HIV-1 encoded virus protein U (Vpu) solution structure of the 41-62 hydrophilic region containing the phosphorylated sites Ser52 and Ser56.
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. To promote CD4 degradation Vpu has to be phosphorylated on a motif DSGXXS, which is conserved in several signalling proteins known to be degraded by the proteasome upon phosphorylation. Such phosphorylation is required for the interaction of Vpu with the ubiquitin ligase SCF-beta-TrCP that triggers CD4 degradation by the proteasome. In the present work, we used two peptides of 22 amino acids between residues 41 and 62 of Vpu. Vpu41-62 was predicted to form an alpha-helix-flexible-alpha-helix including the phosphorylation motif DS52GNES56 and Vpu_P41-62 was phosphorylated at the two sites Ser52 and Ser56. We analysed the conformational change induced by the phosphorylation of this peptide on the residues Ser52 and Ser56. Homo- and heteronuclear NMR techniques were used to assess the structural influence of phosphorylation. The spectra of the free peptides, Vpu_P41-62 and Vpu41-62, in both H2O (at pH 3.5 and 7.2) and a 1:1 mixture of H2O and trifluoroethanol were completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide segment (42-49) shows a less well-defined helix propensity. The Vpu_P41-62 domain of residues 50-62 forms a loop with the phosphate group pointing away, a short beta-strand and a flexible extended 'tail' of residues 60-62. Residues 50-60 exhibit alpha-proton NMR secondary chemical shift changes from random coil toward more beta-like structure with the combined (temperature, solvent and pH) NMR and molecular calculation experiments. Differences in this molecular region 50-62 suggest that conformational changes of Vpu_P play an important role in Vpu_P-induced degradation of CD4 molecules.
Coadou
Gaël
G
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.
Evrard-Todeschi
Nathalie
N
Gharbi-Benarous
Josyane
J
Benarous
Richard
R
Girault
Jean Pierre
JP
D000595:Amino Acid Sequence; D001665:Binding Sites; D015704:CD4 Antigens / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054298:Human Immunodeficiency Virus Proteins; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D015728:Receptors, HIV / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D012996:Solutions; D013816:Thermodynamics; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000086082:Viroporin Proteins
D015704:CD4 Antigens; D054298:Human Immunodeficiency Virus Proteins; D010446:Peptide Fragments; D015728:Receptors, HIV; D012694:Serine; D012996:Solutions; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins