Structure of human immunodeficiency virus type 1 Vpr34–51peptide in micelle containing aqueous solution Andrea Engler1, Thomas Stangler2,3and Dieter Willbold3,4 1 Lehrstuhl fu¨r Biopolym
Trang 1Structure of human immunodeficiency virus type 1 Vpr(34–51)
peptide in micelle containing aqueous solution
Andrea Engler1, Thomas Stangler2,3and Dieter Willbold3,4
1
Lehrstuhl fu¨r Biopolymere, Universita¨t Bayreuth, Germany;2Institut fu¨r Molekulare Biotechnologie, Jena, Germany;3Institut fu¨r Physikalische Biologie, Heinrich-Heine-Universita¨t, Du¨sseldorf, Germany,4Forschungszentrum 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 G2 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 incorporalocaliza-tion 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 accessory 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 G2-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)
Trang 2M A T E R I A L S A N D M E T H O D S
Peptide
The synthetic peptide CH3-CO-FPRIWLHNLGQHIY
ETYG-NH2 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 3JHNNa 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
R E S U L T S A N D D I S C U S S I O N
Recently, a secondary structure prediction for HIV-1 Vpr was reported [22] employing PHD NETWORKfor 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
interresidue sequential (|i ) j| ¼ 1) 64 41 53
interresidue medium range (1 < |i ) j| £ 5) 42 17 28
X - PLOR energies (kcalÆmol)1)
total 20.76 ± 1.36 20.04 ± 0.64 22.48 ± 2.32
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
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
F,Y angles consistent with Ramachandran plot (%)
Trang 3prediction 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 mM
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) and dbN(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, which is
typical for a cis-aminoacyl-proline peptide bond In each of
the chloroform/methanol and 100 mMdodecyl-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 mMdodecyl-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
DihedralF 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 mMdodecyl-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 Summaryof 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].
Trang 4chloroform/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.
Trang 5however, 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 a helix 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
Therefore, the use of small micelles may be a better way
around these technical problems than the use of solvents,
which are known to interfere with tertiary structure
formation, e.g trifluoroethanol Another useful approach
may be specific rational mutation of residues that are
essential for self-aggregation properties, leaving the
conse-quences for biological function aside
A C K N O W L E D G E M E N T S
We thank Dr Karl-Heinz Gu¨hrs for carrying out mass spectroscopy.
This work was supported by a grant from the Deutsche
Forschungs-gemeinschaft (DFG) to D W (SFB 466, A4).
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