Tài liệu Báo cáo khoa học: NMR structural characterization of HIV-1 virus protein U cytoplasmic domain in the presence of dodecylphosphatidylcholine micelles doc

NMR structural characterization of HIV-1 virus protein U
cytoplasmic domain in the presence of
dodecylphosphatidylcholine micelles
Marc Wittlich
1,2
, Bernd W. Koenig
1,2
, Matthias Stoldt
1,2
, Holger Schmidt
1,2,
* and Dieter Willbold
1,2
1 Institut fu
¨
r Strukturbiologie und Biophysik (ISB-3), Forschungszentrum Ju
¨
lich, Germany
2 Institut fu
¨
r Physikalische Biologie, Heinrich-Heine-Universita
¨
tDu
¨
sseldorf, Germany
Introduction
VpU (virus protein U) is an 81 amino acid transmem-
brane protein encoded by HIV-1 and some simian
immunodeficiency virus strains, e.g. SIV
CPZ
. VpU is

not essential for virus replication in cell culture, and is
thus often called accessory protein.
The most well-defined function of VpU is downregu-
lation of CD4 in the endoplasmic reticulum, which is
mediated by the cytoplasmic region of the protein [1,2].
This function involves binding and recruitment of the
b-transducin repeat-containing protein (b-TrCP) [3,4]
Keywords
CD4; DPC micelle; HIV-1 VpU; NMR;
solution structure
Correspondence
D. Willbold, Forschungszentrum Ju
¨
lich
GmbH, ISB-3, 52425 Ju
¨
lich, Germany
Fax: +49 2461612023
Tel: +49 2461612100
E-mail:
*Present address
Max-Planck-Institute for Biophysical
Chemistry, NMR-based Structural Biology,
Go
¨
ttingen, Germany
Database
Resonance assignment tables have been
deposited at the Biological Magnetic
Resonance Data Bank (BMRB) under the

accession code 15513
(Received 31 May 2009, revised 2
September 2009, accepted 7 September
2009)
doi:10.1111/j.1742-4658.2009.07363.x
The HIV-1 encoded virus protein U (VpU) is required for efficient viral
release from human host cells and for induction of CD4 degradation in the
endoplasmic reticulum. The cytoplasmic domain of the membrane protein
VpU (VpUcyt) is essential for the latter activity. The structure and dynam-
ics of VpUcyt were characterized in the presence of membrane simulating
dodecylphosphatidylcholine (DPC) micelles by high-resolution liquid state
NMR. VpUcyt is unstructured in aqueous buffer. The addition of DPC
micelles induces a well-defined membrane proximal a-helix (residues I39–
E48) and an additional helical segment (residues L64–R70). A tight loop
(L73–V78) is observed close to the C-terminus, whereas the interhelical lin-
ker (R49–E63) remains highly flexible. A 3D structure of VpUcyt in the
presence of DPC micelles was calculated from a large set of proton–proton
distance constraints. The topology of micelle-associated VpUcyt was
derived from paramagnetic relaxation enhancement of protein nuclear spins
after the introduction of paramagnetic probes into the interior of the
micelle or the aqueous buffer. Qualitative analysis of secondary chemical
shift and paramagnetic relaxation enhancement data in conjunction with
dynamic information from heteronuclear NOEs and structural insight from
homonuclear NOE-based distance constraints indicated that micelle-associ-
ated VpUcyt retains a substantial degree of structural flexibility.
Abbreviations
DHPC, dihexanoyl phosphatidylcholine; DPC, dodecylphosphatidylcholine; DPC-d38, perdeuterated DPC; HSQC, heteronuclear single
quantum coherence; PRE, paramagnetic relaxation enhancement; TFE, trifluoroethanol; VpU, virus protein U; VpUcyt, C-terminal,
cytoplasmic domain of VpU (residues 39–81) plus N-terminal Gly-Ser dipeptide; b-TrCP, b-transducin repeat-containing protein; TASK,
TWIK-related acid-sensitive K

+
channel; TWIK, tandem of P domains in a weak inwardly rectifying K
+
channel.
6560 FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS
and depends on casein kinase II-mediated phosphoryla-
tion of two serines in VpU [5]. VpU binding to b-TrCP
does not induce its own degradation. Instead, VpU
degradation is reported to be b-TrCP independent and
involves phosphorylation of residue 61 [6].
VpU enhances virus particle release from infected
cells [1,7–10]. The underlying mechanism to enhance
virus particle release was suggested to be based on the
ability of VpU to negatively regulate cellular antiviral
factors, e.g. the potassium ion channel protein TWIK
(tandem of P domains in a weak inwardly rectifying
K
+
channel)-related acid-sensitive K
+
channel
(TASK) [11,12]. More recent studies have reported
that VpU even redirects nascent viral particles to the
cytoplasmic membrane [13,14].
VpU was shown to be required for efficient replica-
tion of chimeric simian–human immunodeficiency
viruses in macaques, underscoring its critical role in
viral pathogenesis [15,16].
The 81 amino acid sequence of VpU can be divided
into three distinct domains. A short stretch of basic res-

idues (Y27–K38, notation according to strain HV1S1)
connects the transmembrane part (I6–V26) and the
extremely acidic cytoplasmic domain (I39–L81). The
transmembrane domain consists of a well-characterized
and defined a-helix (referred to as helix 1) [17–20]. The
structure of the cytoplasmic domain was investigated
by various groups under diverse solution conditions
and at different levels of sophistication. VpU-derived
peptides were studied in native buffer [21], in trifluoro-
ethanol (TFE) solution [22,23], under high salt condi-
tions [24], in the presence of detergent micelles of
dihexanoyl phosphatidylcholine (DHPC) [25] or dod-
ecylphosphatidylcholine (DPC) [26], and associated to
phospholipid membranes [1,27,28]. There is consensus
on the formation of two cytoplasmic helices (helices 2
and 3) in various solvent conditions, but the extension
of helix 3 varies substantially [23–26]. The observation
of additional structural elements and, possibly, a ter-
tiary fold of the cytoplasmic domain of VpU remains
highly debated. To date, the most detailed descriptions
of the soluble VpU region are based on proton–proton
distances derived from solution NMR. Unfortunately,
these studies have been conducted in 50% TFE [22,23]
or in buffer containing 500 mm sodium sulfate [24],
conditions that might induce artificial conformations.
TFE stabilizes the secondary structure and supports the
formation of a-helices [29]. Furthermore, TFE may
weaken the tertiary structure by destabilizing hydro-
phobic interactions [30–33]. Very high ionic strength
appeared to induce a tertiary fold in the cytoplasmic

region of VpU, as indicated by a small number of
observed long-range NOEs [24].
Another important aspect is the topology of the
cytoplasmic domain of VpU on a membrane. Solid
state NMR suggests an orientation of helix 2 parallel
to the membrane surface; data on helix 3 are again
contradictory [1,27,28].
The current study combined diverse solution NMR
experiments and addressed the structure, dynamics and
topology of membrane-associated VpUcyt, a polypep-
tide representing the cytoplasmic domain of VpU.
DPC micelles provided a membrane-like environment
that avoided the shortcomings of organic solvents or
high salt conditions.
Results
CD spectroscopy
CD spectra of VpUcyt (53 lm) were recorded in the
presence and absence of membrane-mimicking DPC
micelles (Fig. 1). The spectrum obtained in detergent-
free buffer showed a pronounced minimum at 199 nm
indicative of a predominantly unordered protein. The
addition of DPC micelles caused local minima near 220
and 205 nm and a maximum around 195 nm, reminis-
cent of the extreme values at 222, 208 and near 190 nm
expected for a regular a-helix [34]. The detergent con-
centration was varied from 5 to 100 mm, well above the
critical micelle concentration of DPC (1.5 mm in H
2
O).
The initial addition of 5 mm DPC caused the most pro-

nounced change in the CD spectrum, whereas increasing
the detergent concentration further enhanced the helical
character only moderately and a clear saturation of the
effect was observed. In particular, the amount of unor-
dered secondary structure elements could be estimated
to be clearly more than 80% in the absence of DPC.
Upon the addition of 100 mm DPC, a substantial
Fig. 1. CD spectra of VpUcyt (53 lM) in sodium phosphate buffer
with and without membrane-mimicking DPC micelles.
M. Wittlich et al. NMR structure of micelle-associated HIV-1 VpUcyt
FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS 6561
fraction of  30% a-helical secondary structure and
 16% turn formed, whereas only  40% of unordered
conformations remained.
NMR spectroscopy and resonance assignment
The experimental conditions for the NMR study of
VpUcyt in the presence of membrane-mimicking
micelles were carefully optimized. First, various combi-
nations of buffer composition, choice of detergent and
temperature were tested. High-quality
15
N-
1
H-hetero-
nuclear single quantum coherence (HSQC) spectra
with enhanced spectral dispersion and the expected
number of cross-peaks of VpUcyt were obtained with
perdeuterated DPC (DPC-d38) at 30 °C. A series of
HSQC spectra of 1 mm VpUcyt was recorded with
varying amounts of DPC-d38 in the sample (from 0 to

200 mm). Many protein resonance positions changed
in a continuous manner with increasing detergent con-
centration and approached new final values in an
asymptotic manner. No further chemical shift variation
occurred above 100 mm DPC-d38. The observed chem-
ical shift changes reflected modifications in the local
environment of the corresponding nuclei, perhaps due
to conformational changes, including hydrogen bond
formation or intermolecular contact with detergent
molecules. Taking into account the aggregation num-
ber of DPC in water ( 50–60 DPC molecules per
micelle [35]), we assumed that micelle-associated
VpUcyt was present at 100 mm DPC. A single set of
VpUcyt NMR signals was observed at 100 mm DPC
(Fig. 2A, red contours), indicating either a uniform
protein conformation or rapid exchange on the chemi-
cal shift time scale between different VpUcyt con-
formational states.
Virtually complete assignment of
1
H,
15
N and
13
C
resonances of VpUcyt at 30 °C in buffer with 100 mm
DPC-d38 was accomplished on the basis of a series of
3D NMR experiments recorded on uniformly
15
N- and

13
C-labelled VpUcyt. Resonance assignment tables
were deposited at the BMRB (accession code: 15513).
An overlay of
15
N-
1
H-HSQC spectra of VpUcyt
recorded in detergent-free buffer (black) and in the
presence of 100 mm DPC-d38 (red) is shown in
Fig. 2A. The corresponding chemical shift changes in
backbone amide
1
H and
15
N spins of VpUcyt, together
with a weighted average of the absolute changes, are
presented as a function of the amino acid sequence
position in Fig. 2B–D. Continuous stretches with
prominent chemical shift changes were observed for
residues 39–51 and 64–78. In contrast, amide chemical
shifts of residues 52–63 were virtually unaffected by
the presence of DPC micelles.
Local helix propensity derived from chemical
shifts
The difference between the observed chemical shifts of a
protein and the corresponding amino acid residue-spe-
cific random coil chemical shifts is referred to as second-
ary chemical shift. In particular,
13

C
a
,
1
H
a
and
13
CO
secondary shifts are sensitive indicators of a-helix and
b-sheet elements [36–38]. The formation of a regular
A
B
C
D
Fig. 2. (A)
1
H-
15
N-HSQC spectra of VpUcyt in the presence (red)
and absence (black) of micelles (100 m
M DPC-d38). Displacement
of selected resonances upon detergent addition is indicated by bro-
ken lines. Side chain amide correlations of glutamine and aspara-
gine residues are connected by a continuous line. Chemical shift
changes of backbone amide H
N
(B) and
15
N (C) resonances upon

the addition of DPC are shown as a function of sequence position.
A measure of the total chemical shift change {D
total
d =[(Dd
1
H)
2
+ (0.1 · Dd
15
N)
2
]
1 ⁄ 2
} [88] is presented in (D). No amide corre-
lation of L42 was observed in the detergent-free sample.
NMR structure of micelle-associated HIV-1 VpUcyt M. Wittlich et al.
6562 FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS
a-helix is indicated by downfield shifts of
13
C
a
and
13
CO and upfield shifts of
1
H
a
resonances with average
changes of 2.6, 1.7 and 0.37 p.p.m., respectively,
whereas b-sheet conformation is indicated by shifts in

the opposite direction [36,37]. The chemical shifts of a
flexible peptide undergoing rapid exchange between
several states are linear combinations of the popula-
tion-weighted conformation-specific chemical shifts.
Secondary
13
C
a
,
1
H
a
and
13
CO shifts of VpUcyt
determined in the absence of detergent did not provide
any indication of secondary structure (Fig. 3, left col-
umn). In contrast, characteristic secondary shifts of
VpUcyt observed in the presence of 100 mm DPC
clearly indicated the formation of two helices (Fig. 3,
right column). They will be referred to as helices 2 and
3 in accordance with the helix nomenclature of full-
length VpU, where helix 1 designates the N-terminal
transmembrane helix of the protein. On the basis of
the three sets of secondary shift data in Fig. 3 (right),
the helices probably range from I39 to E48 (helix 2)
and from L64 to R70 (helix 3).
The fractional helicity of amino acid stretches 39–48
(helix 2) and 64–70 (helix 3) was estimated by compar-
ing the observed average secondary shifts with the

values expected for a regular helix. This procedure
provided fractional helicities of  80% (Dd
13
C
a
) for
helix 2 and 40% (Dd
1
H
a
) for helix 3.
NOE-derived secondary structure and tertiary fold
of VpUcyt in the presence of DPC micelles
2D
15
N-edited NOESY spectra of VpUcyt recorded
with and without DPC micelles in the sample were
very different (Fig. 4). The number of cross-peaks was
rather limited in DPC-free buffer, but strongly
increased upon the addition of micelles. In particular,
a substantial number of d
NN
(i,i + 1) cross-peaks
emerged near the diagonal, indicating helical segments.
Extensive signal overlap in 2D NOESY spectra in
combination with rather broad lines was overcome by
Fig. 3. Secondary chemical shifts of VpUcyt
observed in the absence (left) and presence
(right) of detergent micelles (100 m
M DPC-

d38). Solid bars represent amino acid resi-
dues 39–81 of VpU. The dotted lines mark
the theoretical average values of the down-
field shift of
13
C
a
(top) and
13
CO (bottom) as
well as of the upfield shift of
1
H
a
reso-
nances (middle) characteristic of a 100%
helical secondary structure. VpUcyt appears
to be unstructured in buffer. The addition of
DPC micelles induced helical characteristics
in two regions of the peptide. The most
probable extension of the two helices is
indicated by a grey background.
Fig. 4. Sections of
15
N-edited
1
H-
1
H NOESY spectra of VpUcyt
recorded in the absence (left) and presence (right) of 100 m

M
DPC-d38 using identical acquisition and processing parameters.
M. Wittlich et al. NMR structure of micelle-associated HIV-1 VpUcyt
FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS 6563
the acquisition of heteronuclear-edited 3D NOESY
experiments of VpUcyt in DPC-containing buffer.
Secondary structure-specific short- and medium-
range NOEs are summarized in Fig. 5. Strong
d
NN
(i,i + 1) cross-peaks in conjunction with less
intense d
aN
(i,i + 1) peaks and continuous stretches of
d
aN
(i,i + 3), d
ab
(i,i + 3), and perhaps d
aN
(i,i +4) or
d
aN
(i,i + 2) peaks are indicative of helices. The two
helices deduced from the chemical shift data (Fig. 3)
are also clearly discernable in the NOE diagram. Helix
2 exhibits all classes of NOE cross-peaks expected.
Helix 3 displays several d
aN
(i,i + 2) peaks in addition

to a complete series of d
ab
(i,i + 3) cross-peaks. Only
unambiguously identified cross-peaks are displayed in
Fig. 5, which explains the absence of a few signature
cross-peaks in the helical regions. Indeed, ambiguous
NOE cross-peaks were present at all d
aN
(i,i + 3) and
d
aN
(i,i + 4) positions that would be predicted if resi-
dues 64–68 of VpUcyt formed an a-helix. However,
the lack of unambiguous d
aN
(i,i + 4) peaks in con-
junction with the detection of d
aN
(i,i + 2) cross-peaks
in the sequence region of helix 3 may indicate that
helix 3 is not as regular as a-helix 2 and may even be
of the 3
10
kind. Most residues in the interhelical linker
(R49–E63) and the C-terminal region of VpUcyt from
G71 to L81 exhibited more intense d
aN
(i,i + 1) than
d
NN

(i,i + 1) cross-peaks, a feature that is incompatible
with a rigid regular helical structure [39].
Calculation of the VpUcyt structure in micelle solu-
tion employed 604 upper distance limits derived from
unambiguous NOESY cross-peaks. The set of
1
H-
1
H
distances consisted of 147 intraresidue, 223 sequential,
219 medium-range (2 £ |i ) j| £ 5), and 15 long-range
(|i ) j| > 5) constraints. All 15 long-range connectivi-
ties were encoded by weak NOEs that gave rise to upper
distance limits of 0.55 nm (Table 1). Stripes from a
13
C-
resolved NOESY experiment exemplifying long-range
NOEs of VpUcyt in the presence of DPC micelles are
shown in Fig. 6. Long-range NOEs are crucial for delin-
eating the tertiary fold of VpUcyt. Multiple long-range
Fig. 5. Summary of
1
H-
1
H connectivities of VpUcyt in DPC micelle solution derived from 3D NOESY spectra. The amino acid sequence of
VpUcyt is shown at the top. Capital letters denote residues 39–81 of VpU. The N-terminal Gly-Ser dyad in lower case remains on VpUcyt
after thrombin cleavage of the fusion protein. In case of sequential HN(i) ⁄ HN(i + 1) and H
a
(i) ⁄ HN(i + 1) cross-peaks, the height of the boxes
is proportional to the estimated NOE intensities. Observation of additional classes of NOE interactions is visualized in the six rows below.

Ambiguous or strongly overlapped cross-peaks have been omitted. Helices 2 and 3, as well as a tight loop of VpUcyt, are indicated by grey
stripes and an open rectangle, respectively. The two helices are connected by an interhelical linker. Structural elements are denoted at the
bottom of the diagram.
Table 1. Long-range NOEs of VpUcyt in the micellar environment.
Observed NOE
Chemical shifts of
cross-correlated protons (p.p.m.)
I43-CH
3
c
-E69-CH
2
b
1.205 2.139; 2.064
D44-H
a
-R70-CH
2
c
4.458 1.774
T47-H
b
-Q61-CH
2
b
4.430 2.109; 2.232
T47-H
b
-Q61-CH
2

c
4.430 2.457; 2.461
A50-CH
3
b
-P75-CH
2
d
1.509 1.846; 1.940
G54-CH
2
a
-W76-H
a
4.070 4.777
H72-H
d
-V78-H
a
7.332 4.207
H72-H
d
-V78-CH
3
c
7.332 1.017
H72-H
e
-V78-CH
3

c
8.641 1.017
H72-H
e
-L81-H
c
8.641 1.725
H72-H
e
-L81-CH
3
d
8.641 0.933
NMR structure of micelle-associated HIV-1 VpUcyt M. Wittlich et al.
6564 FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS
NOEs suggested spatial proximity between helix 2 and
amino acids just N-terminal of helix 3 (T47-H
b
-E61-
CH
2
b
; T47-H
b
-E61-CH
2
c
) and immediately C-terminal
of helix 3 (I43-CH
3

c
-E69-CH
2
b
; D44-H
a
-R70-CH
2
c
).
This subset of NOEs indicated an approximately anti-
parallel arrangement of helices 2 and 3. Other long-
range NOEs were observed between the interhelical
linker and C-terminal residues (A50-CH
3
b
-P75-CH
2
d
;
G54-CH
2
a
-W76-H
a
) and between C-terminal residues
defining a tight loop (H72-H
d
-V78-CH
3

c
; H72-H
e
-V78-
CH
3
c
; H72-H
d
-V78-H
a
; H72-H
e
-L81-H
c
; H72-H
e
-L81-
CH
3
d
). One would expect observation of multiple
contacts between pairs of residues that give rise to long-
range NOEs. Indeed, several long-range NOEs were
observed between T47 and Q61, between H72 and V78,
and between H72 and L81 (Table 1). The NOESY data
were scrutinized extensively to identify additional long-
range NOEs between the pairs of residues listed in
Table 1. For example, the observed NOE between I43
CH

3
c
and E69 CH
2
b
should be accompanied by a
detectable NOE between I43 CH
3
d
and E69 CH
2
c
. How-
ever, the corresponding cross-peak exists, but is highly
ambiguous due to spectral overlap. The assignment of
all observed long-range NOEs was carefully checked.
Only unambiguously identified long-range NOEs were
used for structure calculation. This conservative
approach explains the limited number of long-range
NOEs that were employed for structure calculation as
listed in Table 1.
A set of 100 VpUcyt conformers was generated by the
program cyana starting from randomized conforma-
tions and using the 604 distance constraints as the only
experimental input. The 20 structures with the lowest
energy were selected for statistical analysis (Table 2).
The entire set of 604 distance restraints was reasonably
well satisfied in all 20 conformers; the maximum dis-
tance violation amounted to 0.021 nm. The geometric
quality of the calculated structures was acceptable; 89%

of the analysed backbone torsions fell into the most
favoured and additionally allowed regions of the Rama-
chandran plot. An additional 6% of residues were
found in the generously allowed region. None of the res-
idues of helices 2 and 3 was found in the disallowed
regions. Instead, Procheck-NMR [40,41] identified
various subsets of one or a few residues in the less well-
defined loop connecting the two helices (N55, D60, E63)
and ⁄ or at both ends of VpUcyt (S38, V78, D79, D80) in
the disallowed region of the Ramachandran plot.
Superposition of the 20 lowest energy conformers
yielded a narrow bundle of backbone traces with the
two helices nicely visible (Fig. 7). The calculated struc-
tures showed high convergence for helices 2 and 3,
whereas both termini and the interhelical linker were
less well defined. Another common structural element
was a tight loop formed by residues L73–V78 with
W76 located at the tip of the loop. The loop gave rise
to three consecutive medium-range d
ab
(i,i + 3) NOEs
(Fig. 5). A prominent hydrogen bond connects A74-
CO and V78-NH.
Variability of individual structural elements is
reflected by the corresponding rmsd values in Table 2.
Fig. 6. Stripes from 3D
13
C-resolved HSQC-NOESY experiment of
VpUcyt recorded in the presence of 100 m
M DPC-d38 showing six

exemplary long-range
1
H-
1
H NOEs.
Table 2. Analysis of the 20 lowest energy VpUcyt structures in
DPC micelles.
Experimental restraints
Total NOE restraints 604
Intraresidue 147
Sequential 223
Medium range (2 £ |i ) j| £ 5) 219
Long range (|i ) j| > 5) 15
CYANA structural statistics
Target function (nm
2
) 0.0025 ± 0.0006
Sum of NOE violations
a
> 0.015 nm 0.25 nm
Maximum NOE violation in the ensemble 0.021 nm
rmsd to mean structure (nm) (backbone only ⁄ all heavy atoms)
VpUcyt (39–80) 0.100 ⁄ 0.149
Helix 2 (39–48) 0.027 ⁄ 0.081
Interhelical linker (49–63) 0.074 ⁄ 0.141
Helix 3 (64–70) 0.030 ⁄ 0.091
Tight loop (73–78) 0.016 ⁄ 0.046
Helix 3 and loop (64–78) 0.049 ⁄ 0.088
Ramachandran analysis
Most favoured 58%

Additionally allowed 31%
Generously allowed 6%
Disallowed 5%
a
The sum of all NOE violations larger than 0.015 nm was calcu-
lated for each structure and the mean value is shown.
M. Wittlich et al. NMR structure of micelle-associated HIV-1 VpUcyt
FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS 6565
In contrast to the well-defined helices and the tight
loop, there was considerable fuzziness in the interheli-
cal linker region. Likewise, the relative position of
helix 3 and the tight loop was poorly defined according
to the relatively high rmsd of the combined helix 3
plus loop fragment.
The 15 long-range NOEs resulted in a defined ter-
tiary fold of the calculated VpUcyt structure family.
Helices 2 and 3 adopted an approximately antiparallel
orientation, whereas the interhelical linker spanned a
plane that was almost perpendicular to the two helix
axes. Residues S53 and S57 of the interhelical linker
constituted the functionally important phosphorylation
motif of VpU. Interestingly, long-range NOEs between
G54 and W76 suggest spatial proximity of the C-termi-
nal region of VpU to the serine motif (Fig. 7).
Dynamic characterization of VpUcyt by
1
H-
15
N-heteronuclear NOE data
Data on VpUcyt dynamics were recorded in order to

investigate whether the reduced structural definition of
the interhelical and C-terminal regions was due to
increased mobility of the respective residues. Hetero-
nuclear
1
H-
15
N NOE data reflect local variations in
protein backbone dynamics on the pico- to nano-
second time scale. Positive
1
H-
15
N NOE values close
to 0.8 are expected in the absence of fast internal
motions of protein backbone N-H bond vectors [42].
Rapid internal motion will reduce the NOE, which
may even become negative for highly mobile residues
exhibiting large amplitude motions on a sub-nano-
second time scale [38].
Figure 8 shows
1
H-
15
N NOEs of VpUcyt backbone
amides in the presence and absence of DPC micelles.
Small and rather consistent
1
H-
15

N NOEs were
observed for VpUcyt in DPC-free solution, indicating
large backbone motions and no preference for a rigid
structure. The two C-terminal amino acids exhibited
the highest mobility. The addition of DPC micelles
resulted in larger heteronuclear NOEs throughout the
entire sequence, suggesting reduced dynamics in virtu-
ally all regions of VpUcyt. In particular, residues in
helix 2 showed
1
H-
15
N NOEs close to the slow motion
limit, indicating a well-defined secondary structure ele-
ment that was rigid in the pico- to nanosecond time
scale. Heteronuclear NOEs of backbone amides of res-
idues immediately following helix 2 and in the
sequence stretch covering helix 3 and the tight loop
had intermediate values. This suggests that the respec-
tive residues have a reduced mobility, although these
regions are not as stiff as helix 2. With the exception
of helix 2, the level of backbone dynamics was consis-
tently higher than expected for a stable and rigid fold.
Chemical exchange between multiple conformations
might explain the observed intermediate values of the
1
H-
15
N NOEs. A particularly high mobility was
A

B
Fig. 8.
1
H-
15
N-hetero-NOE values of backbone amides of VpUcyt in
the absence (A) and presence (B) of 100 m
M DPC-d38. Intensities
of R45 and V68 could not be determined due to heavy signal over-
lap; P75 lacks a backbone amide group. Helical regions and a tight
loop of VpUcyt are denoted by grey stripes and an open rectangle,
respectively.
Helix 3
Loop
Linke
r
Helix 2
N
C
Fig. 7. Backbone line representation of the 20 lowest energy con-
formers of VpUcyt calculated from distance restraints in 100 m
M
DPC-d38 solution (left). The overlay is based on minimizing the
rmsd between amino acid residues 39–78. The ribbon diagram of a
low-energy conformer of VpUcyt is shown on the right. The tight
loop (residues 73–78) is shown as a green worm. Side chains of
the Ser53 and Ser57 in the interhelical linker, forming a highly con-
served phosphorylation motif, are visualized in ball-and-stick format.
NMR structure of micelle-associated HIV-1 VpUcyt M. Wittlich et al.
6566 FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS

retained in the centre of the interhelical linker and at
the C-terminus of micelle-associated VpUcyt.
Position of VpUcyt relative to the micelle
NMR signal intensities of individual VpUcyt regions
were quenched quite differently by the paramagnetic
probes 16-doxylstearic acid and Mn
2+
(Fig. 9). Incor-
poration of the doxyl probe into the micelle reduced
backbone amide cross-peak intensities of residues in
helices 2 and 3 on average to  30% of their original
values (Fig. 9A). Residues in the interhelical linker
experienced only minor reductions. In particular, the
central residues of the linker were almost unperturbed.
Close to complete signal quenching was observed for
residues in the C-terminal tight loop. Also, backbone
amide cross-peaks of residues between helix 3 and the
loop were strongly reduced in the presence of the
doxyl-bearing fatty acid.
Mn
2+
ions quenched the cross-peaks originating
from residues in the interhelical linker almost com-
pletely (Fig. 9B). Strong signal quenching also applied
to the tight loop and the C-terminus of VpUcyt. In
contrast, most cross-peaks originating from helices 2
and 3, as well as from residues between helix 3 and the
tight loop, were least affected and remained at levels
between 20% and 50%.
The paramagnetic relaxation enhancement (PRE)

data indicated a location of helices 2 and 3, as well as
of the residues between helix 3 and the loop, in the
micelle–water interface region. The highly anionic
interhelical linker was solvent exposed and fully acces-
sible to the Mn
2+
ions. Residues at both ends of the
interhelical linker were superficially associated with the
micelle interface and remained easily accessible by
water-soluble Mn
2+
ions. The strongly charged C-ter-
minal end of VpU (D
77
-VDD-L
81
) was partially pro-
tected from quenching by the doxyl probe and the
protection level increased towards the C-terminus. Fur-
thermore, resonances of these last five residues became
almost undetectable in the presence of Mn
2+
. The sol-
vent-exposed C-terminus of VpUcyt probably pointed
away from the surface of the micelle. The three amide
cross-peaks originating from the hydrophobic cluster
L
73
-AP-W
76

in the tight loop were strongly quenched
by both the 16-doxylstearic acid and the Mn
2+
ions.
This unique behaviour might be caused by dynamic
exchange of this residue stretch between micelle-
embedded and water-exposed conformations.
Discussion
VpUcyt is completely unfolded in TFE-free, ‘low’
salt aqueous solution
Secondary chemical shift analysis of VpUcyt in
TFE-free aqueous solution at a physiological salt con-
centration (Fig. 3, left) revealed complete absence of
secondary structure elements. The heteronuclear NOE
data are consistent with a highly flexible protein lack-
ing a well-defined backbone conformation (Fig. 8A).
CD spectra of VpUcyt in detergent-free buffer con-
firmed the absence of a secondary structure (Fig. 1).
The presented data are the most comprehensive
account of the lack of a conformational preference of
VpUcyt in low salt buffer published to date. Previous
studies on peptides from the cytoplasmic domain of
VpU in low salt buffer relied exclusively on CD
spectroscopic data [21,22].
DPC micelles induce well-defined secondary
structure elements and a tertiary fold in VpUcyt
The addition of DPC micelles induced two helices in
VpUcyt covering residues I39–E48 and L64–R70 as
well as a tight loop (L73–V78) close to the C-terminus
A

B
Fig. 9. PRE data of VpUcyt in DPC micelles. Paramagnetic probes
16-doxylstearic acid in the interior of the micelle (A) and Mn
2+
in
the aqueous buffer (B) selectively attenuate distinct regions of VpU-
cyt, reflecting the topology and the dynamics of the micelle-associ-
ated protein. Helical regions and a tight loop of VpUcyt are denoted
by grey stripes and an open rectangle, respectively.
M. Wittlich et al. NMR structure of micelle-associated HIV-1 VpUcyt
FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS 6567
(Fig. 7). The number of residues in helical regions of
the NMR-derived structures is in good agreement with
the  30% a-helical secondary structure content esti-
mated from the CD spectra of VpUcyt in 100 mm
DPC. Helix 2 of VpUcyt in DPC micelles is slightly
shorter at the C-terminal end in comparison with the
corresponding helix in earlier studies on VpU peptides
in 50% TFE (helix 2 spans residues 37–51) [22,23], in
DHPC micelles (helix 2 spans residues 30–49) [25], or
in high salt buffer (helix 2 spans residues 40–50) [24].
The N-terminal start of helix 2 cannot be compared
due to the different lengths of the VpU peptides
studied.
The length and sequence position of helix 3 differ
appreciably between studies. It is shortest in the pres-
ence of DPC micelles (residues 64–70), slightly
extended in high salt buffer (residues 60–68) [24], but
approximately twice as long in DHPC micelles (resi-
dues 58–70) [25] and in 50% TFE (residues 57–72)

[23]. A turn bounded by VpU residues 73 and 78 was
observed in 50% TFE [23], whereas a short helix (resi-
dues 75–79) was detected under high salt conditions
[24]. Both elements bear structural similarity to the
tight loop formed by L73–V78 of VpUcyt in the pres-
ence of DPC micelles. Although the structural motifs
adopted by the cytoplasmic domain of VpU appear to
be qualitatively similar under various membrane-mim-
icking conditions, the extension of helix 3 seems to be
highly sensitive to the local environment of the
protein.
In comparison with the DPC-free solution, the con-
formational flexibility of VpUcyt was heavily reduced
upon the addition of DPC micelles. Heteronuclear
NOE values close to 0.8 suggest a well-structured helix
2 (Fig. 8B). However, reduced
15
N-
1
H NOEs of resi-
dues in the interhelical linker and the intermediate
15
N-
1
H NOEs observed for the C-terminal half of
VpUcyt indicate a substantial amount of remaining
conformational flexibility. PRE and secondary chemi-
cal shift data support the proposed conformational
exchange in the region C-terminal of the interhelical
linker (see above).

NOESY data recorded on a protein undergoing
dynamic exchange contain contributions from different
conformations. A faithful reconstruction of the confor-
mational ensemble that gives rise to the observed spec-
trum is not straightforward. The single tertiary fold of
VpUcyt derived from the experimental NOE data
should therefore be considered as a ‘limit’ structure. A
limit structure does not necessarily represent the time
and population-weighted mean structure of a protein,
but may contain structural motifs from several, more
or less different conformations in dynamic exchange.
The observed secondary structure elements and the ter-
tiary contacts may be present to a different extent in
each individual conformation. Interestingly, the com-
plete set of upper distance constraints extracted from
NOESY experiments on VpUcyt in the presence of
DPC micelles is simultaneously satisfied in the con-
verged low-energy VpUcyt conformers presented in
Fig. 7. We conclude that the tertiary fold described
here is feasible and might be adopted by a substantial
fraction of micelle-bound VpUcyt.
Topology of micelle-bound VpUcyt
The position of VpUcyt relative to the micelle–water
interface was uncovered by selective PRE of protein
nuclear spins. The paramagnetic agents employed were
confined either to the hydrophobic interior of the
Fig. 10. Surface representations of VpUcyt with amino acids colour
coded based on PRE data (top). The green colour indicates residues
that are mainly affected by 16-doxylstearic acid, suggesting spacial
proximity to the interior of the micelle. The red colour indicates res-

idues predominantly affected by Mn
2+
, suggesting exposure of the
amino acid to water. Colour saturation correlates with the extent of
signal attenuation (Fig. 9). Residues strongly affected by both spin
labels are coloured in yellow, which arises from superposition of
red and green intensities. The molecule has been empirically
aligned in such a way that those parts of the protein structure that
are probably immersed in the micelle are pointing downwards. The
vertical arrow represents the normal of the micelle–water interface.
Two faces of the same structure are shown. They are related to
each other by a 180° rotation about the normal. Ribbon diagrams of
the same VpUcyt conformer are shown at the bottom. The orienta-
tion of molecular representations shown in the same column is
identical.
NMR structure of micelle-associated HIV-1 VpUcyt M. Wittlich et al.
6568 FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS
micelle (16-doxylstearic acid) or to the aqueous buffer
(Mn
2+
). Figure 10 shows a surface plot of a represen-
tative VpUcyt structure colour-coded according to the
PRE data. Residues strongly affected by 16-doxylstea-
ric acid but rather insensitive to quenching by Mn
2+
are shown in green. Residues with opposite quenching
characteristics, i.e. strong signal reduction after the
addition of Mn
2+
but very little response to the doxyl

probe, are coloured in red. Intermediate behaviour is
indicated by shades of light green, yellow and orange,
reflecting increasing water accessibility in this order.
Opposite faces of the same VpUcyt structure are dis-
played in the upper row of Fig. 10. The orientation of
the presented molecule was manually adjusted to
reflect the PRE data in the following way: green-col-
oured regions of the protein that appear to be close to
the core of the micelle but distant from water are
pointing downwards; red-coloured elements that
should be highly water exposed are positioned as close
as possible to the upper edge of the drawing area. The
vertical arrow represents the normal vector of the
micelle–water interface. Each surface plot and the cor-
responding ribbon representation shown underneath
depict the same orientation of VpUcyt.
The question arises, can the NOE-derived tertiary
fold of VpUcyt be reconciled with the residue-specific
PRE data in Fig. 9? The orientation of VpUcyt rela-
tive to the micelle normal shown in Fig. 10 is com-
patible with many, but not all, of the PRE data.
Helices 2 and 3, as well as the amino acids located
between helix 3 and the tight loop, partially dive into
the micelle and are largely shielded from water
(green). In contrast, central residues of the interhelical
linker extend away from the detergent–water interface
(red). Both ends of the interhelical linker and the last
three residues of VpUcyt exhibit intermediate quench-
ing characteristics (orange) and may occupy a region
close to both the interior of the micelle and the aque-

ous buffer. NMR signals of residues in the tight loop
are almost completely quenched by both Mn
2+
and
16-doxylstearic acid, symbolized by the yellow colour
in Fig. 10. These loop residues seen in the upper part
of the VpUcyt projections in Fig. 10 show strikingly
different quenching characteristics than the surround-
ing residues. Residues 73–78 appear to be both fully
accessible to the solvent and close to the centre of
the micelle. The observed PRE data of the tight loop
may arise from conformational exchange involving
dynamic relocation of loop residues between micelle
and buffer. We speculate that residues 64–72 remain
in intimate contact with the micelle throughout the
exchange, whereas residues 73–78 sample qualitatively
different environments. Reasonable flexibility of the
amino acid stretch 64–78 is also evident from the het-
eronuclear NOE values observed for this region
(Fig. 8B). Earlier solid state NMR data on short
peptides from the cytoplasmic region of VpU in
lipid membranes indicated that helix 2 is bound to
the membrane and runs parallel to the lipid–water
interface, whereas no preferred orientation could
be detected for helix 3 [27]. We conclude that the
C-terminal half of micelle-associated VpUcyt retains a
certain degree of structural flexibility, which may well
be relevant for at least one of VpU’s reported activi-
ties, e.g. to act as viroporin [43].
Functional role of protein flexibility

Viral proteins such as HIV-1 Vpr and Tat, together
with many others, are often referred to as fully or
partially flexible, intrinsically unstructured, or natively
unfolded proteins. Under standard solution condi-
tions, such proteins show a high degree of conforma-
tional disorder and flexibility. These proteins
frequently possess propensities for various secondary
structure elements that are adopted only temporarily
and ⁄ or in a fraction of the protein population.
Recent data suggest that even proteins that adopt a
well-defined structure by conventional standards may
exhibit minor populations of additional conforma-
tions. Some of those transiently formed conforma-
tions may be perfectly suited for a selected protein
ligand interaction. The distinguished protein confor-
mation is then recognized by the binding partner.
Directed withdrawal of a particular conformational
subpopulation from the equilibrium is counteracted
by a continuous readjustment of the conformational
ensemble [44]. This scenario of ‘conformational selec-
tion’ was recently proposed as an alternative to the
traditional ‘induced fit’ model of protein interactions
[44].
Viral proteins often target numerous cellular factors.
A diversified set of protein conformational subpopula-
tions is required for productive interaction with multi-
ple targets in the frame of the ‘conformational
selection’ model. Proteins referred to as ‘intrinsically
unstructured’ or ‘natively unfolded’ may therefore be
well adapted for interaction with diverse partners. This

is exactly was has been observed and described for
lentiviral Tat [32,45–52] and Vpr [53–56]. A well-
defined and rigid tertiary structure of these proteins is
observed only in complexes with one of their ligands
[51,52,57,58]. VpU also interacts with a variety of
cellular targets. In this respect, it is not surprising that
VpU exhibits a certain degree of structural flexibility
in the absence of ligands.
M. Wittlich et al. NMR structure of micelle-associated HIV-1 VpUcyt
FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS 6569
Biological implications of the observed VpUcyt
structure
One of the most studied activities of VpU is the induc-
tion of CD4 degradation in the endoplasmatic reticu-
lum of infected CD4
+
cells. Only the cytoplasmic
domain of VpU, together with any membrane anchor,
is essential for this activity [59], suggesting that a mem-
brane environment is relevant for the cytosolic
domain. The transmembrane part of VpU is essential
for efficient viral release from human host cell [60].
This transmembrane part is reported to form penta-
mers. The structural study of membrane-inserted pen-
tamers of full-length VpU is certainly a highly
attractive research topic and may be studied by solid
state NMR methods. However, the objective of the
present study was to gather structural and dynamic
data of VpUcyt in the presence of membrane-simulat-
ing DPC micelles by high-resolution liquid state

NMR. Our data show that VpUcyt becomes at least
partially structured in the presence of membrane-mim-
icking DPC micelles. In full-length VpU, the cytoplas-
mic domain is indifferently anchored to a lipid
membrane. We propose that the structure of micelle-
associated VpUcyt is a reasonable approximation of
the physiologically relevant membrane-attached cyto-
plasmic region of VpU. This view is supported by the
experimentally confirmed location of VpUcyt at the
micelle–water interface. The polar headgroup of DPC
is chemically identical to that of the large fraction of
phospholipids in biological membranes that feature a
phosphatidylcholine headgroup. The observed struc-
ture of micelle-bound VpUcyt is obviously very differ-
ent from the completely unordered VpUcyt in plain
buffer. The association of peptides with lipid mem-
branes often has a pronounced influence on peptide
structure and may be a crucial prerequisite for produc-
tive interaction of a peptide or protein domain with its
membrane receptor [61].
VpU-induced proteasomal degradation of newly syn-
thesized CD4 in the endoplasmic reticulum requires
post-translational phosphorylation of VpU residues
S53 and S57 by casein kinase type II [62]. These two
serines are located in the interhelical linker, which
retains a high degree of structural flexibility upon the
addition of DPC micelles. Chemical shifts of residues
in the linker region are almost unaffected (Figs 2 and
3) and their backbone mobility remains high in the
presence of membrane-mimicking micelles (Fig. 8B).

PRE data suggest buffer-exposed side chains of S53
and S57 (Figs 7, 9 and 10) that are fully accessible to
solvent and, hence, to casein kinase type II in mem-
brane-anchored VpU.
Mutational studies indicated that both the mem-
brane proximal cytoplasmic helix 2 and the five C-ter-
minal residues of VpU are essential for CD4 binding
and degradation [63]. In particular, an interaction
between the cytoplasmic domains of CD4 and VpU is
no longer detectable in a yeast two-hybrid assay if
D77 of VpU is replaced by asparagine [63]. Helix 2
and the C-terminal tight loop of VpU are probably
components of a bipartite binding motif, i.e. produc-
tive interaction between VpU and CD4 will rely on an
appropriate tertiary fold of the cytoplasmic VpU
domain. The long-range NOEs of VpUcyt detected in
micelle solution clearly indicate that VpUcyt samples
one or multiple folded conformations. The NOEs
observed between W76-H
a
in the tight loop and G54-
CH
2
a
in the interhelical linker suggest that the
C-terminal loop is part of a structural motif. Notably,
the fluorescence emission maximum of W76 exhibits a
blue shift by 12 nm upon the addition of DPC micelles
(M. Wittlich & H. Schmidt, unpublished data). This
shift might result from participation of the tryptophan

side chain in a folded protein domain. It remains an
open question: does the low-energy structure calculated
from the measured NMR data represent the active
CD4-binding conformation of VpU? Only an eagerly
awaited complex structure of the interacting domains
will finally answer this question.
Materials and methods
VpUcyt expression and purification
The C-terminal cytoplasmic domain of VpU(residues 39–
81; VpU residue numbering refers to HIV-1 strain HV1S1,
Swiss-Prot accession number P19554) was produced as a
recombinant protein fusion with an N-terminal glutathione
S-transferase affinity tag in Escherichia coli. Thrombin
cleavage of the fusion releases the soluble 45-residue VpU-
cyt polypeptide comprising VpU(39–81) and a preceding
Gly-Ser dipeptide. The amino acid sequence of VpUcyt is
given in Fig. 5. The detailed protocol for cloning, expres-
sion and purification of uniformly
15
N- and
13
C-labelled
VpUcyt has been published previously [26].
NMR spectroscopy
Lyophilized VpUcyt was dispensed at a concentration of
1mm in  330 lL NMR buffer [20 mm sodium phosphate,
pH 6.2, 100 mm NaCl, 0.02% (w ⁄ v) NaN
3
, 10% (v ⁄ v)
2

H
2
O]. DPC-d38 was added to some samples at the speci-
fied concentration. Samples for selected NMR experiments
in H
2
O-free buffer were prepared in NMR buffer, lyophi-
lized and redissolved in
2
H
2
O (99.99%
2
H, Sigma-Aldrich,
NMR structure of micelle-associated HIV-1 VpUcyt M. Wittlich et al.
6570 FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS
Steinheim, Germany). Samples were homogenized by vortex
mixing. The pH was readjusted to 6.2 before transferring
the protein solution to a 5 mm Shigemi NMR tube.
NMR spectra were acquired at 30 °C using Varian (Palo
Alto, CA, USA) Unity INOVA 600 or 800 instruments
operating at 14.1 or 18.8 T, respectively. Inverse detection
5mm
1
H(
13
C,
15
N)-probes equipped with z-axis or triple-
axis pulsed field gradient coils were used. The majority of

NMR spectra were recorded with an HCN-cold probe with
cryogenically cooled
1
H-coil and preamplifier circuitry. The
WATERGATE sequence was used for suppression of the
water signal [64]. Proton and
13
C chemical shifts were
referenced directly to sodium 3-(trimethylsilyl)propane-1-
sulfonate that had been added as an internal standard,
whereas
15
N chemical shifts were referenced indirectly to
sodium 3-(trimethylsilyl)propane-1-sulfonate. NMR data
were processed with vnmrj (Varian) or nmrpipe [65] and
analysed with cara [66].
Resonance assignment
Sequential assignment of the
1
H,
13
C and
15
N resonances of
VpUcyt was accomplished using a combination of 2D and
3D NMR spectra:
1
H-
15
N-HSQC [67,68],

1
H-
13
C-HSQC
[69], HNCACB [70], HNCO [71], HNHA [72],
(H)C(CO)NH [73], HCCH-COSY [74] and HCCH-TOCSY
[74]. Assignment of aromatic side chains was based on
1
H-
13
C-HSQC spectra of the aromatic region and 3D
1
H ⁄
15
N and
1
H ⁄
13
C NOESY experiments.
NOE assignment and structure calculation
Interproton distance restraints were derived from 3D
NOESY-HSQC spectra: a
15
N-edited NOESY-HSQC
(250 ms mixing time) [75], and two
13
C-resolved HSQC-
NOESY experiments [76] for aliphatic protons with 200 ms
mixing time recorded in H
2

Oor
2
H
2
O, respectively. To
check for potential spin diffusion, a series of NOESY spec-
tra were recorded with mixing times from 100 to 400 ms.
The intensity of the observed cross-peaks increased linearly
with mixing time. Apart from this intensity increase, the
NOESY cross-peak pattern looked qualitatively almost
identical. No indication of spin diffusion was found in any
of the spectra. In particular, every single pair of proton res-
onances (A and B) that showed a long-range NOE was
checked for the possible existence of a shared relaxation
partner C with strong cross-peaks between C and both
spins A and B. No such common relaxation partner was
found for any of the long-range NOEs in Table 1. Finally,
the NOESY spectra with a mixing time of 200 ms were
used for complete analysis.
A list of upper distance constraints for structure calcula-
tion was derived from the NOESY data using the auto-
mated NOESY analysis software radar developed by the
Wu
¨
thrich group at ETH in Zu
¨
rich (.
biol.ethz.ch/groups/wuthrich_group/software). radar com-
bines the previously described components atnos for
automated NOESY cross-peak picking and NOE signal

identification [77], candid for NOESY cross-peak assign-
ment and calibration of NOE-derived upper distance con-
straints [78], and dyana for calculation of preliminary
protein structures [79]. radar runs in an iterative manner.
The input of the first cycle consisted of the 3D NOESY
data, the amino acid sequence of VpUcyt and the chemical
shift list reflecting the sequence-specific resonance assign-
ment. Intermediate protein structures are calculated at the
end of each cycle and provide additional guidance for the
interpretation of the NOESY spectra in the subsequent
cycle [78]. atnos revaluates the experimental NOESY data
in each cycle [77]. The software employs multiple strategies
for identification and elimination of erroneous NOE cross-
peaks [77,78]. Distance constraints derived from ambiguous
NOEs that are not uniquely assigned to a single pair of
protons at the end of the last cycle are automatically
purged from the output list [78].
The software-generated list of upper distance constraints
was manually re-inspected. Ambiguous NOE restraints
were re-assessed using a 4D
1
H ⁄
13
C ⁄
1
H ⁄
13
C HMQC-
NOESY-HMQC spectrum with 200 ms mixing time. Ques-
tionable constraints were deleted from the automatically

generated list. Newly identified unique NOEs were manu-
ally assigned, calibrated and converted into additional
upper distance constraints.
The 3D structure of micelle-associated VpUcyt was cal-
culated on the basis of the final set of proton–proton dis-
tance constraints using cyana, a software program that
combines simulated annealing with molecular dynamics in
torsion angle space [80]. The calculated protein conforma-
tions were screened for secondary structure using the pro-
gram dssp [81]. The software molmol [82] was employed
for structure visualization. In addition, molmol generates a
Ramachandran plot for assessment of the geometric quality
of protein conformers. The coordinates of the 20 lowest
energy structures have been deposited in the RCSB Protein
Data Bank under accession code 2K7Y.
Secondary chemical shifts
The analysis of secondary chemical shifts presented here is
based on random coil values determined by Schwarzinger
et al. [83,84] using Ac-GGXGG-NH2 peptides in 8 m urea
and additional corrections for sequence effects.
Heteronuclear NOEs
Heteronuclear
1
H-
15
N NOEs were derived from 2D spectra
recorded with the NOE-TROSY pulse sequence [85]. Spectra
were acquired at 18.8 T with or without
1
H saturation dur-

ing the 3 s recycle delay prior to the first pulse of the NOE
pulse sequence. A series of 120° pulses spaced at 5 ms inter-
M. Wittlich et al. NMR structure of micelle-associated HIV-1 VpUcyt
FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS 6571
vals was used for proton saturation. NOE intensities were
obtained by fitting all NOE cross-peaks to a user-defined
but uniform ‘model peak’ shape composed of Gaussian and
Lorentzian functions using cara [66]. Parameters defining
the peak shape (Gauss–Lorentz balance, line width) were
adjusted manually and independently for both spectral
dimensions using representative NOE peaks. Cross-peak
intensity was the only parameter that was allowed to vary
during the actual fit. The heteronuclear NOE of a
1
H-
15
N
spin pair is defined as the ratio of the corresponding cross-
peak intensities measured with and without
1
H saturation.
PRE
The location of VpUcyt with respect to the micelle–water
interface was derived from selective line broadening of
amide resonances in
1
H-
15
N-HSQC spectra caused by
paramagnetic relaxation agents 16-doxylstearic acid and

Mn
2+
. The paramagnetic doxyl moiety is confined to the
hydrophobic interior of the micelle. It predominantly
broadens NMR signals of nuclei buried in the micelle. The
water-soluble Mn
2+
ions preferentially affect NMR signals
of solvent-exposed spins [86]. The effect of a paramagnetic
probe on individual amino acids is quantified in terms of
the percentage of NMR signal retention, which is derived
from comparison of HSQC cross-peak intensities of
VpUcyt in micellar solution observed in the absence or
presence of the paramagnetic agent.
The effect of different concentrations of either 16-doxylstea-
ric acid (between 0.2 and 8 mm)orMn
2+
(between 0.1 and
3mm) on the NMR signals of VpUcyt (1 mm) in NMR buffer
containing 100 mm DPC-d38 was tested in an initial screen.
The results reported here were obtained at the optimal con-
centrations of 0.1 mm Mn
2+
or 5 mm 16-doxylstearic acid.
CD spectroscopy
Samples contained 53 lm VpUcyt and various amounts (0,
5, 10, 100 mm) of DPC-d38 in 20 mm sodium phosphate
buffer (pH 6.2) complemented with 100 mm KF. KF was
used for ionic strength adjustment instead of NaCl in order
to avoid the strong absorption of chloride ions at low

wavelength [34]. Far-UV CD data were collected between
260 and 185 nm using a Jasco J-810 spectropolarimeter
(Jasco, Tokyo, Japan) operating in step-scan mode (step
size 1 nm; bandwidth 1 nm, time constant 4 s, accumula-
tion of eight scans). A rectangular suprasil quartz cell with
a 1 mm optical path length (Hellma, Mu
¨
llheim, Germany)
was used. Background corrected CD spectra were analysed
with the cdpro software package [87] after converting the
data to per residue differential molar absorbance units
(De ⁄ nincm
)1
ÆM
)1
). cdpro fits the experimental data to a
linear combination of CD spectra of proteins with known
crystal structures, referred to as the basis set. The basis set
SDP48 was employed. It combines reference CD spectra of
43 soluble and five denatured proteins covering the wave-
length range from 190 to 240 nm [87].
Acknowledgement
This work was supported by a grant from the Pra
¨
sid-
entenfond der Helmholtzgemeinschaft (HGF, Virtual
Institute of Structural Biology) to DW.
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