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Báo cáo Y học: NMR structure of the HIV-1 regulatory protein Vpr in H2O/trifluoroethanol Comparison with the Vpr N-terminal (1–51) and C-terminal (52–96) domains pot

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NMR structure of the HIV-1 regulatory protein Vpr in H 2 O/trifluoroethanol Comparison with the Vpr N-terminal (1–51) and C-terminal (52–96) domains K. Wecker, N. Morellet, S. Bouaziz and B. P. Roques De ´ partement de Pharmacochimie Mole ´ culaire et Structurale, INSERM U266 CNRS UMR 8600, UFR des Sciences Pharmaceutiques et Biologiques, Paris, France The human immunodeficiency virus type 1, HIV-1, genome encodes a highly conserved regulatory gene product, Vpr (96 amino acids), which is incorporated into virions in quantities equivalent to those of the viral Gag protein. In infected cells, Vpr is believed to function during the early stages of HIV-1 replication (such as transcription of the proviral genome and migration of preintegration nuclear complex), blocks cells in G2 phase and triggers apoptosis. Vpr also plays a critical role in long-term AIDS disease by inducing viral infection in nondividing cells such as monocytes and macrophages. To gain deeper insight of the structure–function relationship of Vpr, the intact protein (residues 1–96) was synthezised. Its three-dimensional structure was analysed using circular dichroism and two-dimensional 1 H- and 15 N-NMR and refined by restrained molecular dynamics. In addition, 15 N relaxation parameters (T 1 , T 2 ) and heteronuclear 1 H- 15 N NOEs were measured. The structure of the protein is char- acterized by a well-defined c turn(14–16)-a helix(17–33)- turn(34–36), followed by a a helix(40–48)-loop(49–54)-a helix(55–83) domain and ends with a very flexible C-terminal sequence. This structural determination of the whole intact Vpr molecule provide insights into the biological role played by this protein during the virus life cycle, as such amphi- pathic helices are believed to be involved in protein–lipid bilayers, protein–protein and/or protein–nucleic acid inter- actions. Keywords:Vpr;NMR;HIV-1;helix;3Dstructure. The genome of the human immunodeficiency virus type 1, HIV-1, the causative agent of AIDS, encodes in addition to Gag, Pol and Env, several regulatory proteins such as Tat and Vpr (Fig. 1), which ensure rapid and efficient replica- tion of the retrovirus in infected cells [1]. Of particular interest is the protein Vpr, which is encoded late during viral replication by an ORF located in the central region of the viral genome. Vpr is essential for efficient viral infection of macrophages and monocytes [2] and plays an important role in the overall pathogenesis of AIDS [3]. Vpr is a small basic protein of 96 amino acids that is highly conserved among HIV-1, HIV-2 and SIV viruses. It is incorporated into viral particles in molar concentration through interactions with the C-terminal domain of Gag, and studies suggested that it plays a role in the immediate events following infection of permissive cells [4,5]. The C-terminal portion of the Gag precursor corresponding to p6 and particularly the motif (LXX) 4 appear to be essential for the incorporation of Vpr, and seem to interact with the N-terminal domain of Vpr [6]. In vitro, the (80–96) domain of Vpr forms a complex with the second zinc finger of the nucleocapsid protein NCp7 [7,8]. In vivo, the incorporation of Vpr into mature HIV-1 particles seems to occur by a process in which NCp7 cooperates with p6 [9]. In infected cells, Vpr is localized to the nucleus and has the ability to interact with several host cellular proteins [1]. Vpr has been implicated in the nuclear translocation of the preintegration complex [10–12]. The precise mechanism by which Vpr influences the transport of the preintegration complex remains unclear, as no classical nuclear localization signal has been clearly identified in Vpr. Recently, it has been shown that Vpr can interact with karyopherin a and the nucleoporin Nsp1, and thus seems to act as an importin- b-like protein [13,14]. The Vpr(1–39) domain of Vpr has also been shown to promote the initiation of HIV-1 reverse transcription by interacting with tRNA Lys,3 synthetase in its native state [15]. Moreover, Vpr has been reported to interact with Tat and perhaps facilitates the transactivating properties of this protein [16]. The ability to induce G2 cell cycle arrest is an additional biological property of Vpr [17–19]. This cytostatic effect of Vpr occurs by inhibiting the activation of p34cdc-cyclin B, and thus contributes to the immunopathogenicity of HIV [20]. Another cytotoxic effect of Vpr is its capacity to induce apoptosis [21], probably by interaction with proteins of the mitochondrial pore [22]. Vpr has been shown to enter in cells easily [23], where it can form cation-selective channels in planar lipid bilayers and induce large inward sodium flux thus resulting in membrane depolarization and eventual cell death as demonstrated in cultured rat hippocampal neuro- nes [24]. However, the exact molecular mechanisms of interaction between Vpr and other retroviral and host cellular proteins, Correspondence to K. Wecker, Unite de RMN des Biomolecules, De ´ partement des Re ´ trovirus et du SIDA, Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris, Cedex 15, France. Fax: 33 1 45 68 89 29, Tel.: + 33 1 45 68 88 73, E-mail: wecker@pasteur.fr Abbreviations: HIV-1, human immunodeficiency virus type 1; Vpr, viral protein of regulation; TFE, trifluoroethanol. (Received 16 January 2002, revised 23 April 2002, accepted 24 June 2002) Eur. J. Biochem. 269, 3779–3788 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03067.x which may requires distinct functional domains of the Vpr protein [19], remain unknown. It is our aim, in this study, to facilitate structural investigation of Vpr and gain a better understanding of structure-function relationships of this protein. Due to its cellular toxicity, Vpr cannot be obtained in large quantities using the classical cell transfection and Escherichia coli expression methods. Then, the structures of two synthetic peptides corresponding to the N- and C-terminal domains portions of Vpr have been previously determined using NMR, and provided valuable insights into the possible role of these two domains in Vpr functions [25,26]. The two isolated fragments were shown in vitro to interact with each other, however, it possessed lower activity compared to the intact Vpr protein as reflected in various interactions studies such as nucleic acid recognition [27], apoptosis [22] and Vpr-induced DNA transfection [23]. This prompted us to analyze the solution structure of the intact Vpr by circular dichroism, homonuclear and heteronuclear NMR techniques. MATERIALS AND METHODS Protein synthesis The entire Vpr protein was synthesized on an Automatic Applied Biosystems 433 A peptide synthesizer using the stepwise solid phase synthesis method and Fmoc amino acids, as described previously [28]. During peptide synthesis, 22 labeled amino acids (95% 15 N, 15% 13 C) were intro- duced: Thr19, Leu20, Leu22, Leu23, Leu26, Ala30, Phe34, Leu39, Gly43, Tyr47, Ala55, Ala59, Leu60, Ile61, Ile63, Leu64, Leu68, Phe69, Phe72, Gly75 and Thr89. Protein purification was carried out using reverse phase HPLC on a semipreparative Vydac C 18 column using a linear gradient of acetonitrile. An experimental mass of 11 431.92 Da was obtained by electrospray mass spectroscopy and a mass of 11 433.02 Da was calculated taking into account the labeled residues. NMR sample preparation Two NMR samples were prepared, as described previously [25,26], one with the native protein and the other with the labeled one. The final concentration of these samples was 1.0 m M at pH 3.4. Circular dichroism measurements Circular dichroism spectra were recorded on a Jobin–Yvon, CD 6 spectrodichrograph (Longjumeau, France), using a 1 mm path length cell. The experiments were recorded at 293 K with a 2 nm wavelength increment and accumulation time of 1 s per step. Each spectrum was obtained with a protein concentration of 2 m M in presence of 10 m M dithiothreitol and increasing trifluoroethanol (TFE) con- centrations (from 0 to 30%) at pH 3.4 or pH 6.0 (sodium phosphate buffer) as already described for (1–51)Vpr [25] and (52–96)Vpr [26]. Each spectrum, resulting from aver- aging of four successive individual spectra, was baseline corrected and smoothed using a third order least-squares polynomial fit. NMR experiments NMR experiments were recorded on a Bruker DRX600 and Bruker DRX800. Two dimensional homonuclear NMR studies were performed at 313 and 323 K. Homonuclear Hartmann–Hahn measurements (HOHAHA) [29] and Nuclear Overhauser Effect spectroscopy (NOESY) [30] were acquired in the phase sensitive mode using the time proportional phase increment (TPPI) method [31] or the states-TPPI method. The carrier frequency was set on the H 2 O resonance. NOESY experiments were recorded with mixing times of 50 and 200 ms. Spectra were processed using XWINNMR (Bruker) and FELIX 98.0 (Biosym/MSI, San Diego) on a Silicon Graphics O2 work station. Heteronuclear experiments were performed at 323 K. Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Single Quantum Coherence (HSQC) were performed using GARP sequence for decoupling during acquisition. Experiments were recorded on the phase sensitive mode using echo/antiecho gradient selection and trim pulses in inept transfer. A total of 256 FIDs (free induction decay) of eight scans were collected for each experiment. Two-dimensional 15 N-HMQC-TOCSY, HSQC-TOCSY and HMQC-NOESY, HSQC-NOESY (s m 200 ms) were recorded on the phase sensitive mode using TPPI method. Decoupling during acquisition with a GARP sequence and presaturation during relaxation delay (1.6 s) were used. A total of 256 FIDs with 128 transients were collected. The spectral width was set to 8 p.p.m. and 23 p.p.m. for 1 Hand 15 N, respectively. All dynamics experiments were performed at 323 K through the 22 15 N-labeled amino acids. Longitudinal relaxation times T 1 were obtained with delays of 5, 10, 50, Fig. 1. Primary sequence and CD spectra at 293 K of (1–96)Vpr. Upper panel: Primary sequence of (1–96)Vpr protein (14 KDa). The 22 15 N- and 13 C-labeled amino acids are in bold. The N-terminal sequence is enriched in negatively charged amino acids (D,E) while the C-ter- minal domain contains K or R positively charged residues. Lower panel: CD spectra at 293 K of a solution (2 · 10 )5 M ) of (1–96)Vpr in water solution (100% H 2 O) (solid line) and with 30% TFE (dotted line) at pH 3.4 (A) and pH 6.0 (B). The two maxima at 208 nm and 222 nm indicate that the protein is structured with a helices. 3780 K. Wecker et al. (Eur. J. Biochem. 269) Ó FEBS 2002 100, 200, 350, 500, 800, 1200, 3000 ms. The Carr Purcell Meilboom Gill (CPMG) sequence was used during the relaxation period with 15 N pulses applied every 460 lsinthe transverse relaxation experiments (T 2 ). T 2 values were obtained with delays 5, 10, 20, 30, 50, 60, 72, 80, 100, 120, 168, 200, 248, 300 and 400 ms. Heteronuclear 1 H- 15 N NOEs were measured from two experiments (24 transients of 128 increments and a 4 s recycling delay), with and without proton saturation. Relaxation rates were obtained from intensity fitting: I(t) ¼ I 8 +(I 0 ) I 8 )exp(–T 1 t). I 0 is the initial value of the resonance intensity and I 8 corre- sponds to the steady state value. Structure calculation Calculations were performed with the DISCOVER / NMRCHI- TECT software package from MSI with the Amber forcefield using a dielectric constant e ¼ 4r in order to diminish in vacuo electrostatic effects. NOE cross-signal volumes were converted into distances either by an r )6 dependency for well-resolved peaks or semi quantitatively by counting levels. The distances between H5 and H6 protons in Trp 18, 38 and 54 were used for calibration. Fifty structures were generated using a three-stage protocol, as described previ- ously [25] and the 20 best energy minimized structures with the lowest values for total energy and NOE restraint violations were analysed with respect to the rmsd values of the backbone. The structural stability has been examined under minimization and dynamics (300 K) without NMR constraints. RESULTS Circular dichroism CD spectra of the protein in 100% H 2 O (Fig. 1) are characteristic of ordered conformations as illustrated by the two molar ellipticity minima at 208 and 222 nm [32] regardless of which pH was used. The addition of TFE, known to stabilize secondary structures and to disrupt aggregates [33], enhanced negative molar ellipticity without modifying general aspect of the spectra, especially for the solution at pH 6 (Fig. 1). The increase in mean molar ellipticity depicts the stabilization of the pre-existing a helical structures. The degree of helicity, estimated from the ratio between the intensities of the bands at 222 and 208 nm, were 81, 85, 83 and 82% for the solution at pH 6.0, 0% TFE; pH 6.0, 30% TFE; pH 3.4, 0% TFE; and pH 3.4, 30% TFE, respectively. Taking into account the small errors found in percentage determinations, the helical folding of the protein does not appear to be significantly different under these conditions. This result confirms that the addition of TFE does not induce a helical formation but rather stabilizes the pre-existing secondary structures. 1 H- and 15 N-NMR experiments of Vpr All attempts to solubilize Vpr at minimal concentrations for NMR studies in H 2 O at pH 6 failed. At this concentration ( 100 times higher than that used for CD experiments), it was impossible to prevent aggregation even in the presence of 30% TFE. In light of these results, we decided to study the structure of Vpr in the following conditions (where the protein was most soluble): 1 m M aqueous solution, pH 3.4 and in the presence of 30% TFE-d 2 . Preliminary 1D proton NMR experiments were performed at different tempera- tures ranging from 293 to 323 K, in order to determine the best conditions for NMR studies (Fig. 2). Two tempera- tures were selected: 313 and 323 K. Proton assignments were obtained using the strategy developed by Wu ¨ thrich and coworkers [34], supplemented with information from heteronuclear experiments. Unambiguous resonance assign- ments of the 22 labeled residues were obtained from 2D HSQC, HSQC-TOCSY and HSQC-NOESY at 313 and 323 K (Figs 3 and 4). Clean TOCSY and E-COSY experiments allowed for spin system identification and NOESY cross peaks, connecting HN, Ha and Hb of residue i with NH of residue i + 1, were used for sequential assignment [35]. Thus, a complete chemical shift assignment of the backbone and side chain protons was achieved for the 96 amino acids of Vpr at 323 K. Further analysis of the HSQC-NOESY experiment based on the observed NOEs, dNN(i, i +1), dNN(i, i +2), daN(i, i +1), daN(i, i +2), daN(i, i +3) and daN(i, i+4) of the 22 labeled amino acids, suggested that all of these residues, except Thr89, are involved in a helical conformation. Nevertheless Thr55 and Leu39 are apparently involved in a more flexible structured domain. A quasi-complete pattern of strong dNN(i, i +1) andweakdaN(i, i +1) NOE connectivities was observed for residues in the regions (17–34) and (55–84), respectively. Theoccurrenceoftypicala helix encompassing these two Fig. 2. 1D spectra of (1–96)Vpr (1m M ) from 6 to 10 p.p.m. (amide and aromatic protons), pH 3.4 in 70% H 2 O/30% TFE mixture at four different temperatures. Signal narrowing, which facilitated proton resonances assignment was observed as a function of the temperature. Ó FEBS 2002 NMR Structure of the HIV-1 protein Vpr (Eur. J. Biochem. 269) 3781 regions was reinforced by the observation of a series of dab(i, i +3),daN(i, i +4)and daN(i, i + 3) correlations. NOE connectivities, particularly within the (Ser41–Glu48) region, characterized by daN(i, i + 3) proximities, showed that the (Arg36–Tyr50) region is also involved in a well- defined a helix (Fig. 5). The (37–48)Vpr segment might be a helix or turn conformation less structured than those encompassing the (17–34) and (55–84) regions, as it does not present all typical NOEs found in the classical a-helix. Tyr47, Tyr50, Asp52, Thr55, Gly56, Glu58 and Ala59 possess long range connectivities of medium intensity. The long range NOEs characteristics of these residues (47, 50, 52, 55, 56 and 59; Table 1) indicate a spatial proximity between the second and third a helices (Fig. 6). These long rangeNOEsbringthearomaticringofTyr50closetothe second helix segment (40–48), with the domain (47–55) forcing the protein to adopt a unusual U shaped confor- mation in presence of 30% TFE. NMR structure analysis of the entire protein Vpr in presence of 30% TFE The structure of Vpr was determined by a simulated annealing protocol and energy minimization using 1420 distances constraints including 317 sequential (|i ) j| ¼ 1), 293 short-range (1 < |i ) j| ¼ 4), 8 long-range (|i ) j|>4) and 802 intraresidual restraints. According to the lowest total energy and number of NOE restraint violations, 20 structures were selected for structural analysis (Table 2). The solution structure of Vpr shows well-structured helical domains (Fig. 6) with amphipathic properties and gamma turns throughout the protein. The structure is characterized by a flexible N-terminal region (Met1–Glu13), followed by a (Pro14–Asn16) c turn, then an a helix of 17 amino acids, encompassing residues Asp17 to His33, then a second c turn (Phe34–Arg36), a second (His40–Glu48) a helix, a (Asp52–Trp54) c turn and a third a helix of 29 amino acids, extending from Thr55 to Ile83, followed by a very flexible C-terminal (Ile84–Ser96) domain (Fig. 6). To analyze the amphipathic properties of the a helices, the amino acids side chains have been classified into two categories, according to their preference for aqueous or nonpolar environment, using their relative hydrophilicity and hydrophobicity [36,37]. The first a helix (Asp17–His33) has the character- istics of an amphipathic helix (Fig. 7I). Its hydrophilic face is formed by the amino acid side chains: Asp17, Glu21, Glu24, Glu25, Lys27, Asn28, Glu29 and Arg32, while the hydrophobic face is constituted by the side chains of: Trp18, Thr19, Leu20, Leu22, Leu23, Leu26, Ala30, and Val31. This region provides an uninterrupted hydrophobic surface, and is well structured as the rmsd calculated using backbone atoms of the 20 best structures (N, Ca,C¢,O) for (Asp17–His33) region is 0.34 A ˚ (0.18–0.68 A ˚ ). Further- more, the calculated ensemble of structures shows that this a helix is stabilized by CO i -NH i+4 hydrogen bonds through- out the (17–33) segment of the molecule. The second a helix, residues His40 to Glu48, also has amphipathic properties as the hydrophilic side chains of Ser41, Gln44 and Glu48 are located on one side of the helix while the hydrophobic side chains of Leu42, Ile46 are on the other (Fig. 7II). This helical conformation is not integrally conserved in the 20 structures as the average rmsd of the backbone atoms (N, Ca,C¢,O)is0.90 A ˚ , while (Leu42–Glu48) region is perfectly welldefinedwithanaveragedrmsdof0.57A ˚ . Also, this helix is stabilized by hydrogen bonds, CO i –NH i+4 ,within the region of residues (41–48). The third a-helix, extending from Thr55 to Ile83, is also well defined in the (55–74) region,witha0.74A ˚ averaged rmsd, and 1.0 A ˚ average rmsd for (55–77) region. Gly75 appears to induce a slight curvature in the helix, which is poorly defined in the (78–83) region. The hydrophobic amino acid side chains Fig. 3. 15 NHSQCof(1–96)Vpr at pH 3.4 and 323 K performed at 600 MHz. All correlations have been identified and are indicated on the 2D spectrum. Fig. 4. Part of the 2D 15 NHSQC-NOESYperformedon(1–96)Vpr at pH 3.4 and 323 K showing the NH resonances correlations. A qualita- tive secondary structure analysis of this 2D heteronuclear experiment based on the 22 labeled amino acids suggested that all these residues, except Thr 89, are involved in a-helix structure formation. Intrare- sidual correlations are coloured black and interresidual ones are col- oured red. 3782 K. Wecker et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (Val57, Leu61, Leu63, Leu64, Leu67, Leu68 and Ile74) are located on one face of the helix (Fig. 7III) and form an uninterrupted hydrophobic face, whereas amino acid side chains (Glu58, Arg62, Glu65, Glu66, Cys76 and Arg77) form the hydrophilic face. This helix is also stabilized by a H-bond network, CO i -NH i+4 . Three other regions of the protein appear relatively well structured and possess turns containing proline residues. The first c turn (Pro14–Asn16) (averagedrmsdof0.54A ˚ ) preceding the first amphipathic a helix (17–33), is less structured than the second turn (Phe34–Arg36), which has a proline in second position and is stabilized by an hydrogen bond NH36–CO34 with an average rmsd of 0.24 A ˚ . The third (52–54) c turn (rmsd of 0.98 A ˚ ) is localized just before the third amphipathic a helix (55–83), and is stabilized by the hydrogen bond NH54– CO52. NMR relaxation The NMR relaxation data were collected for the 22 labeled amino acids in order to analyse the backbone internal motions and to study the structure obtained by molecular dynamics calculation using NMR constraints. T 1 , T 2 relax- ation times and 15 N- 1 H heteronuclear NOE were been obtained from two independent set experiments and data (Fig. 8). The standard deviations were calculated for two Fig. 5. Summary of sequential and short range NOE data. The thickness of the bar for the sequential NOE data is related to the approximate intensity of the NOE (strong, medium and weak NOEs). Fig. 6. Representation of (1–96)Vpr structure. Upper panel: Backbone superimposition of 10 selected structures of (1–96)Vpr, performed on the (15–33) (38–49) (54–74) a helices. The average rmsd of the back- bone for the (17–83) region is 5.0 A ˚ . Helices are coloured light blue, turns in red and flexible domains in dark blue. Lower panel: Stereoview of the (1–96)Vpr 3D structure. a helices are represented by light blue, turns in red and flexible regions in dark blue. The close proximity of a-helices (40–48) and (55–83) can be observed. Table 1. Long range NOEs, allowing the formation of the hydrophobic cluster that bring close to each other the first and second helices on one side, and the second and the third helices on the other side. 2.6H Tyr50 cH Thr55 3.5H Tyr50 cH Thr55 2.6H Tyr50 a 1 Gly56 3.5H Tyr50 a 1 Gly56 2.6H Tyr50 a 2 Gly56 3.5H Tyr50 a 2 Gly56 2.6H Tyr50 a Ala59 3.5H Tyr50 a Ala59 2.6H Tyr50 b Ala59 3.5H Tyr50 b Ala59 b 1 Tyr50 a 1 Gly56 b 2 Tyr50 a 1 Gly56 b 1 Tyr50 a 2 Gly56 b 2 Tyr50 a 2 Gly56 b 1 Tyr47 b Glu58 b 2 Tyr47 b Glu58 3.5H Tyr47 b 1 Asp52 3.5H Tyr47 b 2 Asp52 Table 2. Structural analysis on the 20 selected structures of Vpr. Average rmsd (A ˚ ) between each structure and the best structure calculated using the backbone atoms (N, Ca,C¢,O) Residues rmsd 14–16 0.54 17–33 0.34 34–36 0.24 40–48 0.90 52–54 0.98 55–83 1.20 NOE constraint violations Residual NOE distance constraint violations (A ˚ ) (1420 constraints) 0.07 ± 0.004 Residual bond distortion (a) (A ˚ ) (1632 bonds) 0.07 ± 0.003 Residual angle distortion (a) (deg) (2927 angles) 26 ± 1.3 Conformational energy Total (kcalÆmol )1 ) )273 ± 34 Nonbond (kcalÆmol )1 ) )251 ± 15 Restraint energy (kcalÆmol )1 )65±7 Ó FEBS 2002 NMR Structure of the HIV-1 protein Vpr (Eur. J. Biochem. 269) 3783 successive experiments using identical conditions. All the relaxation constants were collected for the 22 uniformly labeled amino acids introduced into Vpr. In the case of the two leucine residues at positions 64 and 67, 15 Nand 1 H resonances were overlapped and the relaxation data for these two residues have been collected as a unique corre- lation. For Phe34 and Leu39, their resonances allowed correct determination of T 1 , but not for T 2 or NOEs correlations. Therefore, for the above residues, we do not attempt to derive any interpretation of the results. Among all the relaxation constants, Thr89 is the only one amino acid exhibiting a negative NOE ()1.33), which suggests that there is rapid internal motion in this unstruc- tured C-terminal region. T 1 and T 2 relaxation times are in good agreement with the calculated NOE values and correspond approximately to relaxation times longer than the resonances of others residues (T 1 :322ms;T 2 : 268 ms). These results are consistent with the solution structure of Vpr in which the (84–96) C-terminal domain is less well defined and appears to be flexible. The second weak NOE (0.43) corresponds to Gly75. This residue shows a relatively fast relaxation time (338 ms for T 1 and 134 ms for T 2 )when compared to the other labeled amino acids. This result is also in agreement with the structure of (1–96)Vpr, in which the third a helix (55–83) is disrupted by Gly75, and thus induces a bent in the helix axis. All other amino acids present similar profiles corresponding to short relaxation times and high NOEs, which is characteristic of well structured domains. The only striking result, for which we have no explanation, concerns the T 1 relaxation time for Ala30 that is long whereas its T 2 relaxation time and NOE are weak. An interesting observation in these experiments Fig. 7. Representation of the amphipatic helices. (I)Viewofthefirst amphipathic a helix (17–33)Vpr. The backbone is coloured red, hy- drophilic and hydrophobic side chains are green and blue, respectively. (A) Viewed perpendicular to the axis, (B) View along the helix axis. (II) View of the second amphipathic a helix (40–48)Vpr. The backbone is coloured red, hydrophilic and hydrophobic side chains are green and blue, respectively. (A) View perpendicular to the axis; (B) View along the helix axis. (III) View of the third amphipathic a helix of (55– 83)Vpr. The backbone is coloured red, the hydrophilic and hydro- phobic side chains are green and blue, respectively. (A) View perpen- dicular to the axis; (B) View along the helix axis. Fig. 8. Backbone 15 N NMR relaxation results for the (1–96)Vpr at 323 K, pH 3.4. (A) T 1 data, (B) T 2 data, (C) NOE data and (D) T 1 /T 2 ratio. The NMR relaxation data were collected for all of the 22 uniformly labeled amino acids introduced into Vpr. All amino acids present similar profiles corresponding to short relaxation times and high NOEs, characteristic of structured domains, except for principally Thr89, which is the only one amino acid exhibiting a negative NOE and long relaxation time. 3784 K. Wecker et al. (Eur. J. Biochem. 269) Ó FEBS 2002 was the relaxation parameters obtained for Phe34 and Leu39. The T 1 of Leu39 (374 ms) clearly demonstrates that this amino acid is included in a less structured domain. This result is in agreement with the NMR determined structure in which Leu39 (preceding the second (40–48) a helix) is not involved in any secondary or tertiary structural interactions. Phe34hasaweakT 1 value and seems to be involved in structural domain formation consistent with its first position in the c turn (34–36) relatively constrained by the presence of Pro35. The short relaxation times and strong NOEs obtained for Tyr47 and Gly43 corroborate the presence of a a helix motif imperfectly defined from residues 40–48. Another method we used to analyze dynamic parameters was to calculate the T 1 /T 2 ratio (Fig. 8) [35]. A low ratio corresponds to an amino acid involved in a flexible region, while a high value corresponds to a residue implicated in a rigid or well-structured domain. From this study, we can conclude that almost all labeled residues are involved in well-structured regions with a calculated T 1 /T 2 ratio of about 2.5. An initial set of amino acids residues 20, 22, 23, 26, 55, 64, 68, 69 and 71 are the most constrained and are involved in secondary structure. Four other amino acids residues showed high flexibility when compared to the previous ones, these were: Thr89 (which is involved in the very flexible C-terminal domain), Gly43, Tyr47 and Thr55 are all located in imperfectly structured helices. Thus, the relaxation study is in rather good agreement with the solution structure generated by simulated annealing and molecular dynamics using the NMR constraints. DISCUSSION Vpr has been reported to be involved in several steps of the retroviral life cycle and seem critical for efficient nuclear translocation of the pre-integration complex. These processes are dependent upon interactions between Vpr and other viral and nonviral protein targets. The importance of Vpr protein–protein interactions has been clearly demonstrated by the loss of activity of Vpr following certain point mutations [16,38,39]. This is one of the main reasons why the aim of this study was to determine the solution structure of the intact monomeric form of this protein. One of the main problems encoun- tered with (1–96)Vpr, that impeded structural determina- tions by NMR, is its strong tendency to form aggregates in aqueous solutions [40,41]. This problem could only be overcome by dissolving the protein in a mixture of H 2 O/ TFE (ratio of 7 : 3), which helps to prevent interactions between hydrophobic domains without significantly mod- ifying the secondary structural elements of the protein. One consequence of the presence of TFE on the structure of Vpr is the tertiary structure may open up. However, the NMR solution structure of the short fragment (13– 33)Vpr has been determined in presence of dodecylphosphocholine [41], and the (18–32) a-helix region in this molecule was found not very different from that observed in our NMR study of (1–51)Vpr in H 2 O/ TFE [25]. In the present study, we show that the NMR derived structure of Vpr in H 2 O/TFE contains several differences when compared to the isolated N- and C- terminal domain structures determined under the same conditions [25,26], that may account for the differences in biological activity of the isolated Vpr fragments [22,23,27]. CD experiments on Vpr in aqueous solution at pH 3.4 and pH 6.0 showed two characteristic minima at 208 and 222 nm, and the presence of two maxima at 190 and 212 nm (Fig. 1). This result suggests that Vpr possess a high a helical content [34,36,42]. It is also important to note that TFE can stabilize a helices in regions that have already a high propensity to form this secondary structural arrangement [43–48]. The presence of large a helical segments in (1– 96)Vpr has been confirmed by our NMR data, as reflected by the number of medium range connectivities (i, i +3) and (i, i + 4) throughout the polypeptide chain in the NOESY spectra. This study provides evidence of structural modifications when comparing the isolated domains of (1–51)Vpr [25] and (52–96)Vpr [26] with the intact Vpr protein. The differences in structure are localized to the N-terminal domain. The secondary structures elements observed in (1–51)Vpr i.e. a helix(17–29)-turn(30–33)-a helix(35–46)-turn(47–49), are slightly different in the intact Vpr protein [a helix(17–33)- turn(34–36)-flexible(37–39)-a helix(40–48)] (Figs 9 and 10). In (1–96)Vpr, the first a helix is extended by four amino acids on its the C-terminal side, followed by a turn centered on Pro35, an usual position for this type of amino acid. Moreover, Phe34 is involved in a secondary structure element in the intact protein, but not in the (1–51)Vpr [25]. The amphipathic (54–78) a helix observed at the C-terminal domain of (52–96)Vpr, is extended by a further five amino acids in (1–96)Vpr (Figs 9 and 10) and also stabilized by several hydrogen bonds. In addition, we have studied the protein fold of Vpr around the loop region (49–54) which presents some long range NOEs including the c turn (52–54) region. This local structural arrangement is reinforced by interactions between Fig. 9. Comparison of the (1–96)Vpr structure (in blue) with the (1–51)Vpr (in pink) and (52–96)Vpr (in green) domain structures. The (17–46) domain backbone of the N-terminal (1–51)Vpr (pink) is superimposed with the (17–46) domain backbone of the intact protein (1–96)Vpr (blue) (rmsd of 2.6 A ˚ ). The (54–78) domain backbone of the C-terminal (52–96)Vpr (green) is superimposed with the (54–78) region backbone of the (1–96)Vpr protein (blue) (rmsd of 1.5 A ˚ ). Ó FEBS 2002 NMR Structure of the HIV-1 protein Vpr (Eur. J. Biochem. 269) 3785 amino acids of the loop and residues of the third helix (Fig. 6). Accordingly, amino acids, such as Tyr50 and Ala59, distant in the primary sequence show spatial proximity. The structural organization around the (49–54) region induces an unusual U-shaped conformation. The long-range connectivities seen in the NMR data, impose a relative orientation between the second amphipathic and third a helices. This three-dimensional spatial organization is maintained in the well-defined secondary structures through interactions of hydrophobic clusters. The first hydrophobic cluster, including the amino acid side chains 43, 46, 47, 55, 56, 59, 60 and 63, is observed between the second and the third helices. This cluster strongly stabilizes this region and is responsible for the relative orientation of the two a helices in segments (40–48) and (55–83), respec- tively (Fig. 10). It is possible that the U-shaped conforma- tion of (1–96)Vpr in our study could be due to the presence of 30% TFE, which is known to reduce hydrophobic interactions. The structure of intact Vpr in pure H 2 O could therefore be slightly different to our solution structure. The presence of a succession of amphipathic a helices, with well-defined hydrophilic and hydrophobic faces, is commonly observed in structural motifs that involve protein–protein, protein–lipid or protein–nucleic acid inter- actions. The incorporation of Vpr into the viral particles requires its interaction with a leucine triplet repeat sequence (LXX) 4 found in p6 at the C-terminal domain of Gag [6]. Mutagenesis experiments, have shown that incorporation of Vpr into the maturing virion, is dependent on the Vpr N-terminal domain, particularly the (17–34) region. Thus, mutations in the a helix region covering residues 17–33 led to a reduced incorporation of Vpr into new viral particles [49–52]. These findings are supported by our NMR structure, as these mutations are expected to disrupt the first amphipathic a helix, subsequently preventing protein– protein interactions. The helix-turn-helix region of Vpr could also explain the capacity of the protein to participate in nuclear transport of the proviral DNA [10], as this type of motif is commonly found in DNA binding domains [53]. Moreover, the nucleocapsid protein NCp7 has been shown to promote (70–96)Vpr–RNA interactions and complex formation with (80–96)Vpr [27]. As the structure of Vpr possesses a characteristic a helix at region (70–83) followed by a very flexible domain (84–96), a reorganization of this region could be triggered by a complex formed with both nucleic acids and NCp7. Thus, during the early stages of HIV-1 replication, binding of NCp7 to the C-terminal domain (80–96)Vpr [8] may expose the N-terminal (1–39) portion of the protein and allow its tight binding to Lys-tRNA synthetase [15]. The C-terminal domain (52–96)Vpr and not the N-terminal domain (1–51)Vpr, has been shown to bind viral proteins such as NCp7 [8] or RNA [27]. In both cases, the intact protein (1–96)Vpr, was also found to be less efficient in forming these complexes compared to (52–96)Vpr. It has been suggested that these findings could be explained by an interaction between the N- and C-terminal domains in the intact Vpr protein resulting in steric hindrance in the C-terminal recognition motif [8,27]. Likewise, it has been demonstrated that (1–96)Vpr and particularly (52–96)Vpr, but not the N-terminal sequence (1–51), can induce apoptosis in cells through interactions with the adenine nucleotide translocator (ANT) of the mitochondrial pore [22]. Interestingly, in the short active fragment of Vpr, corresponding to the residues 71–82, mutations of Arg73 or Arg77 to Ala located on the same face of the a helix (55–83) in Vpr, or 54–78 in (52–96)Vpr, completely inhibited the interaction with ANT and subse- quent apoptosis [22]. These findings suggests that at least a part of the Vpr/ANT binding interface is stabilized by electrostatic interactions, a feature that could be exploited in designing inhibitors or activators of apoptosis. Moreover, the importance of the a-helical structure of the 71–82 domain in Vpr for ANT recognition and apoptosis is highlighted by the loss of Vpr-induced apoptosis when Ser79 is replaced by Pro [39]. The solution structure of Vpr that we propose in this paper shows, very well defined secondary structure ele- ments, all along the molecule. Long range NOEs permit the formation of hydrophobic clusters (Fig. 10), that maintain the relative orientation of the first and second helices, on one side, and the second and third helices on the other side of the molecule. It is reasonable to suggest that these local structure elements do not have to be disrupted by the use of 30% TFE. The regions around the second a helix could act as hinges, allowing the first and third helices to be closer in space, under certain conditions or in response to complex Fig. 10. Representation of the (26–67) region of the (1–96)Vpr protein. Hydrophobic side chains of residues 33, 34, 35, 37, 39 and 40 form a cluster between the first (17–33) region and second (40–48) a helices. Hydrophobic side chains of residues 43, 47, 50, 55, 56, 59, 60 and 63 form a cluster between the second (40–48) and third (55–83) a-helices. These two clusters are thought to maintain the relative orientation of the a-helices in the 3D structure of the protein. The backbone is represented in blue and the hydrophobic amino acid side chain coloured pink. 3786 K. Wecker et al. (Eur. J. Biochem. 269) Ó FEBS 2002 formation or protein–protein interactions. The similar T 1 / T 2 values for the 15 N labeled amino acids distributed all along Vpr reinforce the proposed existence of continuously well-structured domains for this protein. Nevertheless, it is very likely that depending of the conditions of the physi- ological medium (pH, ionic strength, etc), this small and intrinsically flexible protein could adopt different confor- mations allowing for example the insertion of the third a helix (54–78) in the mitochondrial bilayer in which ANT (adenine nucleotide translocator), is embedded [22]. Such incorporation mechanism in phospholipid bilayer has been described for Vpu, a linear peptide closely related to Vpr and also characterized by a succession of three a helices [54]. In conclusion, this solution NMR structure of intact Vpr provides new insights into several important properties of this viral protein. A worthwhile further step in the understanding of the roles played by Vpr in the virus life cycle would require structural determination of complexes between Vpr and the domains of other interacting target proteins such as NCp7, ANT or Tat and nucleic acids. ACKNOWLEDGEMENTS We thank C. Lenoir and P. Petitjean for peptide synthesis, C. Vitta for his technical assistance in circular dichroism experiments, C. Dupuis for her assistance in drafting this manuscript and R. Fredericks for English corrections. This work was supported by ANRS and SIDACTION (ECS), the anti-AIDS French programs. REFERENCES 1. Emermann, H. (1996) HIV-1 and the cell cycle. Curr. 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SUPPLEMENTARY MATERIAL The following material is available from http://www.black well-science.com/products/journals/suppmat/EJB/EJB3067/ EJB3067sm.htm Table S1. NMR chemical shifts observed in the (1–96)Vpr protein in 30% TFE-d 2 70% H 2 O at 323 K and pH 3.4, calibrated to HMDS. Table S2. T1, T2 relaxation times T1/T2. 15 N- 1 H hetero- nuclear NOEs ratios observed in the (1–96)Vpr on the 22 labelled amino acids. . modifications when comparing the isolated domains of (1–51 )Vpr [25] and (52–96 )Vpr [26] with the intact Vpr protein. The differences in structure are localized to the N-terminal domain. The secondary structures. portion of the protein and allow its tight binding to Lys-tRNA synthetase [15]. The C-terminal domain (52–96 )Vpr and not the N-terminal domain (1–51 )Vpr, has been shown to bind viral proteins such. NMR structure of the HIV-1 regulatory protein Vpr in H 2 O/trifluoroethanol Comparison with the Vpr N-terminal (1–51) and C-terminal (52–96) domains K. Wecker, N. Morellet, S. Bouaziz and

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