NMR studies for identifying phosphopeptide ligands of the HIV-1 protein Vpu binding to the F-box protein b-trcp

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1 peptides 27 (2006) available at journal homepage: NMR studies for identifying phosphopeptide ligands of the HIV-1 protein Vpu binding to the F-box protein b-trcp Nathalie Evrard-Todeschi a, Josyane Gharbi-Benarous a, Gildas Bertho a, Gaël Coadou a, Simon Megy a, Richard Benarous b, Jean-Pierre Girault a, * a Université René Descartes-Paris V, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (UMR 8601 CNRS), 45 rue des Saint-Pères, Paris Cedex 06, France b U567-INSERM, UMR 8104 CNRS, Institut Cochin-Département des Maladies Infectieuses, Hôpital Cochin Bat. G. Roussy, 27 rue du Faubourg St-Jacques, Paris, France article info Article history: Received 16 June 2005 Received in revised form 22 July 2005 Accepted 25 July 2005 Published on line 13 September 2005 Keywords: Human immunodeficiency virus type 1 Vpu Phosphorylated peptide P-Vpu STD-NMR TRNOESY Restrained molecular dynamics Bound structure Binding fragment abstract The human immunodeficiency virus type 1 (HIV-1) Vpu enhances viral particle release and, its interaction with the ubiquitin ligase SCF-b-TrCP triggers the HIV-1 receptor CD4 degradation by the proteasome. The interaction between b-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 b-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 b- TrCP protein when bound. An analysis of saturation transfer difference (STD) spectra provided clear evidence that the two peptides efficiently bound b-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 b-trcp binding pocket. The upstream LIER hydrophobic region was also essential in binding to a hydrophobic pocket of the b-trcp WD domain. These findings are in good agreement with a recently published X-ray structure of a shorter b-catenin fragment with the b-trcp complex. # 2005 Elsevier Inc. All rights reserved. 1. Introduction The human immunodeficiency virus type 1 (HIV-1) Vpu protein, an integral membrane protein of 81 amino acids (aa) (Fig. 1a), acts as an adaptor for the proteasomal degradation of CD4. It was shown recently that Vpu exerts a positive effect on HIV-1 infectivity by down-modulating CD4 receptor molecules at the surface of HIV-1-producing cells [19]. CD4 degradation requires the phosphorylation of the serine residues at positions 52 and 56 of the Vpu cytoplasmic domain by casein kinase II [34]. The Vpu secondary structure (Fig. 1b) consists of one transmembrane helix (h1), connected with a second helix (h2) residing on the bilayer surface, and a flexible linker region, including the two phosphorylation sites (Ser52 * Corresponding author. Tel.: ; fax: address: jean-pierre.girault@univ-paris5.fr (J.-P. Girault) /$ see front matter # 2005 Elsevier Inc. All rights reserved. doi: /j.peptides

2 peptides 27 (2006) Fig. 1 (a) Primary structure sequence of the HIV-1 Vpu protein and (top) sequence of the Vpu fragments (p1 and p2) which were investigated in this work and (P-Vpu ) in a previous work. (b) Domains of the secondary structural regions found in Vpu. The hydrophobic N-terminal membrane anchor (helix h1, residues 1 28) is followed by two amphipathic a-helices (helix h2, residues 32 51; helix h3, residues 57 72). Both helices are joined by a flexible non-structured link, which contains phosphoacceptors Ser52 and Ser56. The C-terminus forms a reverse turn at Ala74 [48]. (c) Part of the backbone (residues 37 69) of the Vpu cytoplasmic domain (residues 37 81) structure [45]. and Ser56), connects helix h2 with a third helix toward the C-terminal end [7,10,13,21,37,45,48,49]. Vpu enhances the release of new virus particles from the plasma membrane of cells infected with HIV-1 [40] through its N terminal transmembrane domain (aa 1 27), whereas it induces the degradation of the CD4 receptor in the endoplasmic reticulum [39,46] via its cytoplasmic domain (aa 28 81) (Fig. 1c). Vpu-induced degradation of CD4 requires in fact the formation of multiprotein complexes [11,35]: Vpu binds to the F-box b-transducin repeat-containing protein (b-trcp), the receptor component of the multisubunit SCF b-trcp E3 ubiquitin ligase complex, and connects CD4 to the ubiquitin proteasome machinery [22]. b-trcp is a WD40 family F-box protein (Fig. 2): b-trcp is linked to the SCF complex by binding to Skp1 through its N-terminal F-box motif and interacts with Vpu through its C-terminal WD repeat region [22]. b-trcp is also involved in the ubiquitination and proteasome targeting of: (i) b-catenin, the accumulation of which has been implicated in various human cancers [12,47], (ii) IkBa, the inhibitor of the master transcription factor NFkB [16,47,53] and (iii) ATF4, a member of the family of transcription factors [18]. The signal for the recognition of all these cellular ligands by b-trcp is the phosphorylation of the serine residues present in a conserved motif, DpSGXXpS for Vpu, IkBa, b-catenin (Fig. 2c), and DpSGXXXpS for ATF4. Phosphorylation of Ser52 and Ser56 of Vpu [31,42] is necessary for the interaction with b-trcp [34,44]. The SCF b-trcp complex specifically recognizes a Vpu peptide fragment of 22 amino acids [22], the P-Vpu peptide (Fig. 1a), a 19-amino acid motif in IkBa, and a 22-residue-b- Catenin polypeptide in a phosphorylation-dependent manner [47]. To elucidate the basis of b-trcp recognition, the bound structure of the P-Vpu peptide to the F-box protein b-trcp was previously determined by using NMR and MD [8]. This previous work [8] has allowed us to determine which sequence requirements are playing a role in interaction with b-trcp. The bend region (51 56) corresponding to the DpSGXXpS motif associated with the hydrophobic cluster (45 46) characterizes the phosphorylation-dependent recognition by the WD domain (Fig. 2a). A similar kinked-like DpSGXXpS motif also plays a central role in the crystal structure of the human b- TrCP1-Skp1 complex bound to a fragment b-catenin substrate peptide (Fig. 2c) [50]. This study examines the binding and conformation of phosphopeptides from HIV protein Vpu P1 (P-Vpu ) and P2 (P-Vpu )(Fig. 1a) to b-trcp with the objective of defining the minimum length of peptide needed for effective binding. The starting models were chosen with some arbitrary constraints. The P1 peptide (LIERAEDpS 52 G), consists of the Vpu residue pser52 which was previously found to make the most

3 196 peptides 27 (2006) Fig. 2 (a) The full-length human protein b-trcp and below the fusion GST b-trcp protein which was investigated in the present work. This recombinant protein includes the 218 residues of the glutathione S-transferase (GST) protein fused to the seven WD repeats (residues ) of the full-length protein b-trcp by an 18 residues linker. (b) The WD repeats were identified in the primary sequence of b-trcp from their alignment with the regular expression. The F-box protein b-trcp binds the doubly phosphorylated consensus motif DpSGXXpS in IkBa and b-catenin in an analogous manner to Vpu. (c) Ribbons diagram of the seven b-propeller structure of the WD40 repeat region of the yeast F-box protein b-trcp. Surface representation of the top face of the b-trcp WD40 domain with the bound DpSGIHpS motif from the crystal structure of the human b-trcp-skp1 complex bound to a b-catenin substrate peptide [50]. extensive b-trcp contact, and additionally of the amino acids Leu45 and Ile46 which were found experimentally to be in close contact with the b-trcp protein [8]. The P2 peptide (EDpS 52 GNEpS 56 E) contains the majority of the Vpu residues that were found to be embedded in the b-trcp WD 40 domain [50], and located in the bend connecting the helix h2 at the C- terminal. The peptides contain one or two phosphorylated sites (52 or 52 and 56) for P1 and P2, respectively, and the residues previously identified for P-Vpu to be relevant for binding. The role of these peptides can be investigated by using STD-NMR epitope mapping and TRNOE-based conformational analysis. These two NMR experiments have taken an increasing importance as tools in the investigation of biomolecular recognition phenomena [30]. Saturation transfer difference (STD) NMR spectroscopy is really well adapted for analyzing b-trcp binding processes [14,23,24] by screening various peptides and mapping of ligand epitopes [25], and the TRNOESY experiment [28] a fast method to probe the conformation of a ligand bound to a protein receptor. Finally, the conformation of the b-trcp-bound peptides was elucidated by molecular dynamics simulation, an approach combining dynamical annealing and refinement protocols. These techniques, described to identify binding events of ligands to receptors by looking at the resonance signals of the

4 peptides 27 (2006) ligands, allow screening of compounds as well as a detailed identification of the groups involved in the binding to b-trcp. 2. Materials and methods 2.1. Peptides HIV-1 encoded virus protein U (Vpu) fragments (residues 45 53) and (residues 50 57) (numbers refer to Vpu protein), the peptides P-Vpu (Ac-Leu-Ile-Glu-Arg-Ala-Glu-Asp-Ser (PO 3 H 2 )-Gly-NH 2 ) named as P1 and P-Vpu (Ac-Glu-Asp- Ser(PO 3 H 2 )-Gly-Asn-Glu-Ser(PO 3 H 2 )-Glu-NH 2 ) named as P2, containing one or two phosphorylated sites (ps52 or ps52 and ps56), with the LIERAEDpS 52 G and EDpS 52 GNEpS 56 E amino acid sequences, respectively, were purchased from Neosystem Lab. (Strasbourg, France). The purity of the peptides (95%) was tested by analytical HPLC and by mass spectrometry NMR experiments For the preparation of peptide-b-trcp samples for NMR studies, purification of the WD repeat region from human protein b-trcp as a fusion protein with the glutathione S- transferase (GST) was described previously [8]. The purified recombinant protein was concentrated using Amicon Ultra-15 centrifugal filter units from Millipore with a 30-kDa molecular weight cut-off. The solution was washed in NMR buffer and concentrated several times, performing a buffer exchange in order to remove the exceeding reduced glutathione. Protein concentration was determined using the standard Bradford and Coomassie Brilliant Blue method, following the OD at 595 nm. The final yield of purified GST b-trcp from 1 l of Luria Bertani medium (LB) was about 1.2 mg. This amount of purified recombinant protein was used to prepare the NMR samples. NMR samples contained the P1 or P2 peptides with the GST b-trcp protein. They were prepared in NMR phosphate-buffered saline (20 mm phosphate, 5% D 2 O and 0.02% NaN 3 ) at ph 7.2. The protein concentration was estimated to be 0.05 mm and determined by optical density measurement at 595 nm on the visible absorption. A 40-fold ligand excess (2 mm) over binding sites was used throughout the studies. One control sample was prepared at the same w/v concentration as was used for the GST b-trcp sample, and containing the binding peptide with the glutathione S- transferase lacking the seven WD domain of b-trcp protein (Fig. 2a). The latter corresponds to the 218 residues of the GST protein and an 18 residues linker. 1 H NMR spectra were recorded at 280 K on a Bruker AMX- 500 spectrometer using a z-axis gradient. Chemical shifts assignments (Table 1 and Table S4 in Supplementary data) referred to internal 3-(trimethylsilyl)propionic acid-2,2,3,3-d 4, sodium salt (TSP-d 4 ). Two-dimensional NMR spectra were recorded in the phase-sensitive mode using the States TPPI method [38]. All experiments were carried out using the WATERGATE pulse sequence for water suppression [32]. Twodimensional 1 H, 1 H TOCSY spectra were recorded using a mlev-17 spin lock sequence [1] with a mixing time (t m )of35 and 70 ms, respectively. Typically, spectra were acquired with 256 t 1 increments, 2048 data points, and a relaxation delay of 1.5 s. Spectra were processed using XWIN-NMR software. All spectra were zero-filled in F 1 spectral dimension to 1024 data points and a sine bell window function was applied in both dimensions prior to Fourier transformation. The one-dimensional (1D) 1 H STD-NMR [14,23,24] spectra of the protein peptide complexes (Figs. 3 and 4) were recorded at 500 MHz with 2048 scans and selective saturation of protein resonances at 3 ppm (30 ppm for reference spectra) showing that by irradiating at d = 3 ppm, the entire protein can be saturated uniformly and can therefore be efficiently used for the STD-NMR technique. For the on-resonance irradiation frequency values around 3 ppm are practical because no ligand nuclei resonances are found in this spectral region whereas the significant line width of protein signals still allows selective saturation. Investigation of the time dependence of the saturation transfer with saturation times from 0.2 to 4.0 s showed that 2 s was needed for efficient transfer of saturation from the protein to the ligand protons. In order to achieve the desired selectivity and to avoid side-band irradiation, shaped pulses are employed for the saturation of the protein signals. STD-NMR spectra were acquired using a series of 40 equally spaced 50 ms Gaussian-shaped pulses for selective saturation (then, a total saturation time was of approximately 2.0 s) [25], with 1 ms delay between the pulses. With an attenuation of 50 db, the radiofrequency field strength for the selective saturation pulses in all STD-NMR experiments was 190 Hz. The irradiation yields full saturation of the protein by efficient spin diffusion. Subtraction of FID values with onand off-resonance protein saturation was achieved by phase cycling. For STD experiments (Figs. 3 and 4) the ligand to protein ratio was raised to 40:1 (0.2 mm peptide, 50 mm GST b- TrCP protein). Transferred nuclear Overhauser effect (TRNOESY) [6] spectra of the b-trcp peptide complex were recorded with 4 K points and 512 t 1 increments, and a relaxation delay of 1.5 s. Data processing was performed by zero filling to 1 K points in F 1 to give a final 4 K 1 K matrix followed by multiplication with a squared cosine function and Fourier transformation. The bound and free states exist in the same regime, namely vt c > 1; thus the cross-relaxation rates of the P1 and P2 peptides (MW = 1110 and 1066 g/mol) in the free and bound state are negative in sign. The free molecules exhibit very small negative NOEs. The optimal conditions for the TRNOESY measurements were determined by considering a peptide:b-trcp molar ratio ranging from 10 to 100 with mixing times (t m ) of 100, 200, 400 and 500 ms. The buildup curve [17] for different NOE correlations showed that spin diffusion was negligible for a t m of 200 ms. After optimization of the peptide: protein ratio, the final NMR sample was prepared with 3 mg of GST b-trcp protein (0.05 mm) and 1.1 mg of peptide (2 mm), which corresponds to a peptide:binding site ratio of 40:1, in 500 ml of buffer solution at ph 7.2. The observed TRNOE correlations with mixing times of 100 and 200 ms were large and negative. They were assigned to the b-trcp-bound ligand [9] as a sample of the peptide without the presence of b-trcp protein exhibited only intra-residue and sequential NOE intensities with a mixing time of 200 ms. Integration of cross-peak volumes was performed using FELIX software (Accelrys). Cross-peak intensities were converted to interproton distances using the distance between the Ile C b H 2

5 198 peptides 27 (2006) Table 1 1 H and 13 C NMR chemical shifts of the P1 and P2 peptides bound to the protein b-trcp in ppm from TSP-d 4 a Residue dnh dh a dh b dh g dh d dothers dc a dc b dc g dc d (CH 3 CO) 2.03 Leu ; ; 23.3 Ile ; ; ; 12.2 Glu ; Arg ; ; 6.99; ; 42.1 Ala Glu ; Asp ; pser ; Gly (CONH 2 ) 7.20; 7.45 Residue dnh dh a dh b dh g dothers dc a dc b dc g (CH 3 CO) 2.06 Glu ; Asp ; pser Gly Asn ; ; Glu ; pser ; Glu ; ; (CONH 2 ) 7.17; 7.68 a Spectra were recorded at 280 K, ph 7.2, P-Vpu/b-TrCP = 10 and 20 mm sodium phosphate buffer, H 2 O: 2 H 2 O 9:1 (v/v). Resonances marked by a dash were not visible. protons (1.76 Å) and between the Asn NH 2 protons (1.7 Å) asa reference for P1 and P2, respectively (Table S5 in Supplementary data). TRNOE cross-peaks classified as strong, medium, and weak were converted into distance restraints of , and Å, respectively Structure calculations Distance restraints used in the structure calculations were derived from TRNOESY experiments performed with mixing times of 100 and 200 ms, and as for the free ligand, we obtained a blank TRNOESY spectrum at 100 ms and only intra-residue and sequential NH-Ha correlations at 200 ms. The final list of distant restraints (Table 2) containing 75 unambiguous and 12 ambiguous (42 intra-residue, 26 sequential, 10 medium-range and 9 long-range) restraints for P1 and 86 unambiguous and 6 ambiguous (36 intra-residue, 13 sequential, 20 medium-range and 17 long-range) restraints for P2 was incorporated for structure calculation with the standard protocol of ARIA 1.2 [4,20]. Modifications of the phosphorylated residues (pser) were introduced with CHARMM [3] for molecular dynamics calculations using the PARALLHDG 5.3 force field. During the calculations, non-glycine residues were restrained to negative f values (usually the only range considered in NMR-derived structures) [33]. The simulated annealing protocol consisted of four stages: a high-temperature torsion angle simulated annealing phase at 10,000 K (30 ps), a first torsion angle dynamics cooling phase from 10,000 to 50 K (15 ps), a second Cartesian dynamics cooling phase from 2000 to 50 K (27 ps), and a final minimization phase at 50 K. ARIA enabled the incorporation of ambiguous distance restraints and calibration of the NOE restraints using automated matrix analysis as implemented by the program. To differentiate between the probable contributions for each of the ambiguous NOEs, the automated assignment program ARIA was used. Various runs were performed to utilize as many unambiguous and ambiguous restraints as possible from the 500 MHz H 2 O TRNOESY spectra. ARIA runs were performed using the default parameters with eight iterations. Twenty structures were generated each round, and the 10 lowest-energy structures were carried on to the next iteration. In the final iteration, the 20 lowest-energy structures were retained as the final structures. For the free peptides only, a procedure with explicit solvent molecules incorporated during the final run was selected using a box of water molecules with ARIA software (OPLS force field). It was decided that due to the presence of the bound peptide in the hydrophobic recognition sites this step was not applicable in the bound state. The set of P1 and P2 structures was selected for correct geometry and no distances restraint violations of >0.5 Å. Analysis of the structures was performed within Aqua, procheck-nmr (30) programs (Table 2). MOLMOL [15] was used for the analysis and presentation of the results of the structure determination (Table 3). 3. Results The recombinant protein includes the 218 residues of the GST protein fused to the seven WD repeats (residues ) of full-length protein b-trcp by an 18 residues linker. The GST fusion partner, indeed, enhances the solubility and stability of

6 peptides 27 (2006) Fig. 3 (a) 1D 1 H spectrum (bottom) and 1D 1 H STD-NMR spectrum (top) of the P1 peptide in association with the GST b-trcp protein, showing enhancements of resonances of protons making close contacts with the protein combining site; the GST b-trcp protein displayed few broad signals (asterisk) at ppm. (b) Expansion of the region containing resonances of the amide protons. the WD domain and provides a method of immobilizing the protein on a glutathione-derived matrix. The obtained protein, which was used to perform NMR interaction experiments, is hereafter referenced as GST b-trcp, and its molecular weight is approximately 60 kda NMR resonance assignments 1 H chemical shifts and resonance assignments were established using two-dimensional 1 H, 1 H TOCSY and NOESY experiments [51] and are reported in Table 1 and Table S4 in Supplementary data. Sequential assignments of 1 H resonances were based on characteristic sequential NOE connectivities observed between the a-proton of residue i and the amide proton of residue i + 1, i.e. d an (i, i + 1) in NOESY data set. Upon binding of a ligand to a receptor protein, the chemical shifts of both the ligand and protein proton resonance signals are affected. Addition of GST b-trcp caused a line broadening of the P1 and P2 signals, and a chemical shift difference relative to the free peptides (Fig. S11 in Supplementary data), thus providing a clear indication of the existence of binding. Interestingly, in the presence of b-trcp protein, a slight lowfrequency (shielded) shift of the Ha protons was observed in the N-terminal region of P1 and in the region of the P2 peptide. An opposite shift was observed with a large highfrequency (unshielded) shifted HN resonance, particularly for pser52 in both peptides, and a slight high-frequency shift for Glu47, Arg48, Asp51 in P1 peptide and for pser56, Glu57 in the P2 peptide. These shifts may be an indicator of intermolecular contact of the peptides with the binding site. The interaction caused environmental changes on the peptide protein interfaces and hence, affected the chemical shifts of the nuclei in this area. Fast exchange was measured for the interaction of the phosphorylated peptides to the GST b-trcp protein with a dissociation constant estimated between 500 mm and 1 mm [28,43]. This range of binding affinity made the peptides likely to be suitable for TRNOESY NMR experiments, which require fast exchange between the free and bound states b-trcp binding site for P1 and P2 To provide additional information regarding the peptide mode of binding, STD-NMR experiments were performed [14,23,24]. The STD-NMR experiment was initially applied to screen small peptides of Vpu protein for binding activity towards the b-trcp binding protein. Resonances of the protein are selectively saturated, and in a binding ligand, enhancements are observed in the difference (STD-NMR) spectrum resulting from subtraction of this spectrum from a reference spectrum in which the protein is not saturated [14,23,24]. Protons of the

7 200 peptides 27 (2006) Fig. 4 (a) 1D 1 H spectrum (bottom) and 1D 1 H STD-NMR spectrum (top) of the P2 peptide in association with the GST b-trcp protein, showing enhancements of resonances of protons making close contacts with the protein combining site; the GST b-trcp protein displayed few broad signals (asterisk) at ppm. (b) Expansion of the region containing resonances of the amide protons. ligand, which are in close contact with the protein, can easily be identified from the STD-NMR spectrum, because they are saturated to the highest degree. They should have stronger STD, and this allows direct observation of areas of the ligand that comprises the epitope. With regards to the spin diffusion, in the range of the saturation times that we used (from 0.2 to 4.0 s), we observed similar relative results for all the protons. The global intensity of all the signals was modified consistently, indicating that there was no visible spin diffusion under these conditions. It has also been shown that epitope mapping is possible if the ligand can leave the binding site before all magnetization has been equally distributed among all spins in the ligand [24]. In this case, we can say that there is no visible spin diffusion within the peptide. This condition is fulfilled for ligands with a high turnover number. Since (i) we work with a large excess of peptide, and (ii) our peptide is a weakly binding ligand with dissociation constant in the range of the mm, the turnover number, or dissociation rate constant, is high enough to map the interaction. For this reason, we do not observe spin diffusion within the peptide. We investigated the interaction of the P1 and P2 peptides with b-trcp protein. The resulting STD-NMR spectra in the presence of the protein clearly indicated the binding of the two peptides. Figs. 3 and 4 shows 1D STD spectra and normal 1 H spectra of the complexes of P1 and P2 with the b-trcp protein. 1D 1 H NMR spectrum of P1 and P2 in the presence of the protein displayed few broad signals at 3.0 and ppm, normal for a protein. The different signal intensities of the individual protons are best analyzed from the integral values in the reference (I 0 ) and STD spectra (I 0 I sat ), respectively. The integral value of the largest signal of P1 and P2 peptides, the Asp51 H N proton, was set to 100% (Fig. 5). The relative degree of saturation for the individual protons normalized to that of the Asp51, can be used to compare the STD effect [24].The1D spectra of the P2 peptide in a 40-fold excess over the GST b- TrCP protein show clearly that the pser52 (41%) and pser56 (77%) H N resonances, and other DpSGNEpS motif resonances belonging to Glu50 (52%), Asp51 (100%), Gly53 (60%), Asn54 (87%), Glu55 (60%), or Glu57 (51%) have STD intensities between 50 and 100% (Fig. 5). Thus, they are distinctly involved in binding. On the other hand, the functional groups involved in the binding of the ligand P1 to the GST b-trcp protein, the H N resonances of Leu45 (62%), Ile46 (56%), Arg48 (58%) and Ala49 (74%) have similar larger STD intensities, ranging from 50 to 75%, indicating that these residues are involved in direct binding to the protein, in addition to Glu50 (84%), Asp51 (100%), pser52 (78%) and Gly53 (68%). Interestingly, for P1 and P2 peptides, the amino acids in common, Asp51, pser52 and Gly53 received a similar fraction

8 peptides 27 (2006) Table 2 Structural statistics of the final 10 NMR structures of P-Vpu peptides, P1 and P2 bound to the b-trcp protein ARIA output P1 P2 No. of experimental distance restraints Unambiguous NOEs Ambiguous NOEs 12 6 Total NOEs Intra Sequential Medium-range Long-range 9 17 No. of experimental broad dihedral restraints 8 6 RMS differences from distance restraints a Unambiguous NOE (Å) Ambiguous NOE (Å) All NOEs (Å) NOE violations >0.5 Å 0 0 NOE violations >0.3 Å 0 0 RMS differences from mean structure b (Å) Backbone Heavy Ramachandran plot of residues c In most favored regions In additional allowed regions In generously allowed regions 0 0 In disallowed regions 0 0 a Calculated by ARIA. b Calculated by MOLMOL. c Calculated by PROCHECK. of saturation. The saturation transfer also presents maximum intensity for the acetyl protons of N-terminal and the NH amide protons of C-terminal (Figs. 3 and 4). This indicated the proximity of these protons to the protein surface. For the side chains of Arg48 (Fig. 3), and Asn54 (Fig. 4), the signals of the NH protons have similar large STD intensities. The signals observed in the 1D STD-NMR spectra revealed that the whole P1 and P2 peptides could be in intimate contact with the protein. Optimization of the experimental set-up for STD-NMR spectroscopy was achieved using samples without GST b- TrCP protein. In that case, STD spectra did not contain ligand Table 3 Secondary structure analysis and dihedral angles for the bound structures with lowest energy calculated by MD calculations with ARIA, for the P1 and P2 peptides in complex with the protein b-trcp a Residue Structure f c x 1 Leu45 Coil Ile46 Turn Glu47 Turn (helix_3_10, helix1) Arg48 Turn (helix_3_10, helix1) Ala49 Turn (helix_3_10, helix1) Glu50 Bend Asp51 Bend pser52 Coil Gly53 Coil 83.4 Glu50 Coil Asp51 Bend pser52 Bend Gly53 Bend Asn54 Bend Glu55 Bend pser56 Coil Glu57 Coil a The program MOLMOL was used for the analysis and presentation of the results of the structure determination.

9 202 peptides 27 (2006) Fig. 5 Mean STD values (in percent) of the amide protons of the individual amino acids calculated for each amino acid of P1 and P2 from the 1D spectrum. signals, because saturation transfer does not occur without the protein. Another experimental way to distinguish here between specific effects of binding peptide to its target and non-specific interactions between ligand and macromolecular complex, was to use in control experiments, the binding peptides, P1 and P2 with the glutathione S-transferase lacking the seven WD domain of b-trcp protein. However, the C- terminal fragment of b-trcp with the seven WD repeats was required for binding to Vpu. Thus, the negative control provided by recording STD-NMR experiments in the presence of GST protein show clearly the specific binding of the peptide ligands, P1 and P2 to the GST b-trcp protein. Fig. 6 (a) Sequential dnn(i, i + 1), dan(i, i + 1), dbn(i, i + 1), dab(i, i + 1) and medium-range dan(i, i + 2), dan(i, i + 3), dab(i, i + 3) and dan(i, i +4) 1 H 1 H TRNOE connectivities in the peptides P1 and (b) P2 (sequence at the top) in the presence of the GST b-trcp protein at 280 K and ph 7.2. The thickness of the lines reflects the relative intensities of the NOEs within the individual plots Bound conformation of P1 and P2 TRNOESY experiments [5,6] were used to investigate the bound conformation of the peptides. The free peptides exhibited only small negative intra-residue and sequential NH Ha correlations, when the mixing times was 200 ms and negative NOE connectivities only for long mixing times (t m 500 ms) in aqueous solution and in the absence of b- TrCP protein. This fact is in agreement with their conformational studies showing that these peptides do not adopt any favored structures at all, free in solution. The molar ratio of P1 and P2 ligand molecules to b-trcp receptor protein was investigated from 10 to 100, and the optimal conditions for the TRNOESY measurements were observed for a ratio near 40:1. Multiple connectivities are negative (TRNOEs) and can be assigned to the bound conformations of low-molecular weight peptides. Since the peptides are in fast exchange on the cross-relaxation time scale of the bound peptides, the observed TRNOESY intensities are sums of the bound and free peptide NOEs. Fast exchange was measured for the interaction of the phosphorylated peptides to the GST b-trcp protein with a dissociation constant estimated between 500 mm and 1 mm. In this study, analysis of TRNOEs was simplified since NOEs, which were present for the free peptides, were smaller than those for the bound state. With short mixing times ( ms), free peptide NOEs represent little uncorrected NOEs because of the longer mixing times ( ms) used in the free state analysis. A negative control with only sequential NH Ha correlations provided by recording NOESY spectra of the peptides in the presence of GST protein showed that the large number of negative NOEs observed in the presence of the fusion GST b- TrCP protein was due to transfer from the bound peptides (transferred NOE). A summary of sequential d(i, i + 1) and medium range d(i, i + 2) and d(i, i +3) 1 H 1 H TRNOE connectivities is presented in Fig. 6 and Table S5 in Supplementary data. In the P1 TRNOESY spectra, it is interesting to show the propensity for a turn I46 A49 region. The pattern of TRNOE connectivities in the bound P1 TRNOESY spectra suggests that a turn is present in the N- terminal portion of the peptide. Turn structure in this segment is implicated by the density of d an, I46 E47, E47 R48, R48 A49 and d NN, E47 R48, sequential TRNOEs, and the strong longrange TRNOE such as an(i, i + 3), an(i46, A49). Side chain-side chain TRNOEs between the bch 2 protons of Glu47 and gch 2 protons of Leu45 and Ile46 and between the bch 2 protons of Arg48 and bch 2 protons of Glu47 and Ala49 have been observed. On the other hand, the presence of medium an(i, i + 1), an(r48, A49), an(a49, E50), an(e50, D51), an(d51, ps52), an(ps52, G53) cross-peaks, intense bn(i, i + 1), bn(a49, E50) connectivities and medium long-range an(i, i + 4), an(a49, G53), cross-peak indicated that the region presents predominantly a bend structure. The P2 TRNOE spectrum exhibits a great number of NOEs including intense NN(E55, ps56), medium NN(pS52, G53),

10 peptides 27 (2006) NN(G53, N54) NN(N54, E55) and weak NN(D51, ps52), NN(i, i + 1) connectivities (Fig. 6) suggesting the presence of secondary structures (a- or b-turns) for the DpSGNEpS motif. The presence of intense an(i, i + 1), an(i, i + 3) and intense ab(i, i + 3), TRNOE connectivities, in addition to medium range bn(i, i + 1), an(i, i + 2) and an(i, i + 4), TRNOE connectivities also denote the presence of secondary structures. These peaks argue in favor of a folded structure for the DpSGNEpS sequence, which includes the pser-phosphorylated site. The structure was well defined in the region from Glu50 to Glu57, Fig. 7 NMR TRNOE-derived structures of the bound peptides in the presence of GST b-trcp protein. (a) Superimposition of 10 structures generated after eight iterations with ARIA software for P1 and (b) for P2. (c) Energy minimized conformer with the best fit of proton distance constraints for P1 and (d) for P2.

11 204 peptides 27 (2006) where a bend was apparent. Strong an contact between E50- D51, D51-pS52, G53-N54, N54-E55, ps56-e57, G53-pS56, medium an contact G53-E55, D51-E55, ps52-ps56 and other interresidue contacts such as ab(g53, ps56), and bn(n54,e55), bn(e55,s56), helped to define this bend. To study the conformation of the bound state of P1 or P2 in the presence of the GST b-trcp protein, the distance restraints were incorporated into a simulated annealing protocol using ARIA. The structures that were generated resulted in NOE restraint files consisting of 87 and 92 restraints, for P1 or P2, respectively (Table 2). A set of 20 structures produced by simulated annealing was subjected to energy minimization, followed by checks for correct geometry and agreement with the distance restraints (Fig. 7). The structural models fit the NMR data well, with no violations of experimental distance restraints greater than Å. The positions of the backbone (Table 3) and most side chain atoms were well defined by the NMR restraints. Structural statistics are presented in Table 2. Structure calculations by simulated annealing using NOE constraints followed with refinement and energy minimization led to the family of 10 structures shown in Fig. 7a and b for P1 and P2, respectively. TRNOE NMR studies of P1 peptide in the presence of GST b-trcp protein showed, for the bound conformation of the peptide, a propensity for turn formation in N-terminal residues 46 49, and a short bend including the EDpS 52 part of the phosphorylation motif with the phosphate group pointing away (Fig. 7c; Table 3). The P2 calculated structures (Fig. 7d; Table 3) comprise a large bend from Asp51 to Glu55. These structures would expose the pser side chains for interaction with the GST b-trcp protein, a hypothesis consistent with the STD- NMR data. The average root mean square difference for superimposition of the 10 structures with the lowest NOE restraint energy was 0.4 Å for the backbone atoms (N, C a,c,o) of residues The 10 P2 structures superimposed with RMSD = Å for backbone atoms considering the entire peptide. The 10 structures of the P1 peptide (Table 2) superimposed with the same RMSD. 4. Discussion By interfering with cellular proteins such as b-trcp, Vpu probably has a major effect on various functions and signaling pathways in HIV-1-infected cells. As b-trcp also controls essential cellular signaling pathways by degrading b-catenin and IkBa substrates via the ubiquitin proteasome system, it was recently shown that Vpu is a competitive inhibitor of b- TrCP that impairs the degradation of SCF b-trcp substrates as long as Vpu has an intact DpSGNEpS phosphorylation motif and can bind to b-trcp protein [2]. The two ligands with moderate affinity, P2 peptide, centered on the doubly phosphorylated motif and P1 peptide, are essential tools in defining the interactions of Vpu Bound conformation of the P1 and P2 peptides to the protein b-trcp From TRNOE NMR studies and the P1 simulations it can be concluded that the residues involved in the peptide retain a reasonable turn motif (Fig. 7a and c). The motif L 45 IERA 49 has a stabilizing effect on the helical conformation within distances of less than 0.2 nm between each of the residues, and with two hydrophobic residues, Leu45, Ile46 preceding the charged Glu 47 Arg 48 motif. Arg48 is further able to support the loop via the formation of salt bridge with Glu50 and Asp51. On the other hand, TRNOE NMR studies of P2 peptide in the presence of GST b-trcp protein showed that the bound conformation of the peptide is a bend conformation from residues Asp51 to Glu57 (Fig. 7b and d). This bend would expose the side chains of residues for specific interactions with the b-trcp protein combining site that is consistent with the STD-NMR data, showing enhancements of the residues in the same region, between 50 and 100% (Fig. 5), and with the chemical shift variation involving residues of this loop region (Fig. S11 in Supplementary data) Comparison with the longer P-Vpu peptide The SCF b-trcp complex specifically recognizes a Vpu peptide fragment of 22 amino acids [22], the P-Vpu peptide (Fig. 8a), of which the bound structure to the b-trcp protein was previously determined by NMR and MD [8]. After superimposition of the two bound P1 and P2 peptides and the corresponding fragments in the b-trcp-bound P-Vpu peptide (Fig. 8b and c), the RMSD values of their backbone coordinates are 0.35 and 0.31 Å, respectively. The bend (DpSGNEpS) is similarly found in the b-trcp-bound P2 and P-Vpu peptides [8], as shown in Fig. 8c. The phosphorylated pser 52 residues would then be able to dock to b-trcp (Fig. 8b and c) while the pser 56 phosphate groups are pointing away involving probably large dynamical motion (Fig. 8c). In the same way, the turn (LIERAEDpSG) was observed in the P1 and P-Vpu peptides bound to b-trcp (Fig. 8b). The bound conformation of the P1 peptide falls partly into the same regular a-helix h2 of the bound phosphorylated peptide, P-Vpu Then, their pser 52 residues are fitted and are free to point into protein combining site Comparison with the crystal structure of the complex b-trcp-b-catenin peptide We highlighted that interaction of the P2 small peptide relies on the DpSGXXpS motif, similar to that found in the other substrates of b-trcp (entire P-Vpu, IkBa and b-catenin). Indeed, the b-turn motif plays a central role in the crystal structure (Fig. 9a) of the human b-trcp1-skp1 complex bound to a fragment b-catenin substrate peptide (Fig. 2c) [50];oftheb-Catenin motif in the crystals, only an 11 residue segment (residues 30 40), centered on the doubly phosphorylated motif (DpS 33 GIHpS 37 ), makes the largest number of b-trcp contacts (Fig. 9a). As the hydrophobic region upstream of this motif is not present in the crystal structure, its possible participation in the binding was not observed. Therefore, it was interesting to highlight the similarity between the two bound P1 and P2 peptides and the corresponding fragment in the b-trcp-bound b-catenin peptide (Fig. 9b and c); after superimposition of the DpSG motif of the two P1 and P2 peptides and the corresponding X-ray fragment, the RMSD values of their backbone coordinates are 0.42 and 0.40 Å, respectively. In the P1 and P2

12 peptides 27 (2006) Fig. 8 (a) NMR TRNOE-derived structure of the P-Vpu bound peptide in the presence of GST b-trcp protein. (b) Superimposition of the NMR bound peptides P-Vpu and P1. The structures are fitted from residue 46 to 52. (c) Superimposition of the NMR bound peptides P-Vpu and P2. The structures are fitted from residue 51 to 55. peptides, the phosphoserine, aspartic acid, and hydrophobic residues of the DpSG motif are recognized directly by contacts from b-trcp. Again, it appears that the DpSG segment forms a bend very preserved with a reduced mobility (Fig. 9b and c) whereas the final part of the phosphorylation motif (XXpS) seems more mobile b-trcp binding site for P-Vpu P1 and P2 binding region The STD-NMR studies of the P2 peptide in the presence of b- TrCP showed the involvement of the NH groups of the sixresidue sequence DpSGXXpS in binding. The NH and aliphatic groups interact strongly with the corresponding amino acids inside the paratope (STD intensities between 50 and 100%). In addition, for P1 peptide, the NH and aliphatic groups of Leu45, Ile46 residues are recognized (STD intensities, ranging from 55 to 65%). This result shows clearly that the P1 also carries a small portion of binding specificity of the entire Vpu ligand. The signal intensity of the P1 protons is compared to the increase of the signals for P2. The relatively similarity in STD effect (STD intensities between 50 and 100%) shows clearly that P1 and P2 ligands bind strongly to the receptor protein while making a distinction in the binding pocket between the proton involved from the different residues (Fig. 10). The known factor in binding is the phosphate group of the pser residue but the Asp, Glu, Arg and Asn residues also have a strong contact with the protein. In the case of Asp and Glu, the contact is through the carboxyl groups whereas in the case of the Leu or Ile, it is a hydrophobic contact. The Gly also participates in binding for the two peptides with similar larger STD intensities, ranging from 60 to 68% (Fig. 5).

13 206 peptides 27 (2006) Fig. 9 (a) Close-up view of the doubly phosphorylated DpSGIHpS motif bound, from the crystal structure of the human b-trcp-skp1 complex bound to a b-catenin substrate peptide and recognized by the F-box protein b-trcp [50]. Superimposition of the DpSG motif of the P-b-catenin X-ray crystal structure and the corresponding residues (b) of the P1 bound structure and (c) of the P2 bound structure. The NMR data described above show that the epitope comprises a surface extending over residues of the bend DpS 52 GNEpS 56 motif of the bound P2 peptide associated with the hydrophobic cluster (Leu45 Ile46). Leu45 and Ile46, whose hydrophobic nature is conserved in the P1 peptide fragment, are able to make van der Waals contacts with a hydrophobic pocket that would be composed of the aliphatic portion of the Val516 and Phe523 side chains in b-trcp (Fig. 10a). This turn hydrophobic region is able to project Asp51 and pser52 into a three-sided pocket on the WD40 surface (Fig. 10a). This is clearly important here as is shown by the epitope mapping data (STD-NMR) where Leu45 and Ile46 hydrophobic side chains along with negative charged (Asp, pser and Glu) side chains contact the site. The fact that a Leu or Ile was close to the known DpSGXXpS binding fragment enhanced interaction of the Vpu ligand to b-trcp protein The DpSG motif in the binding site of the b-trcp as WD domain protein The C-terminal domain of b-trcp contains seven WD repeats (Fig. 2b), known to form interfaces for protein protein interaction [27], and required for optimal binding to Vpu. Modeling of the F-box protein b-trcp [52] reveals an extensive basic region on the front face of the propeller, which may engage substrate phosphoepitopes. The b-trcp WD domain has the ability to recognize pser epitopes in the context of the

14 peptides 27 (2006) Fig. 10 A portion of the seven b-propeller structure of the WD40 repeat region of the yeast F-box protein b-trcp is shown based on the crystal structure of the complex (b-trcp-skp1 b-catenin peptide) [50]. The residues of the protein surface are colored in yellow according to positive electrostatic potential and hydrogen bonding. (a) Superimposition of the DpSG fragment from P1 peptide (in orange) and the X-ray crystal structure of the b-catenin peptide (in green) also highlights similarities for the b-trcp combining site protein between Vpu and b-catenin protein concerning the first phosphate group of pser residue of the DpSGXXpS bound motif, pser52 and pser33. Interestingly, Leu45 and Ile46, whose hydrophobic nature is conserved in the P1 peptide fragment, made van der Waals contacts with a hydrophobic pocket composed of the aliphatic portion of the Phe523, and Val516 (WD7) side chains in b-trcp (in blue). (b) Superimposition of the DpSG fragment from P2 peptide (in magenta) and the X-ray crystal structure of the b-catenin peptide (in green) highlights similarities for the b-trcp combining site protein between Vpu and b-catenin protein concerning the first phosphate group of pser residue of the DpSGXXpS bound motif. pser52 and pser33 are able to make electrostatic interaction with the positive Arg285 (WD1) and some hydrogen bonds with the side chain hydroxyl groups of Tyr271, Ser309 and Ser325 (WD1), while pser56 and pser37 have a different location, pser56 is close to Lys365 (WD3) and pser37 to Arg431 (WD5). A second broader site is formed by a positive charge distribution on the surface (Arg367, Arg390, Arg431 and Lys365) able to bind the second pser. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

15 208 peptides 27 (2006) adjacent Gly residue while other WD domain employs a pthr Pro peptide [26,29]. The Gly or the Pro binding pocket is able to accommodate other residues with a same propensity to form b turns. The specificity of phosphorylation-recognition by the WD domain of b-trcp is characterized by a dedicated pser Gly binding pocket that selects residues N-terminal to the phosphorylation site. The X-ray crystallographic analysis of b-trcp protein complexed with a fragment b-catenin substrate peptide [50] reveals the binding site specific for a phosphopeptide complex bound to an seven-bladed WD40 propeller domain (Fig. 9a), and that nearly all of the b-trcp contacts are made by the six residue psgihps motif. However, the fragment in the complex is too short to highlight a hydrophobic interaction upstream (Fig. 9b). Interaction of HIV-1 Vpu with b-trcp relies on motif DpSGNEpS similar to that found in the other substrates of b- TrCP (IkBa and b-catenin). It is interesting to note that the first serine residue of this motif seems to play an essential role in this interaction. The second serine residue is involved in the case of Vpu and b-catenin but not required for the interaction of ATF4 with b-trcp (ATF4, a member of the family of transcription factors). Interaction of ATF4 with b-trcp relies on motif DSGXXXS [18]. The P2 bound structure corresponds to the model s prediction of substrate recognition by the b-trcp1 WD40 domain. b-trcp could associate with P2 peptide via chargebased interactions. In the P2 peptide, except for Gly53, all the chain residues, in the EDpSGNEpSE bend form a negatively charged surface that would provide a plausible binding region in contact with the positive protein b-trcp surface. The WD domain of b-trcp has positive amino acids, Arg and Lys (Fig. 10b) that could accept the diphosphorylated segment, DpSGXXpS present in the Vpu, b-catenin and IkBa protein. In Vpu, the phosphate group of pser52 is able to make the largest number of contacts, in forming direct hydrogen bond with the side chain hydroxyl groups of Tyr271, Ser309 and Ser325 in the WD domain of b-trcp protein. The phosphate group forms also direct electrostatic interactions with the guanidium group of Arg285 (Fig. 10b). This creates a hydrogen bond network in the protein around the phosphate group, and can explain the high saturation transfer towards the phosphate group of pser52. On the basis of previous mutational analysis [26,36,41], the Arg residues are essential for function. Study of mutations made it possible to identify the binding site of b-catenin and IkBa for b-trcp [54]. The three Arg285Glu, Arg474Glu and Arg521Glu mutations block the interaction with b-catenin. Asp51, which is an invariant binding motif residue, is also able to make an extensive contact as its side chain allows a hydrogen bond with Arg521, Arg474 and Tyr271 in the WD domain of b-trcp protein. The Asp51 residue gives the higher relative saturation transfer (STD signal 100%) for the two P1 and P2 bound peptides (Fig. 5) corresponding to a tight contact to the b-trcp protein surface. Gly53, also an invariant binding motif residue, is able to pack with the b-trcp receptor in an environment with little space for a non-glycine residue. The conservation of the Gly53 residue is also justified by the need for compact side chains not to disturb the position of Leu331 and Leu351 of b-trcp protein. The position of the XX residues in the DpSGXXpS motif, located above the central channel of the seven-bladed WD40 propeller domain and, without particular implication of the side chains, can explain the variability of the residues to these positions in the phosphorylation motif. The second pser, is less embedded what can explain its role perhaps less essential in the interaction, but its original orientation in the bound Vpu can explain a better interaction of this ligand (Fig. 10b). The binding site seems to be enough restricted to select the DpSG motif (Arg285, Arg474, Arg521 and some hydrogen bonds) since this part of the motif is relatively hidden and a second broader site formed by a positive charge distribution on the surface (Arg367, Arg390, Arg431 and Lys365) laid out around the second pser. The multiplicity and the proximity of these charges can also explain the existence of longer phosphorylation motifs (DpSGXXXpS or DpSGXXXXpS). The lengthening of the phosphorylation motif introduced a great mobility of the terminal part of the motif, what can attenuate the affinity of the ligand for the receptor. Nevertheless, it is possible that the second pser carries out an electrostatic interaction with one of the other positive charges accessible on the b-trcp protein surface. This positive charge distribution can also highlight on the best affinity of P-Vpu compared to the other ligands. Indeed P-Vpu is the only protein containing a negative charge (Glu) after the second pser among the other ligands (Fig. 10b). Its implication in the binding is consistent with the STD-NMR data (Fig. 5) and the variation of the chemical shift of the Glu57 residue (Fig. S11 in Supplementary data). The negative potential generated by the phosphorylated Ser52 and Ser56 increases with Glu57 (and Glu55). A second negative pole around pser56 reinforces the Vpu binding to the b-trcp protein, which was mainly characterized by the first pser, pser52 and Asp51 in close proximity. 5. Conclusion The approaches discussed in this study allow screening of compounds as well as a detailed identification of the fragments involved in the binding events. The Vpu analog peptides, P1 and P2 were characterized efficiently by NMR to bind to the b-trcp WD domain. The two peptides exhibit different secondary structure characteristics in interaction with b-trcp. P2 exhibits a structure with a large bend at DpSGNEpS and P1 exhibits a turn type of structure at IERAED. The differences in the structures of peptides observed may contribute to the selectivity of the b-trcp receptor for Vpu analog peptides. In conclusion, we have shown that interaction in b-trcp protein of Vpu analogs leads to different possible structures but with the DpSG fragment conformation very conserved. A different orientation of the second pser for Vpu which is the only protein with the presence of one Glu residue containing a negative charge after the second pser emphasizes the interaction with the b-trcp protein, and this can also highlighted on the best affinity of P-Vpu compared to the other ligands. These structural differences may be important for the development of novel b-trcp receptor selective (ligands or inhibitors). This study indicates the important structural features responsible for a ligand s ability