The corresponding chemical shift changes in backbone amide1H and15N spins of VpUcyt, together with a weighted average of the absolute changes, are presented as a function of the amino ac
Trang 1cytoplasmic domain in the presence of
dodecylphosphatidylcholine micelles
Marc Wittlich1,2, Bernd W Koenig1,2, Matthias Stoldt1,2, Holger Schmidt1,2,* and Dieter Willbold1,2
1 Institut fu¨r Strukturbiologie und Biophysik (ISB-3), Forschungszentrum Ju¨lich, Germany
2 Institut fu¨r Physikalische Biologie, Heinrich-Heine-Universita¨t Du¨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 SIVCPZ 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: d.willbold@fz-juelich.de
*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.
Trang 2and 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 H2O) 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 l M ) in sodium phosphate buffer with and without membrane-mimicking DPC micelles.
Trang 3fraction 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 15N-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 30C 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 schemi-cale between different VpUcyt
con-formational states
Virtually complete assignment of 1H, 15N and 13C
resonances of VpUcyt at 30C in buffer with 100 mm
DPC-d38 was accomplished on the basis of a series of
3D NMR experiments recorded on uniformly15N- and
13C-labelled VpUcyt Resonance assignment tables
were deposited at the BMRB (accession code: 15513)
An overlay of 15N-1H-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 amide1H and15N 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, 13Ca, 1Ha and 13CO 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.
Trang 4a-helix is indicated by downfield shifts of 13Ca and
13CO and upfield shifts of1Haresonances 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 13Ca, 1Ha and 13CO 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 13Ca) for
helix 2 and 40% (Dd1Ha) for helix 3
NOE-derived secondary structure and tertiary fold
of VpUcyt in the presence of DPC micelles 2D 15N-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 dNN(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 of13C a (top) and13CO (bottom) as
well as of the upfield shift of 1 Ha
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.
Trang 5the 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
dNN(i,i + 1) cross-peaks in conjunction with less
intense daN(i,i + 1) peaks and continuous stretches of
daN(i,i + 3), dab(i,i + 3), and perhaps daN(i,i + 4) or
daN(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 daN(i,i + 2) peaks in addition
to a complete series of dab(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 daN(i,i + 3) and
daN(i,i + 4) positions that would be predicted if
resi-dues 64–68 of VpUcyt formed an a-helix However,
the lack of unambiguous daN(i,i + 4) peaks in
con-junction with the detection of daN(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 310kind Most residues in the interhelical linker
(R49–E63) and the C-terminal region of VpUcyt from
G71 to L81 exhibited more intense daN(i,i + 1) than
dNN(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 1H-1H 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 a13 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 of1H-1H 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.)
Trang 6NOEs suggested spatial proximity between helix 2 and
amino acids just N-terminal of helix 3 (T47-Hb
-E61-CH2b; T47-Hb-E61-CH2) and immediately C-terminal
of helix 3 (I43-CH3-E69-CH2b; D44-Ha-R70-CH2 )
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-CH3b-P75-CH2 ;
G54-CH2a-W76-Ha) and between C-terminal residues
defining a tight loop (H72-Hd-V78-CH3; H72-He
-V78-CH3; H72-Hd-V78-Ha; H72-He-L81-Hc; H72-He
-L81-CH3) One would expect observation of multiple
contacts between pairs of residues that give rise to
long-range NOEs Indeed, several long-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
CH3 and E69 CH2b should be accompanied by a
detectable NOE between I43 CH3 and E69 CH2
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 dab(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 3D13C-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
Medium range (2 £ |i ) j| £ 5) 219
CYANA structural statistics
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)
Interhelical linker (49–63) 0.074 ⁄ 0.141
Ramachandran analysis
a The sum of all NOE violations larger than 0.015 nm was calcu-lated for each structure and the mean value is shown.
Trang 7In 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
1H-15N-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 1H-15N NOE data reflect local variations in
protein backbone dynamics on the pico- to
nano-second time scale Positive 1H-15N 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 1H-15N NOEs of VpUcyt backbone amides in the presence and absence of DPC micelles Small and rather consistent 1H-15N 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 1H-15N 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
1H-15N 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 Linker
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.
Trang 8retained 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 Mn2+ (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
Mn2+ 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 Mn2+ ions Residues at both ends of the interhelical linker were superficially associated with the micelle interface and remained easily accessible by water-soluble Mn2+ions The strongly charged C-ter-minal end of VpU (D77-VDD-L81) 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 Mn2+ 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
L73-AP-W76 in the tight loop were strongly quenched
by both the 16-doxylstearic acid and the Mn2+ 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 Mn2+ 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.
Trang 9(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 (resi(resi-dues 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 15N-1H NOEs of
resi-dues in the interhelical linker and the intermediate
15N-1H 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 Mn2+, 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 representaorienta-tions shown in the same column is identical.
Trang 10micelle (16-doxylstearic acid) or to the aqueous buffer
(Mn2+) 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 Mn2+
are shown in green Residues with opposite quenching
characteristics, i.e strong signal reduction after the
addition of Mn2+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 Mn2+ 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 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