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Jean-pierre Girault - One of the best experts on this subject based on the ideXlab platform.

  • Epitope Mapping of the Phosphorylation Motif of the HIV-1 Protein Vpu Bound to the Selective Monoclonal Antibody Using TRNOESY and STD NMR Spectroscopy
    Biochemistry, 2004
    Co-Authors: J. Gharbi-benarous, G. Bertho, N. Evrard-todeschi, G. Coadou, S. Megy, T. Delaunay, R. Benarous, Jean-pierre Girault
    Abstract:

    The conformational preferences of a 22-amino acid peptide (LIDRLIERAEDpSGNEpSEGEISA) that mimics the phosphorylated HIV-1-encoded virus protein U (Vpu) antigen have been investigated by NMR Spectroscopy. Degradation of HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at sites Ser52 and Ser56 on the DSGXXS motif is required for the interaction of Vpu with the ubiquitin ligase SCF(beta)(-TrCP) which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. The interaction of the P-Vpu(41-62) peptide with its monoclonal antibody has been studied by transferred nuclear Overhauser effect NMR Spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) Spectroscopy. The peptide was found to adopt a bend conformation upon binding to the antibody; the peptide residues (Asp51-pSer56) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. The three-dimensional structure of P-Vpu(41-62) in the bound conformation was determined by TRNOESY spectra; the peptide adopts a compact structure in the presence of mAb with formation of several bends around Leu45 and Ile46 and around Ile60 and Ser61, with a tight bend created by the DpS(52)GNEpS(56) motif. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentale association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of Vpu with the SCF(beta)(-TrCP) protein.

  • nmr studies of the phosphorylation motif of the hiv 1 protein vpu bound to the f box protein β trcp
    Biochemistry, 2003
    Co-Authors: G. Coadou, G. Bertho, S. Megy, R. Benarous, Josyane Gharbibenarous, Nathalie Evrardtodeschi, Emmanuel Segeral, Jean-pierre Girault
    Abstract:

    A protein-protein association regulated by phosphorylation of serine is examined by NMR studies. Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at two sites, Ser52 and Ser56, on the motif DSGXXS is required for the interaction of Vpu with the ubiquitin ligase SCF-betaTrCP which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. To elucidate the basis of beta-TrCP recognition, the bound conformation of the P-Vpu(41-62) peptide was determined by using NMR and MD. The TRNOE intensities provided distance constraints which were used in simulated annealing. The beta-TrCP-bound structure of P-Vpu was found to be similar to the structure of the free peptide in solution and to the structure recognized by its antibody. Residues 50-57 formed a bend while the phosphate groups are pointing away. The binding fragment was studied by STD-NMR Spectroscopy. The phosphorylated motif DpS(52)GNEpS(56) was found to make intimate contact with beta-TrCP, and pSer52 displays the strongest binding effect. It is suggested that Ser phosphorylation allows protein-protein association by electrostatic stabilization: an obvious negative binding region of Vpu was recognizable by positive residues (Arg and Lys) of the WD domain of beta-TrCP. The Ile46 residue was also found essential for interaction with the beta-TrCP protein. Leu45 and Ile46 side chains lie in close proximity to a hydrophobic pocket of the WD domain.

Jesus Angulo - One of the best experts on this subject based on the ideXlab platform.

  • Exploring Multi-Subsite Binding Pockets in Proteins: DEEP-STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin B**
    Chemistry (Weinheim an der Bergstrasse Germany), 2020
    Co-Authors: Serena Monaco, Ridvan Nepravishta, Samuel Walpole, Hassan Doukani, Macarena Martínez-bailén, Ana T. Carmona, Javier Ramos-soriano, Maria Bergström, Inmaculada Robina, Jesus Angulo
    Abstract:

    Ligand-based NMR techniques to study protein–ligand interactions are potent tools in drug design. Saturation transfer difference (STD) NMR Spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi-frequency STD NMR approach, differential epitope mapping (DEEP)-STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so-called DEEP-STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunit B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR Spectroscopy. Remarkably, the combination of DEEP-STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi-subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin.

  • ligand receptor binding affinities from saturation transfer difference std nmr Spectroscopy the binding isotherm of std initial growth rates
    Chemistry: A European Journal, 2010
    Co-Authors: Jesus Angulo, Pedro M Enriqueznavas, Pedro M. Nieto
    Abstract:

    The direct evaluation of dissociation constants (KD) from the variation of saturation transfer difference (STD) NMR Spectroscopy values with the receptor–ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of KD, as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR Spectroscopy for the direct measurements of receptor–ligand dissociation constants (KD) from single-ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein–ligand affinities by STD NMR Spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein–ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein–ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well-studied protein–ligand systems: the binding of the saccharides GlcNAc and GlcNAcs1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L-tryptophan to bovine serum albumin (BSA). In all cases, the experimental KD measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein–ligand dissociation constants, extending the applicability of the STD NMR Spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.

Pedro M. Nieto - One of the best experts on this subject based on the ideXlab platform.

  • Chondroitin Sulfate Tetrasaccharides: Synthesis, Three-Dimensional Structure and Interaction with Midkine.
    Chemistry (Weinheim an der Bergstrasse Germany), 2016
    Co-Authors: Cristina Solera, Giuseppe Macchione, Susana Maza, M. Mar Kayser, Francisco Corzana, José L. De Paz, Pedro M. Nieto
    Abstract:

    The biological activity of midkine, a cytokine implicated in neuro- and tumourigenesis, is regulated by its binding to glycosaminoglycans (GAGs), such as heparin and chondroitin sulfate (CS). To better understand the molecular recognition of GAG sequences by this growth factor, the interactions between synthetic chondroitin sulfate-like tetrasaccharides and midkine were studied by using different techniques. Firstly, a synthetic approach for the preparation of CS-like oligosaccharides in the sequence GalNAc-GlcA was developed. A fluorescence polarisation competition assay was then employed to analyse the relative binding affinities of the synthetic compounds and revealed that midkine interacted with CS-like tetrasaccharides in the micromolar range. The 3D structure of these tetramers was studied in detail by a combination of NMR Spectroscopy experiments and molecular dynamics simulations. Saturation transfer difference (STD) NMR Spectroscopy experiments indicate that the CS tetrasaccharides bind to midkine in an extended conformation, with similar saturation effects along the entire sugar chain. These results are compatible with docking studies that suggest an interaction of the tetrasaccharide with midkine in a folded structure. Overall, this study provides valuable information on the interaction between midkine and well-defined, chemically synthesised CS oligosaccharides and these data can be useful for the design of more active compounds that modulate the biological function of this protein.

  • ligand receptor binding affinities from saturation transfer difference std nmr Spectroscopy the binding isotherm of std initial growth rates
    Chemistry: A European Journal, 2010
    Co-Authors: Jesus Angulo, Pedro M Enriqueznavas, Pedro M. Nieto
    Abstract:

    The direct evaluation of dissociation constants (KD) from the variation of saturation transfer difference (STD) NMR Spectroscopy values with the receptor–ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of KD, as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR Spectroscopy for the direct measurements of receptor–ligand dissociation constants (KD) from single-ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein–ligand affinities by STD NMR Spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein–ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein–ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well-studied protein–ligand systems: the binding of the saccharides GlcNAc and GlcNAcs1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L-tryptophan to bovine serum albumin (BSA). In all cases, the experimental KD measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein–ligand dissociation constants, extending the applicability of the STD NMR Spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.

G. Coadou - One of the best experts on this subject based on the ideXlab platform.

  • Epitope Mapping of the Phosphorylation Motif of the HIV-1 Protein Vpu Bound to the Selective Monoclonal Antibody Using TRNOESY and STD NMR Spectroscopy
    Biochemistry, 2004
    Co-Authors: J. Gharbi-benarous, G. Bertho, N. Evrard-todeschi, G. Coadou, S. Megy, T. Delaunay, R. Benarous, Jean-pierre Girault
    Abstract:

    The conformational preferences of a 22-amino acid peptide (LIDRLIERAEDpSGNEpSEGEISA) that mimics the phosphorylated HIV-1-encoded virus protein U (Vpu) antigen have been investigated by NMR Spectroscopy. Degradation of HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at sites Ser52 and Ser56 on the DSGXXS motif is required for the interaction of Vpu with the ubiquitin ligase SCF(beta)(-TrCP) which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. The interaction of the P-Vpu(41-62) peptide with its monoclonal antibody has been studied by transferred nuclear Overhauser effect NMR Spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) Spectroscopy. The peptide was found to adopt a bend conformation upon binding to the antibody; the peptide residues (Asp51-pSer56) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. The three-dimensional structure of P-Vpu(41-62) in the bound conformation was determined by TRNOESY spectra; the peptide adopts a compact structure in the presence of mAb with formation of several bends around Leu45 and Ile46 and around Ile60 and Ser61, with a tight bend created by the DpS(52)GNEpS(56) motif. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentale association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of Vpu with the SCF(beta)(-TrCP) protein.

  • nmr studies of the phosphorylation motif of the hiv 1 protein vpu bound to the f box protein β trcp
    Biochemistry, 2003
    Co-Authors: G. Coadou, G. Bertho, S. Megy, R. Benarous, Josyane Gharbibenarous, Nathalie Evrardtodeschi, Emmanuel Segeral, Jean-pierre Girault
    Abstract:

    A protein-protein association regulated by phosphorylation of serine is examined by NMR studies. Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at two sites, Ser52 and Ser56, on the motif DSGXXS is required for the interaction of Vpu with the ubiquitin ligase SCF-betaTrCP which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. To elucidate the basis of beta-TrCP recognition, the bound conformation of the P-Vpu(41-62) peptide was determined by using NMR and MD. The TRNOE intensities provided distance constraints which were used in simulated annealing. The beta-TrCP-bound structure of P-Vpu was found to be similar to the structure of the free peptide in solution and to the structure recognized by its antibody. Residues 50-57 formed a bend while the phosphate groups are pointing away. The binding fragment was studied by STD-NMR Spectroscopy. The phosphorylated motif DpS(52)GNEpS(56) was found to make intimate contact with beta-TrCP, and pSer52 displays the strongest binding effect. It is suggested that Ser phosphorylation allows protein-protein association by electrostatic stabilization: an obvious negative binding region of Vpu was recognizable by positive residues (Arg and Lys) of the WD domain of beta-TrCP. The Ile46 residue was also found essential for interaction with the beta-TrCP protein. Leu45 and Ile46 side chains lie in close proximity to a hydrophobic pocket of the WD domain.

G. Bertho - One of the best experts on this subject based on the ideXlab platform.

  • Epitope Mapping of the Phosphorylation Motif of the HIV-1 Protein Vpu Bound to the Selective Monoclonal Antibody Using TRNOESY and STD NMR Spectroscopy
    Biochemistry, 2004
    Co-Authors: J. Gharbi-benarous, G. Bertho, N. Evrard-todeschi, G. Coadou, S. Megy, T. Delaunay, R. Benarous, Jean-pierre Girault
    Abstract:

    The conformational preferences of a 22-amino acid peptide (LIDRLIERAEDpSGNEpSEGEISA) that mimics the phosphorylated HIV-1-encoded virus protein U (Vpu) antigen have been investigated by NMR Spectroscopy. Degradation of HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at sites Ser52 and Ser56 on the DSGXXS motif is required for the interaction of Vpu with the ubiquitin ligase SCF(beta)(-TrCP) which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. The interaction of the P-Vpu(41-62) peptide with its monoclonal antibody has been studied by transferred nuclear Overhauser effect NMR Spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) Spectroscopy. The peptide was found to adopt a bend conformation upon binding to the antibody; the peptide residues (Asp51-pSer56) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. The three-dimensional structure of P-Vpu(41-62) in the bound conformation was determined by TRNOESY spectra; the peptide adopts a compact structure in the presence of mAb with formation of several bends around Leu45 and Ile46 and around Ile60 and Ser61, with a tight bend created by the DpS(52)GNEpS(56) motif. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentale association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of Vpu with the SCF(beta)(-TrCP) protein.

  • nmr studies of the phosphorylation motif of the hiv 1 protein vpu bound to the f box protein β trcp
    Biochemistry, 2003
    Co-Authors: G. Coadou, G. Bertho, S. Megy, R. Benarous, Josyane Gharbibenarous, Nathalie Evrardtodeschi, Emmanuel Segeral, Jean-pierre Girault
    Abstract:

    A protein-protein association regulated by phosphorylation of serine is examined by NMR studies. Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at two sites, Ser52 and Ser56, on the motif DSGXXS is required for the interaction of Vpu with the ubiquitin ligase SCF-betaTrCP which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. To elucidate the basis of beta-TrCP recognition, the bound conformation of the P-Vpu(41-62) peptide was determined by using NMR and MD. The TRNOE intensities provided distance constraints which were used in simulated annealing. The beta-TrCP-bound structure of P-Vpu was found to be similar to the structure of the free peptide in solution and to the structure recognized by its antibody. Residues 50-57 formed a bend while the phosphate groups are pointing away. The binding fragment was studied by STD-NMR Spectroscopy. The phosphorylated motif DpS(52)GNEpS(56) was found to make intimate contact with beta-TrCP, and pSer52 displays the strongest binding effect. It is suggested that Ser phosphorylation allows protein-protein association by electrostatic stabilization: an obvious negative binding region of Vpu was recognizable by positive residues (Arg and Lys) of the WD domain of beta-TrCP. The Ile46 residue was also found essential for interaction with the beta-TrCP protein. Leu45 and Ile46 side chains lie in close proximity to a hydrophobic pocket of the WD domain.