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

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:
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.
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SUPPLEMENTARY MATERIAL
The following material is available from ck
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.