NMR studies of the phosphorylation motif of the HIV-1 protein Vpu bound to the F-box protein beta-TrCP.
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.
Coadou
Gaël
G
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université René Descartes, Paris V, France.
Gharbi-Benarous
Josyane
J
Megy
Simon
S
Bertho
Gildas
G
Evrard-Todeschi
Nathalie
N
Segeral
Emmanuel
E
Benarous
Richard
R
Girault
Jean-Pierre
JP
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015704:CD4 Antigens / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods; D015497:HIV-1 / Q000737:chemistry* / Q000378:metabolism; D054298:Human Immunodeficiency Virus Proteins; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry* / Q000378:metabolism; D044844:beta-Transducin Repeat-Containing Proteins / Q000737:chemistry* / Q000378:metabolism; D000086082:Viroporin Proteins
D015704:CD4 Antigens; D054298:Human Immunodeficiency Virus Proteins; D010446:Peptide Fragments; D054334:Viral Regulatory and Accessory Proteins; D044844:beta-Transducin Repeat-Containing Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins