Báo cáo Y học: Structure of human immunodeficiency virus type 1 Vpr(34–51) peptide in micelle containing aqueous solution pptx

Structure of human immunodeficiency virus type 1 Vpr(34–51) peptide in micelle containing aqueous solution Andrea Engler 1 , Thomas Stangler 2,3 and Dieter Willbold 3,4 1 Lehrstuhl fu ¨ r Biopolymere, Universita ¨ t Bayreuth, Germany; 2 Institut fu ¨ r Molekulare Biotechnologie, Jena, Germany; 3 Institut fu ¨ r Physikalische Biologie, Heinrich-Heine-Universita ¨ t, Du ¨ sseldorf, Germany, 4 Forschungszentrum Ju ¨ lich, IBI-2, Germany Human immunodeficiency virus type 1 protein R (HIV-1 Vpr) promotes nuclear entry of viral nucleic acids in nondividing cells, causes G 2 cell cycle arrest and is involved in cellular differentiation and cell death. Vpr subcellular localization is as variable as its functions. It is known, that consistent with its role in nuclear transport, Vpr localizes to the nuclear envelope of human cells. Further, a reported ion channel activity of Vpr is clearly dependent on its localization in or at membranes. We focused our structural studies on the secondary structure of a peptide consisting of residues 34–51 of HIV-1 Vpr. This part of Vpr plays an important role in Vpr oligomerization, contributes to cell cycle arrest activity, and is essential for virion incorporation and binding to HHR23A, a protein involved in DNA repair. Employing NMR spectroscopy we found this part of Vpr to be almost completely a helical in the presence of micelles, as well as in trifluoroethanol containing and methanol/ chloroform solvent. Our results provide structural data suggesting residues 34–51 of Vpr to contain an amphi- pathic, leucine-zipper-like a helix, which serves as a basis for oligomerization of Vpr and its interactions with cellular and viral factors involved in subcellular localiza- tion and virion incorporation of Vpr. Keywords: HIV-1; Vpr; solution structure; dodecylphos- phocholine micelles; NMR. Human immunodeficiency virus type 1 (HIV-1) is a member of the lentivirus family. In addition to the gag, pol and env genes present in all retroviruses, HIV-1 encodes two regulatory and four so called ÔaccessoryÕ proteins, that are dispensable for viral replication in cell culture but are known to be decisive for viral infectivity, replication and pathogenesis in vivo. One of these accessory proteins is virus protein R (Vpr). Vpr seems to be required at various steps of the HIV replication cycle and is therefore an interesting target for the development of antiviral agents. This 96-amino-acid protein is an important factor for the pathogenesis of HIV [1,2]. Vpr is an integral part of viral particles suggesting an important role in early stages of infection [3–6]. Vpr is involved in the transport of the preintegration complex into the host cell nucleus, which is an important feature for infection of nondividing cells [7,8]. Vpr arrests mammalian and yeast cells in G 2 -phase of the cell cycle [9–12]. Further, Vpr has been proposed to have ion-channel activity [13,14]. Different cellular proteins are reported to interact with Vpr: transcription factor Sp1 [15], uracil DNA glycosylase (UNG) [16], HHR23A, a protein implicated in DNA repair [17], importin-a, nuclear pore protein Nsp1p [18], and many others. Former structural studies of Vpr fragments by NMR were performed in a (30%) trifluoroethanol-containing solution and, not surprisingly, revealed a long amphipathic a helix-turn-a helix (amino acids 17–46) motif ended by a turn for Vpr(1–51) [19]. The structure of Vpr(52–96) fragment, also in trifluoroethanol-containing solution, is characterized by an amphipathic a helix from residue 53 to residue 78 and a less defined C-terminal domain [20]. Another fragment of Vpr, residues 50–82, was shown to contain a helix from residues 53–81 in 50% trifluoro- ethanol. Trifluoroethanol, however, is well known to induce a helical secondary structures in peptides [21]. Structural studies of Vpr fragment 13–33, known to be essential for ion channel activity and virion incorporation, showed that this part of Vpr is almost completely a helical in the presence of dodecylphosphocholine (dodecyl-PCho) micelles [22]. Several different functions of Vpr take place in or at membranes, such as ion channel activity [14] and virion incorporation of Vpr [23–25]. This suggests most of the various cellular and viral proteins that are reported to directly interact with Vpr may form hydrophobic environ- ments for Vpr interaction. To avoid self aggregation and to take into account a rather hydrophobic environment that may be present in vivo, we determined the solution structure of Vpr(34–51) peptide in micelle-containing solutions and some additional solvents, often referred to as membrane-mimicking. Only recently, it was shown that this region of the protein is important for oligomerization, virion incorporation, and subcellular localization of Vpr [26]. Correspondence to D. Willbold, Forschungszentrum Ju ¨ lich, IBI-2, 52425 Ju ¨ lich, Germany. Fax: + 49 2461612023, Tel.: + 49 2461612100, E-mail: dieter.willbold@uni-duesseldorf.de Abbreviations: HIV, human immunodeficiency virus; HIV-1, HIV type 1; rmsd, root mean square deviation; SIV, simian immunodeficiency virus; Vpr, virus protein R. (Received 13 February 2002, revised 13 May 2002, accepted 17 May 2002) Eur. J. Biochem. 269, 3264–3269 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03005.x MATERIALS AND METHODS Peptide The synthetic peptide CH 3 -CO-FPRIWLHNLGQHIY ETYG-NH 2 with the amino-acid sequence of HIV-1 Vpr (34–51) was purchased as a purified product (Interac- tiva, Ulm, Germany). N- and C-termini were modified by an acetyl and an amide group, respectively, to remove charges, that are not present in the full length Vpr protein either. The peptide was more than 95% pure as judged from reversed phase HPLC analysis. Mass spectroscopy proved the product to have a mass of 2286 Da, close to the theoretical value (2285.5 Da). NMR spectroscopy All NMR spectra were collected at 298 K on a Varian INOVA 600 spectrometer equipped with a triple-axis pulsed field gradient probe. Proton resonances were assigned by standard procedures using DQF-COSY and TOCSY (80 and 90 ms mixing time) experiments. Proton–proton dis- tance constraints were obtained from NOESY (100 and 200 ms mixing time) experiments. NOE cross peaks were classified as strong, medium and weak and converted into upper limit distance constraints of 2.7, 3.5 and 5.0 A ˚ , respectively. In the spectra of Vpr(34–51) in chloroform/ methanol, a total of 10 residues showed 3 J HNNa scalar couplings of less than 6.0 Hz and were therefore restrained to adopt backbone torsion angles between )80° and )40°. All NMR data were processed and analyzed with the program package NDEE (SpinUp Inc., Dortmund, Germany). Structure calculation was performed using XPLOR 3.851 and a modified ab initio simulated annealing protocol including floating assignment of prochiral groups and a conformational database potential during all but the last 200 cooling steps. Of the 60–97 structures resulting from the final round of structure calculation, for each of the three solvents those 20 structures showing the lowest overall energies were selected for further characterization. No NOE distance violation was larger than 0.016 nm. No dihedral constraint was violated more than one degree in the chloroform/methanol-derived structures. The calculated structures were analyzed using the PROCHECK [27] and PROMOTIF [28] software. The coordinates have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY, with accession codes 1KZS, 1KZV, and 1KZT for the resulting structures obtained in trifluoroethanol/water, chloroform/ methanol, and dodecyl-PCho micelles, respectively. Chem- ical shifts have also been deposited at the BioMagResBank, University of Wisconsin, with accession no. 5283. RESULTS AND DISCUSSION Recently, a secondary structure prediction for HIV-1 Vpr was reported [22] employing PHD NETWORK for secondary structure prediction [29]. PHD NETWORK has a reported Table 1. Statistics of Vpr(34–51) structure calculations. Trifluoroethanol/water CHCl 3 /methanol Dodecyl-PCho Number of experimental distance restraints total number of assigned NOEs 122 82 106 intraresidue (|i ) j| ¼ 0) 16 24 25 interresidue sequential (|i ) j| ¼ 1) 64 41 53 interresidue medium range (1 < |i ) j| £ 5) 42 17 28 long range (|i ) j| > 5) 0 0 0 X - PLOR energies (kcalÆmol )1 ) total 20.76 ± 1.36 20.04 ± 0.64 22.48 ± 2.32 bond 0.49 ± 0.08 0.42 ± 0.04 0.57 ± 0.14 angle 16.91 ± 0.65 16.80 ± 0.41 17.95 ± 1.29 impropers 1.95 ± 0.15 1.92 ± 0.07 2.12 ± 0.23 repel 0.60 ± 0.39 0.35 ± 0.23 0.94 ± 0.56 NOE 0.80 ± 0.51 0.55 ± 0.15 0.90 ± 0.54 RMS deviations to the mean structure (nm) backbone (residues 34–51) 0.079 ± 0.027 0.115 ± 0.029 0.187 ± 0.048 heavy (residues 34–51) 0.145 ± 0.038 0.191 ± 0.027 0.248 ± 0.039 backbone (residues 38–50) 0.033 ± 0.018 0.033 ± 0.008 0.059 ± 0.023 heavy (residues 38–51) 0.110 ± 0.023 0.147 ± 0.019 0.117 ± 0.028 RMS deviations to experimental constraints and idealized geometry bonds (pm) 0.12 ± 0.01 0.11 ± 0.02 0.13 ± 0.02 angles (degree) 0.44 ± 0.01 0.44 ± 0.01 0.45 ± 0.02 impropers (degree) 21.58 ± 2.68 18.48 ± 3.37 20.80 ± 4.76 NOE (pm) 1.1 ± 0.3 1.2 ± 0.2 1.3 ± 0.4 F,Y angles consistent with Ramachandran plot (%) most favored regions 98.6 100 94.6 additionally allowed regions 1.4 0 2.9 generously allowed regions 0 0 1.4 disallowed regions 0 0 1.1 Ó FEBS 2002 Vpr(34–51) peptide structure (Eur. J. Biochem. 269) 3265 prediction accuracy of greater than 70%. As a result, three amphipathic helices were predicted with the first helix ranging from amino acid Asn16 to His33, the second helix from Arg36 to Thr49 and the third from Trp54 to Ile74 [22]. This is in fairly good agreement with NMR structural work in trifluoroethanol-containing solutions [19,20]. According to these secondary structure prediction results we used synthetic Vpr(34–51) peptide, which comprises the amino acids for the second putative helix, to investigate its behavior under various solvent conditions. For structural characterization of the Vpr(34–51) peptide structure we recorded homonuclear one- and two-dimen- sional NMR spectra of Vpr(34–51) in water, 100 m M dodecyl-PCho, chloroform/methanol (1 : 1, v/v) and tri- fluoroethanol/water (1 : 1, v/v). Possibly due to self-aggre- gation of the peptide, only a few broad resonances could be detected in water under several pH and ionic conditions. This is in agreement with the finding that residues in Vpr(34–51) are responsible for Vpr oligomerization [26]. Evaluation of DQF-COSY and TOCSY spectra resulted in the identifica- tion of all spin systems in all three other solvents. All resonances could be assigned sequence specifically according to dNN(i,i +1),daN(i,i +1)anddbN(i,i +1)NOEs. Interestingly, in trifluoroethanol/water two sets of reso- nances for Phe34 to Asn41 were detected. This indicates the presence of a minor population of Vpr(34–51) peptide resulting from a cis configuration of the Phe34-Pro35 peptide bond as shown by a dNa(i ) 1,i)NOE,whichis typical for a cis-aminoacyl-proline peptide bond. In each of the chloroform/methanol and 100 m M dodecyl-PCho solu- tions, however, only one set of resonances could be detected. An overview of the distance constraints used for structure calculations and structural statistics is shown in Table 1 and Fig. 1. Backbone rmsd values of 0.115 nm and 0.079 nm for methanol/chloroform and trifluoroethanol/water, respectively, show the overall structures to be well defined. Also, Vpr(34–51) peptide in 100 m M dodecyl-PCho shows a well defined a helical region from Trp38 to Tyr50 as shown by a backbone rmsd of 0.059 nm, whereas the rmsd value for the entire peptide was 0.187 nm. This is probably due to higher flexibility of the residues outside the a helix. Dihedral F and Q angles of more than 99% of all residues in the final converged structures in each of the solvents fall in either the most favorable or additionally allowed regions. Analysis with the PROCHECK [27] and PROMOTIF [28] programs revealed regularly a helical secondary structure for Vpr(34–51) peptide in each of the solvents studied. a helix was deduced for residues Ile37 to Tyr50 in chloroform/ methanol, Trp38 to Tyr50 in 100 m M dodecyl-PCho, and Pro35 to Tyr50 in trifluoroethanol/water. In the previously reported structure of Vpr(1–51)in 30% trifluoroethanol (v/v) the second helix also started at Pro35, but ended one turn earlier at residue Ile46 [19]. Comparison of the structures obtained in the different solution conditions elucidates good conformity among the structures of Vpr(34–51) peptide for residues Trp38 to Tyr50 in all three solvents (Fig. 2A). Differences can be observed only for the N-terminal residues. Not surprisingly, in trifluoroethanol/water solution the a helix content was highest among all solvent conditions. Chloroform/methanol was used previously for NMR studies of peptides and proteins to mimic hydrophobic environments [30,31]. Because the part of Vpr investigated in the present study may be relevant for the reported ion channel activity of Vpr [14], we also determined its structure in chloroform/methanol. The detected a helix content of Vpr(35–51) increased from 76 to 88 and 94% in micelle-containing solution, Fig. 1. Summary of the NOE connectivities and chemical shift index analysis of Vpr (34–51) in water/trifluoroethanol (1 : 1, v/v, A), chloroform/methanol (1 : 1, v/v, B) and 100 m M dodecyl-PCho (C). Amino acids are labeled according to the one-letter convention. NOESY connectivities relevant for secondary structure are represented by horizontal bars connecting two residues that are related by the NOE specified to the left. The height of the bars symbolizes the relative strength (weak, medium, strong) of the cross peaks in a qualitative way. Overlapping and therefore ambiguous cross-peaks are marked by an asterisk. Ha chemical shift index (CSI) is given below [34]. 3266 A. Engler et al. (Eur. J. Biochem. 269) Ó FEBS 2002 chloroform/methanol and trifluoroethanol/water, respect- ively. This is in agreement with other studies comparing peptide structures in trifluoroethanol-, chloroform- and micelle-containing solutions [35–40], where trifluoroethanol was also found to induce the highest content of a helical secondary structure. Our rationale to employ micelle-containing and non- aqueous solvents to study the structure of Vpr(34–51) is based on Vpr functions that clearly take place in or at membranes, such as the reported ion channel activity [14] and virion incorporation of Vpr [23,25], suggesting most of the various cellular and viral proteins that are reported to directly interact with Vpr may form hydrophobic environ- ments for Vpr interaction. In this respect, among the solvent conditions used for the present study, the micelle-containing solution most closely resembles a membrane environment; these conditions were able to neutralize the high intrinsic oligomerization propensity of the peptide. Thus, the following discussion is based on the structure found for the micelle-containing solution. The a helix found for residues 38–50 builds up a leucine zipper-like basis for interactions to other proteins. Residues Leu39, Leu42, Gly43 and Ile46, that are described to be essential for virion incorporation [26] are lined up on one side of the helix (Fig. 2C). Multiple sequence alignment of amino acids 34–51 of HIV-1 Vpr with sequences of all HIV-1, HIV-2 and SIV isolates deposited in the SwissProt data base (Fig. 3), shows that almost all residues in this region are highly conserved among the two groups of isolates consisting of HIV-1 and chimpanzee SIV on one side and HIV-1 and the other SIV isolates on the other side. Among all reported isolates only a few residues are 100% conserved: Leu39, Leu42, and Ile46 are 100% conserved and Gly43 is conserved in all but one isolate. The fact that exactly the same residues are involved in oligomerization of Vpr [26] complicates structural studies. The only two other residues that are 100% conserved in the Vpr region studied here are Phe34 and Gly51. Phe34 is essential for nuclear localization [18]. Mutation of Phe34 to Ile abolishes Vpr binding to importin-a and nucleoporins. Vpr residues 25–40 were reported to contain essential interaction sites for HHR23A binding [32]. A Vpr construct with a deletion of the 24 N-terminal residues still bound to HHR23A. Thus, the first amino proximal helix of Vpr does not seem to be necessary for this interaction. A construct with another 15 residues deleted from the N-terminus, Fig. 3. Multiple sequence alignment of HIV-1, HIV-2 and SIV Vpr protein sequences of the part (residues 34–51) studied in the present paper. The sequence numbers given in the top line correspond to the HIV-1 NL4-3 Vpr isolate (VPR_HV1BR). The SwissProt accession no. of the respective isolate is given on the left side. Amino-acid sequences are shown on the right side using the one-letter-code. Asterisks in the bottom line and gray boxes mark identical residues, points mark similar residues among Vpr sequences. Sequences were obtained from the SwissProt data bank. Fig. 2. Representation of Vpr(34–51) structure. (A) Backbone overlay of all structures of Vpr(34–51) peptide in trifluoroethanol/water (blue), chloroform/methanol (green), and dodecyl-PCho micelles (red). Structures were fitted to backbone atoms of residues 38–50. (B) The structure of Vpr(34–51) peptide in presence of 100 m M dodecyl-PCho is shown as surface representation from three different views, rotated by 120 degrees against each other. Orientation of the peptide is N-terminal to the top. Positive and negative electrostatic potentials are indicated by blue and red colors, respectively. (C) Residues Leu39, Leu42, Gly43 and Ile46, that are described to be essential for virion incorporation [26] are lined up on one side of the helix, as shown in a surface view where the surface built up by residues Leu39, Leu42, Gly43 and Ile46 is colored in green. Ó FEBS 2002 Vpr(34–51) peptide structure (Eur. J. Biochem. 269) 3267 however, did not bind HHR23A. This suggests that the helical region studied in the present work contains essential elements for HHR23A interaction of Vpr. The structure of the ubiquitin associated (UBA) domain of HHR23A, reported to be responsible for Vpr interaction, consists of a three helix bundle, and the potential Vpr binding surface has been reported to be a large hydrophobic and uncharged surface [33]. The helix formed by residues Trp38 to Tyr50 of Vpr reported here, is almost completely absent of charged residues (Fig. 2B) and is well suited for a leucine-zipper-like helix–helix interaction. Residues 34–51 of Vpr were barely investigated func- tionally until recently, when it was shown that this region of the protein is important for oligomerization, virion incorporation, and subcellular localization of Vpr [26]. Employing NMR spectroscopy and restrained simulated annealing molecular dynamics calculations, we obtained for Vpr(34–51) under various solution conditions extremely similar three-dimensional structures, with only minor differences for the very N-terminal residues of the peptide. The amphipathic ahelix within Vpr(34–51) with its hydro- phobic leucine-zipper-like surface may form a critical secondary structural element necessary for protein–protein interactions, but may also be responsible for the self- aggregation properties found for Vpr that substantially hinder structural investigations of the full-length protein. 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Structure of human immunodeficiency virus type 1 Vpr(34– 51) peptide in micelle containing aqueous solution Andrea Engler 1 , Thomas Stangler 2,3 and. virion incorporation of Vpr. Keywords: HIV -1; Vpr; solution structure; dodecylphos- phocholine micelles; NMR. Human immunodeficiency virus type 1 (HIV -1) is