NMR Studies of the Phosphorylation Motif of the HIV-1 Protein Vpu Bound to the F-Box Protein -TrCP ² Gae ¨l Coadou, ‡ Josyane Gharbi-Benarous, ‡ Simon Megy, ‡ Gildas Bertho, ‡ Nathalie Evrard-Todeschi, ‡ Emmanuel Segeral, § Richard Benarous, § and Jean-Pierre Girault* ,‡ Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, UniVersite ´ Rene ´ Descartes, Paris V, 45 rue des Saint-Pe ` res, 75270 Paris Cedex 06, France, and De ´ partement des Maladies Infectieuses, Institut Cochin, U567 INSERM, UMR 8104 CNRS, Ho ˆ pital Cochin Bat. G. Roussy, 27 rue du Faubourg St. Jacques, 75014 Paris, France ReceiVed July 9, 2003; ReVised Manuscript ReceiVed September 18, 2003 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-TrCP 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 -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 -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 -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 -TrCP. The Ile46 residue was also found essential for interaction with the -TrCP protein. Leu45 and Ile46 side chains lie in close proximity to a hydrophobic pocket of the WD domain. Vpu 1 is an 81-residue membrane-associated accessory protein encoded in the HIV-1 genome (Figure 1a). Vpu is specific for HIV-1, the major causative agent of the acquired immune deficiency syndrome (AIDS) and is not found in HIV-2 (which is less pathogenic than HIV-1) or in most simian immunodeficiency viruses (SIV) (1, 2). It was shown (Figure 1b) that its secondary structure consists of one transmembrane hydrophobic helix (h1) near the N-terminus and two amphipathic helices (h2 in-plane and h3) in the cytoplasmic C-terminal domain, using a combination of solid-state NMR and solution NMR methods (3). The protein has two biological activities that contribute to the virulence of HIV-1 infections in humans (Figure 2a). It enhances the release of new virus particles from cells infected with HIV-1 and induces the intracellular degradation of the CD4 receptor protein (4, 5) by targeting it for proteolysis by the ubiquitin- proteasome pathway (6). Both activities contribute to in- creased virion production (7). These functions appear to be associated with two different structural domains of Vpu (8, 9). The N-terminal transmembrane helix, which serves as a membrane anchor, is required to regulate virus secretion, most likely by formation of an ion channel. The cytoplasmic domain of Vpu, which has two phosphorylation sites (pSer52 and pSer56), is essential for interaction with CD4 and induction of CD4 degradation in the endoplasmic reticulum (ER). The mechanism by which Vpu mediates CD4 degrada- tion has been elucidated (Figure 2a). It has been demonstrated that, by binding to the F-box WD40 protein -TrCP, the receptor component of the SCF E3 ubiquitin ligase -TrCP, Vpu can subvert the cellular targeting pathways to the proteasome in order to promote CD4 degradation (6). Phosphorylation of Ser52 and Ser56 of Vpu by casein kinase 2(10, 11) is necessary for interaction with -TrCP (12, 13). Vpu-induced degradation of CD4 requires, in fact, the formation of multiprotein complexes containing CD4, Vpu, -TrCP, Skp1, and other subunits of the SCF E3 ubiquitin ligase (6) and the function of the proteasome (14, 15). ² This work was supported by grants from organizations involved in AIDS and cancer research: ANRS (Agence Nationale pour la Recherche contre le SIDA), SIDACTION, ARC, and Ligue Nationale contre le Cancer. E.S. is supported by the ARC. * To whom correspondence should be addressed. Tel: 01 42 86 21 80. Fax: 01 42 86 83 87. E-mail: giraultj@biomedicale.univ-paris5.fr. ‡ LCBPT, CNRS, Universite ´ Rene ´ Descartes, Paris V. § U567 INSERM, UMR 8104 CNRS, Institut Cochin, De ´partement des Maladies Infectieuses, Ho ˆpital Cochin Paris. 1 Abbreviations: TrCP, transducin repeat containing protein; GST, glutathione S-transferase; HIV, human immunodeficiency virus; HSQC, heteronuclear single-quantum correlation; INEPT, insensitive nuclei enhanced by polarization transfer; MD, molecular dynamics; NOESY, nuclear Overhauser effect spectroscopy; PBS, phosphate-buffered saline; rmsd, root mean square deviation; SCF, Skp1-Cullin-FBox; STD, saturation transfer difference; TRNOESY, transferred nuclear Over- hauser effect spectroscopy; TOCSY, total correlation spectroscopy; TPPI, time-proportional phase incrementation; Vpu, HIV-1 encoded virus protein U; WATERGATE, water suppression by gradient-tailored excitation; WD domain, Trp Asp domain. 14741 Biochemistry 2003, 42, 14741-14751 10.1021/bi035207u CCC: $25.00 © 2003 American Chemical Society Published on Web 11/21/2003