Their protonation induces local destabilizations, disrupting the tertiary structure and favoring membrane interaction. We propose that the selection of histidine residues as triggers of membrane interaction enables the T domain to initiate translocation at the rather mild pH found in the endosome, contributing to toxin efficacy.

Differential capacity of T cell priming in naive donors of promiscuous CD4+ T cell epitopes of HCV NS3 and Core proteins.

To understand the inter-individual and virus-independent variability of CD4+ T cell responses to HCV components, we evaluated the effect on these responses of HLA II molecules in uninfected healthy donors. Using HLA II-specific binding assays, we identified, in the Core and NS3 proteins, 21 long fragments and 24 15-mer peptides that bound to four to eight of the most preponderant HLA II molecules. We then evaluated the priming capacity of eight long promiscuous peptides in 12 HLA-unrelated healthy donors. The NS3 1250-1264 peptide primed T cells in all the naive donors, while five others were stimulating in at least half of the individuals. We also report sequences that bind to multiple HLA II molecules but are weakly immunogenic. We therefore conclude that (i) broad HLA II specificity is only a prerequisite for a peptide to be stimulating in multiple individuals, and (ii) promiscuous peptides widely differ in their capacity to prime CD4+ T cells from uninfected healthy donors. We suggest that these priming differences result from inter-individual variations in the peptide-specific T cell repertoire. Interestingly, five of the most immunogenic peptides we identified correspond to frequently targeted T cell epitopes in infected patients.

Behavior of the N-terminal helices of the diphtheria toxin T domain during the successive steps of membrane interaction.

During intoxication of a cell, the translocation (T) domain of the diphtheria toxin helps the passage of the catalytic domain across the membrane of the endosome into the cytoplasm. We have investigated the behavior of the N-terminal region of the T domain during the successive steps of its interaction with membranes at acidic pH using tryptophan fluorescence, its quenching by brominated lipids, and trypsin digestion. The change in the environment of this region was monitored using mutant W281F carrying a single native tryptophan at position 206 at the tip of helix TH1. The intrinsic propensity to interact with the membrane of each helix of the N-terminus of the T domain, TH1, TH2, TH3, and TH4, was also studied using synthetic peptides. We showed the N-terminal region of the T domain was not involved in the binding of the domain to the membrane, which occurred at pH 6 mainly through hydrophobic effects. At that stage of the interaction, the N-terminal region remained strongly solvated. Further acidification eliminated repulsive electrostatic interactions between this region and the membrane, allowing its penetration into the membrane by attractive electrostatic interactions and hydrophobic effects. The peptide study indicated the nature of forces contributing to membrane penetration. Overall, the data suggested that the acidic pH found in the endosome not only triggers the formation of the molten globule state of the T domain required for membrane interaction but also governs a progressive penetration of the N-terminal part of the T domain in the membrane. We propose that these physicochemical properties are necessary for the translocation of the catalytic domain.

The venom of the snake genus Atheris contains a new class of peptides with clusters of histidine and glycine residues.

We investigated venoms from members of the genus Atheris (Serpentes, Viperidae), namely the rough scale bush viper (Atheris squamigera), the green bush viper (A. chlorechis) and the great lakes bush viper (A. nitschei), using mass spectrometry-based strategies, relying on matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with de novo peptide sequencing. We discovered a set of novel peptides with masses in the 2-3 kDa range and containing poly-His and poly-Gly segments (pHpG). Complete primary structural elucidation and confirmation of two sequences by Edman degradation indicated the consensus sequence EDDH(9)GVG(10). Bioinformatic investigations in protein sequence databanks did not show relevant homology with known peptides or proteins. However, a more extensive investigation of data in nucleic acid databases revealed some similarities to the precursor sequences of bradykinin potentiating peptides (BPP) and C-type natriuretic peptides (CNP), agents that are known to affect the cardiovascular system by acting on specific metalloproteases and receptors. The novel pHpG peptides found in Atheris venoms might also act on the cardiovascular system by inhibiting particular metalloproteases, which however remain to be identified.

Selective identification of HLA-DP4 binding T cell epitopes encoded by the MAGE-A gene family.

Because of the high frequency of HLA-DP4 in the Caucasian population, we have selectively delineated HLA-DP4 restricted T cell epitopes in the MAGE-A tumor antigens. We identified 12 good binders to HLA-DP4 and investigated the capacity of the seven best binders to induce in vitro specific CD4+ T cell lines from HLA-DP4 healthy donors. We found that the MAGE-A1 90-104 peptide exhibited a high and constant frequency of CD4+ T cell precursors in all the six tested donors. The MAGE-A1 268-282 peptide was found immunogenic in only two donors but with a high precursor frequency. The MAGE-A12 127-141 peptide was T cell stimulating in six different donors and induced fewer T cell lines. The peptide-specific T cell lines were stimulated by DC loaded with the lysates of cells transfected with MAGE-A1 or MAGE-A12, or loaded with the recombinant protein. We also show that the immunoreactivity of CD4+ T cell epitopes restricted to the same HLA II molecule may vary from one individual to another, as a result of inter-individual variations in the CD4+ T cell repertoire.

How three-finger-fold toxins interact with various cholinergic receptors.

Three-finger-fold toxins, isolated from various snake venoms, are recognized by high affinity and various specificities by different nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs, respectively) present in peripheral, as well as central, nervous systems (Karlsson et al., 2000; Servent and Ménez, 2001; Nirthanan and Gwee, 2004). The goal of our studies is (1) to identify, at the molecular level, the functional determinants involved in the various interaction profiles of nicotinic or muscarinic toxins on their respective receptors subtypes, (2) to model some of these toxin-receptor complexes using distance constraints obtained from cycle-mutant experiments, and (3) to understand how a unique scaffold (the three-finger fold) is able to support these different functional profiles and how molecular determinants have been selected during the evolution process to create these different specific properties. Finally, these structure/function analyses should be exploited to engineer non-natural peptides with new binding and functional properties useful as pharmacological tools or therapeutic agents.

Photocrosslinking/label transfer: A key step in mapping short alpha-neurotoxin binding site on nicotinic acetylcholine receptor.

We developed a novel radioactive short bifunctional photoprobe, which could be coupled through a cleavable bond to an engineered cysteinyl residue on an analogue of a nicotinic acetylcholine receptor-specific alpha-neurotoxin. This cysteine was put on the tip of loop II in place of Arg33, a major residue for the interaction with the receptor. To facilitate the purification of the nAChR labeled subunits, we tagged the ligand with a desthiobiotin moiety. After irradiation of the photosensitive toxin-nAChR complex, gel electrophoresis showed that most of the radioactivity was attached to the alpha subunit (59%), followed by the gamma subunit (28%), with the delta subunit (13%) being less labeled. On a preparative scale, the labeled subunits were purified on streptavidin beads before separation on SDS-PAGE. "In-gel" CNBr cleavage of the labeled alpha subunit followed by Edman degradation of the purified peptides showed that alphaTyr190 and alphaTyr198 were the most labeled residues, with a less important labeling on alphaCys192. We believe that the novel photoactivatable probe will be of great use to identify key residues of ligands interacting with macromolecules.

Prediction of CD4(+) T cell epitopes restricted to HLA-DP4 molecules.

We have set up a method to predict peptide binding to HLA-DP4 molecules. These HLA II molecules are the most frequent worldwide and hence are an interesting target for epitope-based vaccines. The prediction is based on quantitative matrices built with binding data for peptides substituted at anchoring positions for HLA-DP4. A set of 98 peptides of various origins was used to compare the prediction with binding activity. At different prediction thresholds, the positive predictive value and the sensitivity of the prediction ranged from 50% to 80%, demonstrating its efficiency. This prediction method can be applied to the entire genomes of pathogens and large peptide sequences derived from tumor antigens.

Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity.

Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle alpha(1)betagammadelta-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 A by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snake's diet.

Structural studies of human alkaline phosphatase in complex with strontium: implication for its secondary effect in bones.

Strontium is used in the treatment of osteoporosis as a ranelate compound, and in the treatment of painful scattered bone metastases as isotope. At very high doses and in certain conditions, it can lead to osteomalacia characterized by impairment of bone mineralization. The osteomalacia symptoms resemble those of hypophosphatasia, a rare inherited disorder associated with mutations in the gene encoding for tissue-nonspecific alkaline phosphatase (TNAP). Human alkaline phosphatases have four metal binding sites--two for zinc, one for magnesium, and one for calcium ion--that can be substituted by strontium. Here we present the crystal structure of strontium-substituted human placental alkaline phosphatase (PLAP), a related isozyme of TNAP, in which such replacement can have important physiological implications. The structure shows that strontium substitutes the calcium ion with concomitant modification of the metal coordination. The use of the flexible and polarizable force-field TCPEp (topological and classical polarization effects for proteins) predicts that calcium or strontium has similar interaction energies at the calcium-binding site of PLAP. Since calcium helps stabilize a large area that includes loops 210-228 and 250-297, its substitution by strontium could affect the stability of this region. Energy calculations suggest that only at high doses of strontium, comparable to those found for calcium, can strontium substitute for calcium. Since osteomalacia is observed after ingestion of high doses of strontium, alkaline phosphatase is likely to be one of the targets of strontium, and thus this enzyme might be involved in this disease.

Cyclic and acyclic oxorhenium(V)-peptide conjugates as new ligands of the human cyclophilin hCyp-18.

Peptide metalloconstructs display interesting conformations, activities, and resistance to proteolysis. However, introduction of a metal core close to the residues that interact with the protein might strongly affect the binding. We investigated the effects of a coordinated oxorhenium core on the binding of model peptides to cyclophilin hCyp-18, a protein implicated in important biological processes and several diseases. For this purpose, we synthesized a series of linear metalloconstructs bearing an oxorhenium(V) core (ReO3+), as well as a peptide cyclized through oxorhenium(V) coordination. All these peptides contain an Ala-Pro-Xaa-pNA moiety (Xaa = Cys derivative) and are anticipated to bind simultaneously to the S1-S1' and S2'-S3' subsites of hCyp-18. Therefore, the metal core is coordinated to both the cysteine residue and exogenous or endogenous NS2 tridentate systems. Cyclization of the peptide through metal coordination did not affect the affinity whereas bimolecular oxorhenium metalloconstructs bind hCyp-18 with a slightly better affinity than the corresponding nonmetalated peptide. Peptide labeling with a 99mTcO3+ core was also carried out successfully.