Epitope mapping of the phosphorylation motif of the HIV-1 protein Vpu bound to the selective monoclonal antibody using TRNOESY and STD NMR spectroscopy

Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (UMR 8601 CNRS), Université René Descartes-Paris V, 45 rue des Saint-Pères, 75270 Paris Cedex 06, France.
Biochemistry (Impact Factor: 3.02). 12/2004; 43(46):14555-65. DOI: 10.1021/bi0492861
Source: PubMed


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.

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    • "Recent developments in STD-NMR provide additional methods for characterizing compounds bound to target proteins such as S100B [139]. For example, STD data combined with Transfer NOE data was used to monitor the interaction between large complexes such as the HIV-1 protein Vpu (residues 41-62) bound to a selective monoclonal antibody [140]. In addition, STD NMR was used to refine crystal structures using only the strength of the STD signal in the absence of NOE data [141]. "
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