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

Eur. J. Biochem. 269, 3779–3788 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03067.x 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 K. Wecker, N. Morellet, S. Bouaziz and B. P. Roques Departement de Pharmacochimie Moleculaire et Structurale, INSERM U266 CNRS UMR 8600, UFR desSciences Pharmaceutiques et Biologiques, Paris, France The human immunodeficiency virus type 1, HIV-1, genome encodesahighlyconservedregulatorygeneproduct,Vpr(96 aminoacids),whichisincorporatedintovirionsinquantities equivalenttothoseoftheviralGagprotein.Ininfectedcells, Vpr is believed to function during the early stages of HIV-1 replication(suchastranscriptionoftheproviralgenomeand migrationofpreintegrationnuclearcomplex),blockscellsin G2phaseandtriggersapoptosis.Vpralsoplaysacriticalrole 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 1H- and 15N-NMR and 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 interestistheproteinVpr,whichisencodedlateduringviral 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 Correspondence to K. Wecker, Unite de RMN des Biomolecules, Departement des Retrovirus 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) refined by restrained molecular dynamics. In addition, 15N relaxation parameters (T1, T2) and heteronuclear 1H-15N NOEs were measured. The structure of the protein is char-acterized by a well-defined cturn(14–16)-a helix(17–33)-turn(34–36), followed by a ahelix(40–48)-loop(49–54)-a helix(55–83)domainandendswithaveryflexibleC-terminal sequence. This structural determination of the whole intact Vprmoleculeprovideinsightsintothebiologicalroleplayed 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; 3D structure. 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 complexremainsunclear,asnoclassicalnuclearlocalization signal has been clearly identified in Vpr. Recently, it has been shown that Vpr can interact with karyopherin a and thenucleoporinNsp1,andthusseemstoactasanimportin-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 tRNALys,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 effectof 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, 3780 K. Wecker et al. (Eur. J. Biochem. 269) Ó FEBS 2002 semipreparative Vydac C18 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 Dawascalculatedtakingintoaccountthelabeled 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 mM at pH 3.4. Fig. 1. PrimarysequenceandCDspectraat293 Kof(1–96)Vpr.Upper panel: Primary sequence of (1–96)Vpr protein (14 KDa). The 22 15N-and13C-labeledaminoacidsareinbold.TheN-terminalsequence 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% H2O) (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. 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 interactwitheachother,however,itpossessedloweractivity compared to the intact Vpr protein as reflected in various interactions studies such as nucleic acid recognition [27], apoptosis[22]andVpr-inducedDNAtransfection[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,asdescribedpreviously[28].Duringpeptidesynthesis, 22 labeled amino acids (95% 15N, 15% 13C) 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 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 Kwitha2 nmwavelengthincrementandaccumulation time of 1 s per step. Each spectrum was obtained with a protein concentration of 2 mM in presence of 10 mM 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 NMRexperimentswererecordedonaBrukerDRX600and 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 H2O 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 15N-HMQC-TOCSY, HSQC-TOCSY and HMQC-NOESY, HSQC-NOESY (sm 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 1H and 15N, respectively. All dynamics experiments were performed at 323 K through the 22 15N-labeled amino acids. Longitudinal relaxation times T1 were obtained with delays of 5, 10, 50, Ó FEBS 2002 NMR Structure of the HIV-1 protein Vpr (Eur. J. Biochem. 269) 3781 100, 200, 350, 500, 800, 1200, 3000 ms. The Carr Purcell Meilboom Gill (CPMG) sequence was used during the relaxationperiodwith15Npulsesappliedevery460 lsinthe transverse relaxation experiments (T2). T2 values were obtained with delays 5, 10, 20, 30, 50, 60, 72, 80, 100, 120, 168, 200, 248, 300 and 400 ms. Heteronuclear 1H-15N 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 I8 + (I0 ) I8) exp(–T1t). I0 is the initial value of the resonance intensity and I8 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 e¼ 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. protein was most soluble): 1 mM aqueous solution, pH 3.4 and in the presence of 30% TFE-d2. 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 Wuthrich and coworkers [34], supplemented with information from heteronuclearexperiments.Unambiguousresonanceassign-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 NOESYcrosspeaks,connectingHN,HaandHbofresidue i with NH of residue i + 1, were used for sequential assignment [35]. Thus, a complete chemical shift assignment ofthebackboneandsidechainprotonswasachievedforthe 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 ahelical conformation. Nevertheless Thr55 and Leu39 are apparently involved in a more flexible structured domain. A quasi-complete pattern of strong dNN(i, i+1) and weak daN(i, i+1) NOE connectivities was observed for residues in the regions (17–34) and (55–84), respectively. The occurrence of typical ahelix encompassing these two RESULTS Circular dichroism CD spectra of the protein in 100% H2O (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 ratiobetweentheintensitiesofthebandsat222and208 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 ofTFE does notinduce ahelical formation but rather stabilizes the pre-existing secondary structures. 1H- and 15N-NMR experiments of Vpr All attempts to solubilize Vpr at minimal concentrations for NMR studies in H2O 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 Fig. 2. 1D spectra of (1–96)Vpr (1 mM) from 6 to 10 p.p.m. (amide and aromatic protons), pH 3.4 in 70% H2O/30% TFE mixture at four different temperatures. Signal narrowing, which facilitated proton resonances assignment was observed as a function of the temperature. 3782 K. Wecker et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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 ahelices (Fig. 6). These long range NOEs bring the aromatic ring of Tyr50 close to the 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. Fig. 3. 15N HSQC of (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 15N HSQC-NOESY performed on (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. 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 ahelix (Fig. 5). The (37–48)Vpr segment might be ahelix or turn conformation less structured than those encompassingthe(17–34)and(55–84)regions,asitdoesnot 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), 293short-range(1 < |i ) j| ¼ 4),8long-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 byaflexibleN-terminalregion(Met1–Glu13),followedbya (Pro14–Asn16) cturn, then an ahelix of 17 amino acids, encompassing residues Asp17 to His33, then a second c turn (Phe34–Arg36), a second (His40–Glu48) ahelix, a (Asp52–Trp54) c turn and a third ahelix 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 ahelices, 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 ahelix (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 hydrophobicfaceisconstitutedbythesidechainsof: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 ahelixisstabilizedbyCOi-NHi+4 hydrogenbondsthrough-outthe(17–33)segmentofthemolecule.Thesecondahelix, 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)regionisperfectly well defined with an averaged rmsd of 0.57 A. Also, this helix is stabilized by hydrogen bonds, COi–NHi+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, with a 0.74 A 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 Ó FEBS 2002 NMR Structure of the HIV-1 protein Vpr (Eur. J. Biochem. 269) 3783 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). 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 a1 Gly56 3.5H Tyr50 a1 Gly56 2.6H Tyr50 a2 Gly56 3.5H Tyr50 a2 Gly56 2.6H Tyr50 a Ala59 3.5H Tyr50 a Ala59 2.6H Tyr50 b Ala59 3.5H Tyr50 b Ala59 b1 Tyr50 a1 Gly56 b2 Tyr50 a1 Gly56 b1 Tyr50 a2 Gly56 b2 Tyr50 a2 Gly56 b1 Tyr47 b Glu58 b2 Tyr47 b Glu58 3.5H Tyr47 b1 Asp52 3.5H Tyr47 b2 Asp52 (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, COi-NHi+4. Three other regions of the protein appear relatively well structured and possess turns containing proline residues. The first cturn (Pro14–Asn16) (averaged rmsd of 0.54 A) preceding the first amphipathic ahelix (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) cturn (rmsd of 0.98 A) is localized just before the third amphipathic ahelix (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 , T relax-ation times and 15N-1H heteronuclear NOE were been obtained from two independent set experiments and data (Fig. 8). The standard deviations were calculated for two 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) 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) ahelices. The average rmsd of the back-bone for the (17–83) region is 5.0 A. Helices are coloured light blue, turnsinredandflexibledomainsindarkblue.Lowerpanel:Stereoview of the (1–96)Vpr 3D structure. ahelices 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. Residues 14–16 17–33 34–36 40–48 52–54 55–83 NOE constraint violations Residual NOE distance constraint violations (A) (1420 constraints) Residual bond distortion (a) (A) (1632 bonds) Residual angle distortion (a) (deg) (2927 angles) Conformational energy Total (kcalÆmol)1) Nonbond (kcalÆmol)1) Restraint energy (kcalÆmol)1) rmsd 0.54 0.34 0.24 0.90 0.98 1.20 0.07 ± 0.004 0.07 ± 0.003 26 ± 1.3 )273 ± 34 )251 ± 15 65 ± 7 ... - tailieumienphi.vn