NMR studies for identifying phosphopeptide ligands of the HIV-1 protein Vpu binding to the F-box protein beta-TrCP.
The human immunodeficiency virus type 1 (HIV-1) Vpu enhances viral particle release and, its interaction with the ubiquitin ligase SCF-beta-TrCP triggers the HIV-1 receptor CD4 degradation by the proteasome. The interaction between beta-TrCP protein and ligands containing the phosphorylated DpSGXXpS motif plays a key role for the development of severe disease states, such as HIV or cancer. This study examines the binding and conformation of phosphopeptides (P1, LIERAEDpSG and P2, EDpSGNEpSE) from HIV protein Vpu to beta-TrCP with the objective of defining the minimum length of peptide needed for effective binding. The screening step can be analyzed by NMR spectroscopy, in particular, saturation transfer NMR methods clearly identify the residues in the peptide that make direct contact with beta-TrCP protein when bound. An analysis of saturation transfer difference (STD) spectra provided clear evidence that the two peptides efficiently bound beta-TrCP receptor protein. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated peptides was determined using transferred NOESY methods, which gave rise to a well-defined structure. P1 and P2 can fold in a bend arrangement for the DpSG motif, showing the protons identified by STD-NMR as exposed in close proximity at the molecule surface. Ser phosphorylation allows electrostatic interaction and hydrogen bond with the amino acids of the beta-TrCP binding pocket. The upstream LIER hydrophobic region was also essential in binding to a hydrophobic pocket of the beta-TrCP WD domain. These findings are in good agreement with a recently published X-ray structure of a shorter beta-Catenin fragment with the beta-TrCP complex.
Evrard-Todeschi
Nathalie
N
Université René Descartes-Paris V, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (UMR 8601 CNRS), 45 rue des Saint-Pères, 75270 Paris Cedex 06, France.
Gharbi-Benarous
Josyane
J
Bertho
Gildas
G
Coadou
Gaël
G
Megy
Simon
S
Benarous
Richard
R
Girault
Jean-Pierre
JP
D000595:Amino Acid Sequence; D001665:Binding Sites; D015497:HIV-1 / Q000737:chemistry / Q000378:metabolism*; D054298:Human Immunodeficiency Virus Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry* / Q000378:metabolism*; D044844:beta-Transducin Repeat-Containing Proteins / Q000737:chemistry* / Q000378:metabolism*; D000086082:Viroporin Proteins
D054298:Human Immunodeficiency Virus Proteins; D008024:Ligands; D010748:Phosphopeptides; D054334:Viral Regulatory and Accessory Proteins; D044844:beta-Transducin Repeat-Containing Proteins; C471644:BTRC protein, human; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins