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 Wittlich1,2, Bernd W Koenig1,2, Matthias Stoldt1,2, Holger Schmidt1,2,* and Dieter Willbold1,2 Institut fur Strukturbiologie und Biophysik (ISB-3), Forschungszentrum Julich, Germany ă ă Institut fur Physikalische Biologie, Heinrich-Heine-Universitat Dusseldorf, Germany ă ă ¨ Keywords CD4; DPC micelle; HIV-1 VpU; NMR; solution structure Correspondence D Willbold, Forschungszentrum Julich ă GmbH, ISB-3, 52425 Julich, Germany ă 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, Gottingen, Germany ă 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 September 2009, accepted September 2009) 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 dynamics 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 linker (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-associated VpUcyt retains a substantial degree of structural flexibility doi:10.1111/j.1742-4658.2009.07363.x Introduction VpU (virus protein U) is an 81 amino acid transmembrane 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 downregulation 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] 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 M Wittlich et al and depends on casein kinase II-mediated phosphorylation 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 acid-sensitive K+ channel K+ channel)-related (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 replication 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 residues (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 trifluoroethanol (TFE) solution [22,23], under high salt conditions [24], in the presence of detergent micelles of dihexanoyl phosphatidylcholine (DHPC) [25] or dodecylphosphatidylcholine (DPC) [26], and associated to phospholipid membranes [1,27,28] There is consensus on the formation of two cytoplasmic helices (helices and 3) in various solvent conditions, but the extension of helix varies substantially [23–26] The observation of additional structural elements and, possibly, a tertiary 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 hydrophobic 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] NMR structure of micelle-associated HIV-1 VpUcyt Another important aspect is the topology of the cytoplasmic domain of VpU on a membrane Solid state NMR suggests an orientation of helix parallel to the membrane surface; data on helix 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 polypeptide 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 detergentfree 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, reminiscent of the extreme values at 222, 208 and near 190 nm expected for a regular a-helix [34] The detergent concentration was varied from to 100 mm, well above the critical micelle concentration of DPC (1.5 mm in H2O) The initial addition of mm DPC caused the most pronounced 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 unordered 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 CD spectra of VpUcyt (53 lM) in sodium phosphate buffer with and without membrane-mimicking DPC micelles FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS 6561 NMR structure of micelle-associated HIV-1 VpUcyt M Wittlich et al fraction of  30% a-helical secondary structure and  16% turn formed, whereas only  40% of unordered conformations remained A 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 combinations of buffer composition, choice of detergent and temperature were tested High-quality 15N-1H-heteronuclear 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 mm VpUcyt was recorded with varying amounts of DPC-d38 in the sample (from to 200 mm) Many protein resonance positions changed in a continuous manner with increasing detergent concentration and approached new final values in an asymptotic manner No further chemical shift variation occurred above 100 mm DPC-d38 The observed chemical 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 number 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 chemical shift time scale between different VpUcyt conformational states Virtually complete assignment of 1H, 15N and 13C 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 15N- and 13 C-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 amide 1H and 15N 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 6562 B C D Fig (A) 1H-15N-HSQC spectra of VpUcyt in the presence (red) and absence (black) of micelles (100 mM DPC-d38) Displacement of selected resonances upon detergent addition is indicated by broken lines Side chain amide correlations of glutamine and asparagine residues are connected by a continuous line Chemical shift changes of backbone amide HN (B) and 15N (C) resonances upon the addition of DPC are shown as a function of sequence position A measure of the total chemical shift change {Dtotald = [(Dd H)2 + (0.1 · Dd 15N)2]1 ⁄ 2} [88] is presented in (D) No amide correlation of L42 was observed in the detergent-free sample Local helix propensity derived from chemical shifts The difference between the observed chemical shifts of a protein and the corresponding amino acid residue-specific random coil chemical shifts is referred to as secondary 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 FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS M Wittlich et al NMR structure of micelle-associated HIV-1 VpUcyt Fig Secondary chemical shifts of VpUcyt observed in the absence (left) and presence (right) of detergent micelles (100 mM DPCd38) Solid bars represent amino acid residues 39–81 of VpU The dotted lines mark the theoretical average values of the downfield shift of 13Ca (top) and 13CO (bottom) as well as of the upfield shift of 1Ha resonances (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 a-helix is indicated by downfield shifts of 13Ca and 13 CO and upfield shifts of 1Ha 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 population-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 column) 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 and in accordance with the helix nomenclature of fulllength VpU, where helix designates the N-terminal transmembrane helix of the protein On the basis of the three sets of secondary shift data in Fig (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 comparing the observed average secondary shifts with the values expected for a regular helix This procedure provided fractional helicities of  80% (Dd 13Ca) for helix and 40% (Dd 1Ha) for helix Fig Sections of 15N-edited 1H-1H NOESY spectra of VpUcyt recorded in the absence (left) and presence (right) of 100 mM DPC-d38 using identical acquisition and processing parameters 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 FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS 6563 NMR structure of micelle-associated HIV-1 VpUcyt M Wittlich et al Fig Summary of 1H-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 Ha(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 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 the acquisition of heteronuclear-edited 3D NOESY experiments of VpUcyt in DPC-containing buffer Secondary structure-specific short- and mediumrange NOEs are summarized in Fig 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 exhibits all classes of NOE cross-peaks expected Helix 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 residues 64–68 of VpUcyt formed an a-helix However, the lack of unambiguous daN(i,i + 4) peaks in conjunction with the detection of daN(i,i + 2) cross-peaks in the sequence region of helix may indicate that helix is not as regular as a-helix and may even be of the 310 kind 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] 6564 Table Long-range NOEs of VpUcyt in the micellar environment Observed NOE Chemical shifts of cross-correlated protons (p.p.m.) I43-CH3c-E69-CH2b D44-Ha-R70-CH2c T47-Hb-Q61-CH2b T47-Hb-Q61-CH2c A50-CH3b-P75-CH2d G54-CH2a-W76-Ha H72-Hd-V78-Ha H72-Hd-V78-CH3c H72-He-V78-CH3c H72-He-L81-Hc H72-He-L81-CH3d 1.205 4.458 4.430 4.430 1.509 4.070 7.332 7.332 8.641 8.641 8.641 2.139; 1.774 2.109; 2.457; 1.846; 4.777 4.207 1.017 1.017 1.725 0.933 2.064 2.232 2.461 1.940 Calculation of the VpUcyt structure in micelle solution 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 connectivities were encoded by weak NOEs that gave rise to upper distance limits of 0.55 nm (Table 1) Stripes from a 13Cresolved NOESY experiment exemplifying long-range NOEs of VpUcyt in the presence of DPC micelles are shown in Fig Long-range NOEs are crucial for delineating the tertiary fold of VpUcyt Multiple long-range FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS M Wittlich et al NMR structure of micelle-associated HIV-1 VpUcyt Table Analysis of the 20 lowest energy VpUcyt structures in DPC micelles Fig Stripes from 3D 13C-resolved HSQC-NOESY experiment of VpUcyt recorded in the presence of 100 mM DPC-d38 showing six exemplary long-range 1H-1H NOEs NOEs suggested spatial proximity between helix and amino acids just N-terminal of helix (T47-Hb-E61CH2b; T47-Hb-E61-CH2c) and immediately C-terminal of helix (I43-CH3c-E69-CH2b; D44-Ha-R70-CH2c) This subset of NOEs indicated an approximately antiparallel arrangement of helices and Other longrange NOEs were observed between the interhelical linker and C-terminal residues (A50-CH3b-P75-CH2d; G54-CH2a-W76-Ha) and between C-terminal residues defining a tight loop (H72-Hd-V78-CH3c; H72-He-V78CH3c; H72-Hd-V78-Ha; H72-He-L81-Hc; H72-He-L81CH3d) One would expect observation of multiple contacts between pairs of residues that give rise to longrange 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 longrange NOEs between the pairs of residues listed in Table For example, the observed NOE between I43 CH3c and E69 CH2b should be accompanied by a detectable NOE between I43 CH3d and E69 CH2c However, 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 A set of 100 VpUcyt conformers was generated by the program cyana starting from randomized conformations and using the 604 distance constraints as the only experimental input The 20 structures with the lowest Experimental restraints Total NOE restraints Intraresidue Sequential Medium range (2 £ |i ) j| £ 5) Long range (|i ) j| > 5) CYANA structural statistics Target function (nm2) Sum of NOE violationsa > 0.015 nm Maximum NOE violation in the ensemble rmsd to mean structure (nm) (backbone only ⁄ all VpUcyt (39–80) Helix (39–48) Interhelical linker (49–63) Helix (64–70) Tight loop (73–78) Helix and loop (64–78) Ramachandran analysis Most favoured Additionally allowed Generously allowed Disallowed 604 147 223 219 15 0.0025 ± 0.0006 0.25 nm 0.021 nm heavy atoms) 0.100 ⁄ 0.149 0.027 ⁄ 0.081 0.074 ⁄ 0.141 0.030 ⁄ 0.091 0.016 ⁄ 0.046 0.049 ⁄ 0.088 58% 31% 6% 5% a The sum of all NOE violations larger than 0.015 nm was calculated for each structure and the mean value is shown 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 distance 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 Ramachandran plot An additional 6% of residues were found in the generously allowed region None of the residues of helices and was found in the disallowed regions Instead, Procheck-NMR [40,41] identified various subsets of one or a few residues in the less welldefined 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 structures showed high convergence for helices 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 A74CO and V78-NH Variability of individual structural elements is reflected by the corresponding rmsd values in Table FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS 6565 NMR structure of micelle-associated HIV-1 VpUcyt Loop M Wittlich et al A Linker C Helix N Helix Fig Backbone line representation of the 20 lowest energy conformers of VpUcyt calculated from distance restraints in 100 mM 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 conserved phosphorylation motif, are visualized in ball-and-stick format In contrast to the well-defined helices and the tight loop, there was considerable fuzziness in the interhelical linker region Likewise, the relative position of helix and the tight loop was poorly defined according to the relatively high rmsd of the combined helix plus loop fragment The 15 long-range NOEs resulted in a defined tertiary fold of the calculated VpUcyt structure family Helices and 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-terminal region of VpU to the serine motif (Fig 7) Dynamic characterization of VpUcyt by H-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 Heteronuclear 1H-15N NOE data reflect local variations in protein backbone dynamics on the pico- to nanosecond 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-nanosecond time scale [38] 6566 B Fig 1H-15N-hetero-NOE values of backbone amides of VpUcyt in the absence (A) and presence (B) of 100 mM DPC-d38 Intensities of R45 and V68 could not be determined due to heavy signal overlap; P75 lacks a backbone amide group Helical regions and a tight loop of VpUcyt are denoted by grey stripes and an open rectangle, respectively Figure 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 virtually all regions of VpUcyt In particular, residues in helix showed 1H-15N NOEs close to the slow motion limit, indicating a well-defined secondary structure element that was rigid in the pico- to nanosecond time scale Heteronuclear NOEs of backbone amides of residues immediately following helix and in the sequence stretch covering helix and the tight loop had intermediate values This suggests that the respective residues have a reduced mobility, although these regions are not as stiff as helix With the exception of helix 2, the level of backbone dynamics was consistently higher than expected for a stable and rigid fold Chemical exchange between multiple conformations might explain the observed intermediate values of the H-15N NOEs A particularly high mobility was FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS M Wittlich et al NMR structure of micelle-associated HIV-1 VpUcyt A B Fig 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 VpUcyt, reflecting the topology and the dynamics of the micelle-associated protein Helical regions and a tight loop of VpUcyt are denoted by grey stripes and an open rectangle, respectively 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 Mn2+ (Fig 9) Incorporation of the doxyl probe into the micelle reduced backbone amide cross-peak intensities of residues in helices and 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 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 completely (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 and 3, as well as from residues between helix 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 and 3, as well as of the residues between helix and the loop, in the micelle–water interface region The highly anionic interhelical linker was solvent exposed and fully accessible 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-terminal end of VpU (D77-VDD-L81) was partially protected from quenching by the doxyl probe and the protection level increased towards the C-terminus Furthermore, resonances of these last five residues became almost undetectable in the presence of Mn2+ The solvent-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 micelleembedded 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 concentration (Fig 3, left) revealed complete absence of secondary structure elements The heteronuclear NOE data are consistent with a highly flexible protein lacking a well-defined backbone conformation (Fig 8A) CD spectra of VpUcyt in detergent-free buffer confirmed 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 FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS 6567 NMR structure of micelle-associated HIV-1 VpUcyt M Wittlich et al (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 estimated from the CD spectra of VpUcyt in 100 mm DPC Helix 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 spans residues 37–51) [22,23], in DHPC micelles (helix spans residues 30–49) [25], or in high salt buffer (helix spans residues 40–50) [24] The N-terminal start of helix cannot be compared due to the different lengths of the VpU peptides studied The length and sequence position of helix differ appreciably between studies It is shortest in the presence of DPC micelles (residues 64–70), slightly extended in high salt buffer (residues 60–68) [24], but approximately twice as long in DHPC micelles (residues 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 (residues 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 presence of DPC micelles Although the structural motifs adopted by the cytoplasmic domain of VpU appear to be qualitatively similar under various membrane-mimicking conditions, the extension of helix seems to be highly sensitive to the local environment of the protein In comparison with the DPC-free solution, the conformational flexibility of VpUcyt was heavily reduced upon the addition of DPC micelles Heteronuclear NOE values close to 0.8 suggest a well-structured helix (Fig 8B) However, reduced 15N-1H NOEs of residues in the interhelical linker and the intermediate 15 N-1H NOEs observed for the C-terminal half of VpUcyt indicate a substantial amount of remaining conformational flexibility PRE and secondary chemical 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 conformational ensemble that gives rise to the observed spectrum 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 6568 The observed secondary structure elements and the tertiary contacts may be present to a different extent in each individual conformation Interestingly, the complete set of upper distance constraints extracted from NOESY experiments on VpUcyt in the presence of DPC micelles is simultaneously satisfied in the converged low-energy VpUcyt conformers presented in Fig 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 residues 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 orientation of molecular representations shown in the same column is identical FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS M Wittlich et al micelle (16-doxylstearic acid) or to the aqueous buffer (Mn2+) Figure 10 shows a surface plot of a representative VpUcyt structure colour-coded according to the PRE data Residues strongly affected by 16-doxylstearic 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 displayed 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-coloured 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 corresponding 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 relative to the micelle normal shown in Fig 10 is compatible with many, but not all, of the PRE data Helices and 3, as well as the amino acids located between helix 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 quenching characteristics (orange) and may occupy a region close to both the interior of the micelle and the aqueous 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 surrounding 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 NMR structure of micelle-associated HIV-1 VpUcyt amino acid stretch 64–78 is also evident from the heteronuclear 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 is bound to the membrane and runs parallel to the lipid–water interface, whereas no preferred orientation could be detected for helix [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 activities, 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 conditions, such proteins show a high degree of conformational 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 conformations Some of those transiently formed conformations may be perfectly suited for a selected protein ligand interaction The distinguished protein conformation 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 selection’ 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 subpopulations is required for productive interaction with multiple 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 welldefined 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 FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS 6569 NMR structure of micelle-associated HIV-1 VpUcyt M Wittlich et al Biological implications of the observed VpUcyt structure One of the most studied activities of VpU is the induction of CD4 degradation in the endoplasmatic reticulum of infected CD4+ cells Only the cytoplasmic domain of VpU, together with any membrane anchor, is essential for this activity [59], suggesting that a membrane 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 pentamers The structural study of membrane-inserted pentamers 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-simulating DPC micelles by high-resolution liquid state NMR Our data show that VpUcyt becomes at least partially structured in the presence of membrane-mimicking DPC micelles In full-length VpU, the cytoplasmic domain is indifferently anchored to a lipid membrane We propose that the structure of micelleassociated VpUcyt is a reasonable approximation of the physiologically relevant membrane-attached cytoplasmic 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 structure of micelle-bound VpUcyt is obviously very different from the completely unordered VpUcyt in plain buffer The association of peptides with lipid membranes often has a pronounced influence on peptide structure and may be a crucial prerequisite for productive interaction of a peptide or protein domain with its membrane receptor [61] VpU-induced proteasomal degradation of newly synthesized 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 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, and 10) that are fully accessible to solvent and, hence, to casein kinase type II in membrane-anchored VpU 6570 Mutational studies indicated that both the membrane proximal cytoplasmic helix and the five C-terminal 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 and the C-terminal tight loop of VpU are probably components of a bipartite binding motif, i.e productive 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-Ha in the tight loop and G54CH2a 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 VpUcyt polypeptide comprising VpU(39–81) and a preceding Gly-Ser dipeptide The amino acid sequence of VpUcyt is given in Fig The detailed protocol for cloning, expression and purification of uniformly 15N- and 13C-labelled VpUcyt has been published previously [26] NMR spectroscopy Lyophilized VpUcyt was dispensed at a concentration of mm in  330 lL NMR buffer [20 mm sodium phosphate, pH 6.2, 100 mm NaCl, 0.02% (w ⁄ v) NaN3, 10% (v ⁄ v) H2O] DPC-d38 was added to some samples at the specified concentration Samples for selected NMR experiments in H2O-free buffer were prepared in NMR buffer, lyophilized and redissolved in 2H2O (99.99% 2H, Sigma-Aldrich, FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS M Wittlich et al Steinheim, Germany) Samples were homogenized by vortex mixing The pH was readjusted to 6.2 before transferring the protein solution to a 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 mm 1H(13C,15N)-probes equipped with z-axis or tripleaxis pulsed field gradient coils were used The majority of NMR spectra were recorded with an HCN-cold probe with cryogenically cooled 1H-coil and preamplifier circuitry The WATERGATE sequence was used for suppression of the water signal [64] Proton and 13C chemical shifts were referenced directly to sodium 3-(trimethylsilyl)propane-1sulfonate that had been added as an internal standard, whereas 15N 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 1H, 13C and 15N resonances of VpUcyt was accomplished using a combination of 2D and 3D NMR spectra: 1H-15N-HSQC [67,68], 1H-13C-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 H-13C-HSQC spectra of the aromatic region and 3D H ⁄ 15N and 1H ⁄ 13C NOESY experiments NOE assignment and structure calculation Interproton distance restraints were derived from 3D NOESY-HSQC spectra: a 15N-edited NOESY-HSQC (250 ms mixing time) [75], and two 13C-resolved HSQCNOESY experiments [76] for aliphatic protons with 200 ms mixing time recorded in H2O or 2H2O, respectively To check for potential spin diffusion, a series of NOESY spectra 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 resonances (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 Finally, the NOESY spectra with a mixing time of 200 ms were used for complete analysis A list of upper distance constraints for structure calculation was derived from the NOESY data using the automated NOESY analysis software radar developed by the Wuthrich group at ETH in Zurich (http://www.mol ă ¨ NMR structure of micelle-associated HIV-1 VpUcyt biol.ethz.ch/groups/wuthrich_group/software) radar combines the previously described components atnos for automated NOESY cross-peak picking and NOE signal identification [77], candid for NOESY cross-peak assignment and calibration of NOE-derived upper distance constraints [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 assignment 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 crosspeaks [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 1H ⁄ 13C ⁄ 1H ⁄ 13C HMQCNOESY-HMQC spectrum with 200 ms mixing time Questionable constraints were deleted from the automatically generated list Newly identified unique NOEs were manually assigned, calibrated and converted into additional upper distance constraints The 3D structure of micelle-associated VpUcyt was calculated on the basis of the final set of proton–proton distance constraints using cyana, a software program that combines simulated annealing with molecular dynamics in torsion angle space [80] The calculated protein conformations were screened for secondary structure using the program 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 m urea and additional corrections for sequence effects Heteronuclear NOEs Heteronuclear 1H-15N NOEs were derived from 2D spectra recorded with the NOE-TROSY pulse sequence [85] Spectra were acquired at 18.8 T with or without 1H saturation during the s recycle delay prior to the first pulse of the NOE pulse sequence A series of 120° pulses spaced at ms inter- FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS 6571 NMR structure of micelle-associated HIV-1 VpUcyt M Wittlich et al 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 1H-15N spin pair is defined as the ratio of the corresponding crosspeak intensities measured with and without 1H saturation PRE The location of VpUcyt with respect to the micelle–water interface was derived from selective line broadening of amide resonances in 1H-15N-HSQC spectra caused by paramagnetic relaxation agents 16-doxylstearic acid and Mn2+ 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 Mn2+ 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-doxylstearic acid (between 0.2 and mm) or Mn2+ (between 0.1 and mm) 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 concentrations of 0.1 mm Mn2+ or 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 nm; bandwidth nm, time constant s, accumulation of eight scans) A rectangular suprasil quartz cell with a mm optical path length (Hellma, Mullheim, Germany) ă 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 ⁄ n in cm)1ỈM)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 6572 43 soluble and five denatured proteins covering the wavelength range from 190 to 240 nm [87] Acknowledgement This work was supported by a grant from the Prasidă entenfond der Helmholtzgemeinschaft (HGF, Virtual Institute of Structural Biology) to DW References Marassi FM, Ma C, Gratkowski H, Straus SK, Strebel K, Oblatt-Montal M, Montal M & Opella SJ (1999) Correlation of the structural and functional domains in the membrane protein 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