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. 2007 Oct;16(10):2205–2215. doi: 10.1110/ps.073041107

Conformational changes induced by a single amino acid substitution in the trans-membrane domain of Vpu: Implications for HIV-1 susceptibility to channel blocking drugs

Sang Ho Park 1, Stanley J Opella 1
PMCID: PMC2204142  PMID: 17766368

Abstract

The channel-forming trans-membrane domain of Vpu (Vpu TM) from HIV-1 is known to enhance virion release from the infected cells and is a potential target for ion-channel blockers. The substitution of alanine at position 18 by a histidine (A18H) has been shown to render HIV-1 infections susceptible to rimantadine, a channel blocker of M2 protein from the influenza virus. In order to describe the influence of the mutation on the structure and rimantadine susceptibility of Vpu, we determined the structure of A18H Vpu TM, and compared it to those of wild-type Vpu TM and M2 TM. Both isotropic and orientationally dependent NMR frequencies of the backbone amide resonance of His18 were perturbed by rimantadine, and those of Ile15 and Trp22 were also affected, suggesting that His18 is the key residue for rimantadine binding and that residues located on the same face of the TM helix are also involved. A18H Vpu TM has an ideal, straight α-helix spanning residues 6–27 with an average tilt angle of 41° in C14 phospholipid bicelles, indicating that the tilt angle is increased by 11° compared to that of wild-type Vpu TM. The longer helix formed by the A18H mutation has a larger tilt angle to compensate for the hydrophobic mismatch with the length of the phospholipids in the bilayer. These results demonstrate that the local change of the primary structure plays an important role in secondary and tertiary structures of Vpu TM in lipid bilayers and affects its ability to interact with channel blockers.

Keywords: ion channel, viroporin, HIV, channel blocker, micelles, bicelles, solid-state NMR

Virus protein “u” (Vpu) is a small, 81-residue, monotopic membrane protein whose sequence is encoded in the genome of HIV-1 (Cohen et al. 1988; Strebel et al. 1988); it is found in the membranes of infected cells, but not the virus particles themselves. Vpu is responsible for two biological activities that contribute to the pathogenicity of HIV-1 infections in humans (Bour and Strebel 2003): It enhances the release of newly formed virus particles from the cell (Strebel et al. 1988, 1989; Terwilliger et al. 1989) and it accelerates the degradation of CD4 receptors (Willey et al. 1992). There is strong evidence that these activities are associated with separate domains of the protein (Schubert et al. 1996a; Marassi et al. 1999). The phosphorylated cytoplasmic domain of Vpu interacts with beta transducin repeat containing protein (β-TrCP), the F-box protein of the Skp1-Cullin-F-box protein (SCF) complex responsible for recruiting the CD4 receptor bound to Vpu for polyubiquitination and subsequent destruction by the proteasome. The hydrophobic trans-membrane domain of Vpu (Vpu TM) is a helix that forms homo-oligomers, which exhibit ion-channel activity (Ewart et al. 1996; Schubert et al. 1996a,b; Grice et al. 1997; Romer et al. 2004) and are involved in the regulation of the assembly and release of new virus particles. With a few exceptions (Huet et al. 1990; Courgnaud et al. 2002), the gene for Vpu is not found in HIV-2 or simian immunodeficiency virus (SIV) genomes. Interestingly, some isolates of HIV-2 appear to have envelope (Env) proteins with Vpu-like biological activities (Bour and Strebel 1996; Bour et al. 1996; Ritter Jr. et al. 1996; Abada et al. 2005).

Vpu is classified as an accessory protein, along with Tat, Nef, Vpr, and Vif, since its gene is found only in lentiviruses, and it is not required for viral replication in continuous cell lines of CD4-containing T lymphocytes (Trono 1995). None of the accessory proteins, including Vpu, have enzymatic activity. Vpu is also classified as a viroporin (Fischer and Sansom 2002; Gonzalez and Carrasco 2003), since it is a small viral membrane protein that exhibits ion-channel activities and plays a key role in membrane-associated processes. The best-characterized viroporin is the M2 protein from influenza virus (Pinto and Lamb 2006). There is evidence that Vpu and perhaps other viroporins are interchangeable (Gonzalez and Carrasco 2001), suggesting that findings about Vpu may be broadly relevant; for example, drugs targeted to Vpu (Ewart et al. 2002, 2004; Lemaitre et al. 2004; Kim et al. 2006) may also serve as lead compounds for other viroporins (Premkumar et al. 2004; Wilson et al. 2006). It also suggests that domain swapping among viroporins, as well as homology-based site directed mutagenesis (Hout et al. 2006a,b) have the potential to be incisive tools for characterizing the structural basis for their molecular mechanisms of action. Consequently, structural studies of Vpu and its domains have the potential to contribute to the molecular and structural biology of HIV (Turner and Summers 1999) and provide a starting point for the development of antiviral drugs targeted to a novel receptor (Miller and Sarver 1997).

Currently, there are two hypotheses to explain how Vpu enhances the release of virus particles. It is well established that the hydrophobic trans-membrane helix of Vpu forms homo-oligomers in membranes as an isolated domain and as part of the intact protein. These oligomers exhibit ion-channel activity (Ewart et al. 1996; Schubert et al. 1996a,b; Grice et al. 1997; Romer et al. 2004). It has been suggested, although it remains controversial (Lamb and Pinto 1997; Coady et al. 1998), that Vpu's ion-channel activity is responsible for enhancing the release of virus particles (Ewart et al. 1996; Schubert et al. 1996b; Gonzalez and Carrasco 1998). This argument is bolstered by the sequence similarity to the influenza M2 protein, which has definitively been shown to be a channel protein (Pinto et al. 1992). The second hypothesis is that Vpu enhances the release of new virus particles by overcoming a dominant host cell restriction to assembly (Varthakavi et al. 2003). One possible mechanism for this may be that Vpu inhibits the intrinsic channel activity of the TASK-1 ion-channel (Hsu et al. 2004; Strebel 2004) by forming dysfunctional hetero-oligomers with a segment of the TASK-1 protein that is similar to Vpu TM. Alternatively, Vpu's enhancement of virus particle release activity may be linked to a cellular factor associated with the pericentriolar recycling endosome (Varthakavi et al. 2006) or possibly the plasma membrane that results in the trapping of assembled virions at the cell surface (Neil et al. 2006).

The recently reported domain swapping and mutation experiments of Stephens and coworkers (Hout et al. 2006a,b) place the ion-channel hypothesis on firmer ground. Not only does Vpu remain fully functional when its trans-membrane helix is replaced by that of the M2 protein, but also this makes the virus infection sensitive to the well-established M2 channel blocker, rimantadine. They also show that this effect can be replicated when a single alanine residue is substituted by a histidine residue within Vpu TM (Hout et al. 2006a); this places a histidine four residues away from a tryptophan, as in M2 TM (Fig. 1). Electron microscopy of cells infected with a virus whose Vpu gene contains the A18H mutation and treated with rimantadine reveal an accumulation of viral particles at the cell surface and within intracellular vesicles.

Figure 1.

Figure 1.

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Amino acid sequence alignment of wild-type Vpu TM and A18H Vpu TM from HIV-1 and M2 TM from influenza virus A.

Previously, we determined the three-dimensional structure of wild-type Vpu TM in several lipid environments, including weakly aligned micelles, mechanically aligned bilayers on glass plates, and magnetically aligned bilayers (bicelles) (Park et al. 2003, 2006). In this article, in order to describe the influence of the single amino substitution of histidine for alanine at position 18 on the structure and rimantadine susceptibility of Vpu, we determined the structures of a 36-residue polypeptide corresponding to A18H Vpu TM in micelles and bilayers, and compared them to those of wild-type Vpu TM as well as the influenza M2 TM characterized by Cross and coworkers (Wang et al. 2001; Nishimura et al. 2002). We found that the tilt angle of the Vpu TM helix in phospholipid bilayers is significantly altered by the A18H mutation.

Results

Solution-state NMR spectra of wild-type Vpu TM and A18H Vpu TM in micelles

Two-dimensional 1H/15N HSQC spectra of uniformly 15N-labeled 36-residue polypeptides whose sequences correspond to those of wild-type Vpu TM and A18H Vpu TM are plotted with black contours in Figure 2. The samples are protein-containing isotropic DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine) micelles in aqueous solution. We were able to use the previous assignments of the amide resonances of wild-type Vpu TM (Park et al. 2003) to assign the spectrum of A18H Vpu TM because, except for resonances from a few residues near the site of the A18H mutation, nearly all of the correlation resonances of A18H Vpu TM (Fig. 2B) are superimposable on those of wild-type Vpu TM (Fig. 2A). The resonance assignments were confirmed by the sequential NH-NH NOE connectivities obtained from a two-dimensional 1H/15N HMQC-NOESY spectrum obtained on a uniformly 15N-labeled sample (Shon and Opella 1989).

Figure 2.

Figure 2.

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Superimposed solution-state NMR 1H/15N HSQC spectra of uniformly 15N-labeled polypeptides in DHPC micelles in the absence (black contours) and the presence (red contours) of a 20-fold molar excess of rimantadine. (A) Wild-type Vpu TM. (B) A18H Vpu TM. The sequential assignments of the amide resonances are indicated by the residue numbers. The arrow indicates the perturbation of His18 amide resonance by rimantadine binding.

The spectra in Figure 2 represented by the red contours were obtained from identical samples of protein-containing micelles, except for the addition of rimantadine. Rimantadine does not appear to interact specifically with wild-type Vpu TM as evidenced by the lack of sequence-specific changes in the frequencies of the 1H/15N amide resonances in Figure 2A. In contrast, substantial frequency shifts of several resonances are induced by rimantadine in the spectrum of A18H Vpu TM, in particular for Ile15, His18, and Trp22; several additional resonances from residues in the C-terminal region, including Ile27, Glu28, and Gly29, are also affected (Fig. 2B). The spectral changes induced by the interaction of rimantadine have the characteristics of “fast exchange” on the timescales of the 1H and 15N chemical shifts. With increasing concentrations of rimantadine, the amide resonances of affected residues shifted gradually from their frequencies of the apo state to those of the final fully bound state.

Solid-state NMR spectra of wild-type Vpu TM and A18H Vpu TM in aligned bicelles

Figure 3 shows the two-dimensional solid-state NMR 15N chemical shift/1H-15N heteronuclear dipolar coupling separated local field spectra of uniformly 15N-labeled wild-type Vpu TM and A18H Vpu TM in magnetically aligned phospholipid bicelles. The bicelles are characterized by the ratio (q = 3.2) of long-chain (C14; 14-O-PC; 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine) to short-chain (C6; 6-O-PC; 1,2-di-O-hexyl-sn-glycero-3-phosphocholine) phospholipids. The polypeptides are immobilized (on NMR timescales) and aligned along with the phospholipids in planar bilayers with normals perpendicular to the direction of the applied magnetic field. The wheel-like patterns of resonances observed in the spectra of uniformly 15N-labeled samples are characteristic of a tilted trans-membrane helix (Marassi and Opella 2000; Wang et al. 2000). The polarity index slant angle (PISA) wheel patterns correspond to helical wheel projections and are direct indices of secondary structure and its topology.

Figure 3.

Figure 3.

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Solid-state NMR 15N chemical shift/1H-15N dipolar coupling separated local field spectra of uniformly 15N-labeled polypeptides in 14-O-PC/6-O-PC (q = 3.2) bicelles aligned magnetically with their bilayer normals perpendicular to the magnetic field. (A) Wild-type Vpu TM. (B) A18H Vpu TM. The sequential assignments of the amide resonances are indicated by the residue numbers. Superimposed on the experimental spectra are ellipses corresponding to the ideal PISA wheel for an α-helix with uniform dihedral angles (ϕ = −61°, ψ = −45°) tilted at 30° (A) and 41° (B) with respect to the membrane normal.

We have previously assigned and analyzed the 1H-15N separated local field spectra of wild-type Vpu TM in several different phospholipid bilayer environments, including mechanically aligned 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glyceor-3-[phospho-rac-(1-glycerol)] (DOPG) bilayers on glass plates, and magnetically aligned 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (16-O-PC)/6-O-PC and 14-O-PC/6-O-PC bicelles (Park et al. 2003, 2006). In the present study, the separated local field spectra were obtained by applying the SAMPI4 pulse sequence (Nevzorov and Opella 2007), which has improved bandwidth compared to earlier versions of high-resolution separated local field experiments (Wu et al. 1994; Nevzorov and Opella 2003). The separated local field spectrum of wild-type Vpu TM (Fig. 3A) is reproducible on separate samples within ∼2 ppm for 15N chemical shift and ∼300 Hz for 1H-15N dipolar coupling frequencies, which are similar to the experimental line widths of the resonances in their respective frequency dimensions. The resonance assignments of wild-type Vpu TM and A18H Vpu TM were obtained independently using the “shotgun” approach, which takes advantage of the irregular distributions of the amino acid types in the linear sequence and the predictable patterns in helical/PISA wheel diagrams (Marassi and Opella 2003). Spectra from selective (by residue type) Ile, Val, Ala, Trp, and His 15N-labeled samples were utilized in the analysis, and the assignments were confirmed by the results of 2H/1H exchange experiments of uniformly 15N- and selectively 15N Ile-labeled samples.

In contrast to the minimal differences observed between the solution-state NMR spectra of wild-type Vpu TM and A18H Vpu TM (Fig. 2, A vs. B), the substitution of alanine by histidine at position 18 results in very large changes throughout the solid-state NMR spectrum. On average, the 15N chemical shift frequencies of the amide backbone resonances are shifted downfield by 15.4 ppm, and the 1H-15N dipolar couplings are decreased by 1.2 kHz in the spectrum of A18H Vpu TM (Fig. 3B) compared to that of wild-type Vpu TM (Fig. 3A). The analysis of the PISA wheel and corresponding dipolar wave (Mesleh et al. 2003) plots indicate that both of the polypeptides contain an ideal trans-membrane helix, but that the A18H mutation, near the middle of the helix, increases the tilt angle by 11°. This is a remarkably large change in protein structure from a single-site mutation that not only preserves biological activity, but also is in the middle of a stable α-helix.

Effects of the A18H mutation on rimantadine binding

The effects of rimantadine binding on the solid-state NMR spectrum of A18H Vpu TM in bicelles are more subtle than those observed on the solution-state spectrum of the same polypeptide in micelles. The changes in chemical shift and dipolar coupling frequencies as a function of residue number that result from the substitution of histidine for alanine at position 18 and from the addition of rimantadine to samples of wild-type Vpu TM and A18H Vpu TM are summarized in Figure 4. Since the GGKKKK residues, which are added as a solubility tag at the C terminus, are unstructured and mobile (Park et al. 2003), data for residues 33–36 are not included in these plots. Figure 4A identifies the residues that are perturbed by the A18H mutation. In contrast, no significant changes are observed in the spectrum of wild-type Vpu TM upon addition of 20-fold molar excess of rimantadine, as indicated by the plot of the weighted isotropic chemical shift differences in Figure 4B. Minor changes were observed in the resonances from residues in the C- and N-terminal regions (<0.03 ppm), but we consider these to be nonspecific, especially when compared to those observed for A18H Vpu TM, where the addition of rimantadine resulted in shifts >0.06 ppm for the resonances from Ile15 and His18, and >0.04 for Trp22 and several residues near the C terminus (Ile27, Glu28, and Gly29). These results suggest that rimantadine binding involves several specific residues on one face of the trans-membrane helix of Vpu near histidine 18. The changes in isotropic chemical shifts plotted in Figure 4B,C reflect local environmental perturbations at the backbone amide sites of the affected residues.

Figure 4.

Figure 4.

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Perturbation of isotropic and orientationally dependent frequencies as a function of the residue number by the A18H mutation and rimantadine binding. (A) Isotropic chemical shift difference between wild-type Vpu TM and A18H Vpu TM. (B,C) Perturbations of isotropic chemical shift in micelles by addition of rimantadine to wild-type Vpu TM (B) and A18H Vpu TM (C). A combined 1H and 15N isotropic chemical shift perturbation map as defined in the Materials and Methods section was employed. (D,E) Perturbations of 15N anisotropic chemical shift (D) and 1H-15N dipolar coupling (E) of A18H Vpu TM in aligned bicelles by addition of a fivefold molar excess of rimantadine.

Figure 4D,E shows the changes of the orientationally dependent chemical shift and dipolar coupling frequencies upon the interaction of rimantadine with A18H Vpu TM in aligned bicelles. The solid-state NMR spectrum of wild-type Vpu TM was not affected by the addition of excess rimantadine (data not shown). However, in the case of A18H Vpu TM, both frequencies associated with His18 are affected by the polypeptide interacting with rimantadine; the changes are 2.9 ppm for the 15N chemical shift and 220 Hz for the 1H-15N dipolar coupling. The changes of all the other residues in the trans-membrane helix were <0.9 ppm and <90 Hz for the 15N chemical shift and 1H-15N dipolar coupling, respectively. The root mean square (RMS) deviation of frequencies of replicate experiments on the same sample was ∼0.5 ppm for 15N chemical shift and 100 Hz for 1H-15N dipolar couplings, respectively. Taken together, the results in Figure 4 suggest that the histidine is the key residue involved in the interaction with rimantadine. The solution-state NMR data indicate that the local environment for several residues is altered by the presence of rimantadine, and the solid-state NMR data indicate that the orientation of the backbone at residue 18 is changed.

Structural change from the A18H mutation

The separated local field spectra of selectively 15N Ile-labeled wild-type Vpu TM and A18H Vpu TM in bicelles are compared in Figure 5. There are 10 isoleucine residues distributed throughout the sequence of the 36-residue polypeptide (Fig. 1): six residues (8, 15, 16, 17, 19, and 24) in the core of the trans-membrane helix, two residues (4 and 6) in the N-terminal region, and two residues (26 and 27) in the C-terminal region. Eight resonances can be accounted for in the spectrum of wild-type TM (Fig. 5A), two of which are overlapped (8 and 15) and one of which is weak (6). The signals from two of the residues (4 and 27) were not observed in spectra of wild-type Vpu TM obtained using a range of experimental and processing parameters. In our experience, missing backbone amide resonances in solid-state NMR spectra of aligned samples of membrane proteins can almost always be ascribed to the effects of motional averaging. This would be consistent with the absent resonances being from residues near the ends of the helix. It is also consistent with our finding that the amide groups of the next two isoleucine residues in the sequence (6 and 26) undergo more facile 2H/1H exchange, since their signals are present in 1H2O but not 2H2O solution.

Figure 5.

Figure 5.

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Solid-state NMR 15N chemical shift/1H-15N dipolar coupling separated local field spectra of selectively 15N Ile-labeled polypeptides in 14-O-PC/6-O-PC (q = 3.2) bicelles aligned magnetically with their bilayer normals perpendicular to the magnetic field. (A) Wild-type Vpu TM. (B) A18H Vpu TM. The assignments of the amide resonances are indicated by the residue numbers. Superimposed on the experimental spectra are ellipses corresponding to the ideal PISA wheel for an α-helix with uniform dihedral angles (ϕ = −61°, ψ = −45°) tilted at 30° (A) and 41° (B) with respect to the membrane normal. The arrows indicate the residues that disappear in 2H2O exchange experiments.

The separated local field spectrum of 15N Ile-labeled A18H Vpu TM (Fig. 5B) is quite different from that of wild-type Vpu TM (Fig. 5A). Not only are the resonances significantly shifted in both the 15N chemical shift and 1H-15N heteronuclear dipolar coupling frequency dimensions, as noted for the uniformly 15N-labeled samples in Figure 3, but also two additional signals are present. Signal intensity from all 10 isoleucine residues can be observed in the spectrum (Fig. 5B); therefore an effect of the A18H mutation is to extend the length of the helix to include residues 4 and 27. The finding that the resonances from residues 4, 6, and 27 undergo facile exchange with 2H2O is consistent with this conclusion. The 1H/15N HSQC spectrum of uniformly 15N-labeled A18H TM obtained in 2H2O contains resonances from a continuous stretch of amino acids between Val9 and Ile26, indicating that residues in the middle of the polypeptide are resistant to hydrogen exchange in micelles as well (data not shown).

The magnitudes of the residual dipolar couplings measured from the weakly aligned sample in micelles and the full dipolar couplings from the fully aligned sample in bicelles are plotted as a function of residue number in Figure 6. Structural distortions are readily detected using dipolar waves, since the resonance patterns from ideal helices are well fit by sinusoids with the 3.6 period of the secondary structure (Mesleh et al. 2002, 2003; Mesleh and Opella 2003). Dipolar Waves derived from solid-state NMR data give absolute measurements of helix orientations because the polypeptides are aligned along with the phospholipid bilayers with a known alignment in the magnetic field. An automated fitting of a sine wave to the experimental data clearly identifies those residues that contribute to the α-helical region of the polypeptide based on periodicity. The range and average value of the dipolar couplings give precise measures of the helix tilt and rotation. Previously, we have shown that wild-type Vpu TM in various lipid environments including micelles, bicelles, and bilayers on glass plates spans 18 residues, from 8 to 25, and the rotation angle of the helix is unchanged in lipid environments that result in significant changes of the tilt angle (Fig. 6A,C; Park et al. 2003, 2006). A kink is apparent in wild-type Vpu TM in micelles that is also present in thick bilayers with small tilt angle (Park et al. 2003); the kink is not present in thinner bilayers or in A18H Vpu TM in any lipid environment.

Figure 6.

Figure 6.

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Dipolar wave plots of wild-type Vpu TM (A,C) and A18H Vpu TM (B,D). (A,B) 1H-15N residual dipolar couplings obtained from the weakly aligned sample in micelles. (B,D) 1H-15N full dipolar couplings obtained from the sample in 14-O-PC/6-O-PC (q = 3.2) bicelles aligned magnetically with their bilayer normals perpendicular to the magnetic field. The residues that disappeared as a result of exchange in 2H2O are indicated with open circles and fit with dashed lines.

The experimental residual (Fig. 6B) and full (Fig. 6D) dipolar couplings of A18H Vpu TM as a function of residue number are well fit by single sine waves. The RMS error of the residual dipolar couplings is 0.59 Hz for residues 6–27 (0.44 Hz for 8–27). And the RMS error of the full dipolar couplings is 0.36 kHz for the residues 6–27, which is only slightly larger than the experimental line widths in the heteronuclear dipolar coupling dimension of the spectra. These results indicate that the helix of A18H Vpu TM is an ideal, straight α-helix spanning 22 amino acids from Ile6 to Ile27 in C14 bicelles. This is remarkable because it means that the trans-membrane helix in A18H Vpu TM is four residues longer than that in wild-type Vpu TM.

Structural fitting (Nevzorov and Opella 2003) enables the calculation of protein structures from the chemical shift and heteronuclear dipolar coupling frequencies associated with individual resonances in solid-state NMR spectra obtained from uniaxailly aligned samples of proteins. In addition to being able to determine the three-dimensional structures of proteins, the structural fitting algorithm is sensitive enough to detect minor deviations from ideality in helices. A structural fit to a fully assigned spectrum is equivalent to a direct calculation of the protein structure (Park et al. 2003, 2006; Zeri et al. 2003; Thiriot et al. 2004; De Angelis et al. 2006). By performing repeated structural fits to the completely assigned spectrum of A18H Vpu TM shown in Figure 2B, 20 structural solutions were generated. The mean value of the RMSD relative to the average structure was 0.34 Å. A representative structure of A18H Vpu TM is compared with that of wild-type Vpu TM in Figure 7. Both of the structures were determined in C14 bilayers.

Figure 7.

Figure 7.

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Three-dimensional backbone structures of (A) wild-type Vpu TM (PDB code 2GOF) and (B) A18H Vpu TM (PDB code 2jpx) determined in 14-O-PC/6-O-PC (q = 3.2) bicelles by using the method of structural fitting of experimental solid-state NMR data. An average tilt angle of the helix is represented and the side chains of residue 18 and 22 indicate the rotation of the helix. The shaded box indicates the thickness of the hydrophobic region of the lipid bilayer. The images were created using the program MOLMOL (Koradi et al. 1996). The chemical structure of rimantadine is shown in the approximate location predicted by the NMR results.

Discussion

The N-terminal domain of Vpu consists mainly of a hydrophobic trans-membrane helix that affects the assembly and release of virus particles, and the C-terminal cytoplasmic domain is responsible for targeting the CD4 receptors for degradation by the proteasome. Studies of the separate structural and functional domains provide background for understanding the integration of the biological activities in a single polypeptide chain. In our earlier work, we described how the lipid environment affected the properties of the trans-membrane helix (Park et al. 2003, 2006; Park and Opella 2005). In this article, we describe this domain's response to a single-site mutation and to the addition of a channel blocker. A major theme emerging from the NMR studies of the trans-membrane helix of Vpu is that its structure is highly adaptable to changes in both lipid and polypeptide components.

A trans-membrane helix is characterized by its length (number of residues), tilt angle, rotation angle, and any deviations from ideality, such as the presence of a kink or curvature, all of which are relevant to understanding the structure and functions of a membrane protein (Mesleh et al. 2003). Aligned sample solid-state NMR is especially well suited for describing the properties of membrane proteins in phospholipid bilayers (Opella and Marassi 2004), their native environment, because of the direct mapping of their structure and topology onto the spectra by the orientationally dependent frequencies of the chemical shift and dipolar interactions. PISA wheels and dipolar waves provide accurate representations of the periodic variations of the frequencies and secondary structures. These same data also provide input for the calculation of complete three-dimensional protein structures.

In earlier applications of NMR spectroscopy to the trans-membrane helix of Vpu, we were able to show that the tilt angle, but not the rotation angle, is determined by hydrophobic mismatch with the length of the phospholipids (Park and Opella 2005). The effect of the length of the phospholipids provided some of the first evidence of the conformational plasticity of Vpu, and this is likely to be important for its functions, since Vpu is found in cellular membranes with different lipid compositions and hydrophobic thicknesses.

Stephens and coworkers have shown that Vpu with its trans-membrane helix replaced by that of the influenza M2 protein is not only functional but also confers susceptibility to the channel blocker rimantadine to the virus infection (Hout et al. 2006b). Through sequence comparisons, they extended this to a single-site mutation, A18H, that makes the trans-membrane helix of Vpu more similar to that of M2 and has the same properties as the M2 sequence in this role (Hout et al. 2006a). The 36-residue N-terminal construct of Vpu containing this mutation, A18H Vpu TM, has a tilt angle of 41°, which is substantially larger than that observed for wild-type Vpu TM in the same C14 phospholipid bilayers. This is clearly demonstrated by the differences in the experimental PISA wheel patterns in the solid-state NMR spectra (Fig. 3). The patterns have the same sinusoid phase in the dipolar waves derived from these data (Fig. 6), which indicates that the rotation angles of the helices are the same. The difference in tilt angle but not in rotation angle is also apparent in the three-dimensional structures of the wild-type Vpu TM and A18H Vpu TM calculated from the solid-state NMR data and shown in Figure 7.

The structure of the M2 TM domain determined in C14 lipid bilayers using solid-state NMR and computational modeling (Nishimura et al. 2002) is also a nearly ideal α-helix spanning residues 23–45; the configuration of the tetramer structure of the M2 domain places the side chains of His37 and Trp41, which are located on the same face of the helix, on the interior of the pore. The tilt angle of A18H Vpu TM (41°) is similar to that of M2 TM (38°) in C14 phospholipid bilayers. In addition, the rotation angles that we determined for His18 and Trp22 in A18H TM are almost identical to those of His37 and Trp41 in the M2 TM structure (Wang et al. 2001). These results suggest that the structural changes resulting from the substitution of histidine for alanine at residue 18 do indeed make it more similar to the M2 TM structure, as anticipated by Hout et al. (2006a).

By combining results from magic angle spinning and aligned sample solid-state NMR experiments, Cross and coworkers (Hu et al. 2007a,b) have recently shown that the channel blocker amantadine interacts with the side chain of the key histidine residue and induces structural changes throughout the trans-membrane domain of the M2 TM domain. Otherwise, structure–activity relationships by NMR (Hajduk et al. 1997) or more generally NMR target-based screening has only been applied to a few membrane proteins. A solution-state NMR study involving a potassium channel bound to a peptide antagonist (Yu et al. 2005) and several solid-state NMR studies of ligand binding to membrane proteins (Watts 2005; Baldus 2006) have been described. By combining results from solution-state and solid-state NMR, we have investigated the interaction of a small molecule with the thrombopoietin receptor segment in lipid environments (Kim et al. 2007).

The comparison of solution-state NMR spectra obtained in the absence and presence of rimantadine clearly show that rimantadine binds to A18H Vpu TM but not wild-type Vpu TM (Fig. 2). The corresponding solid-state NMR spectra of the trans-membrane domains in aligned bicelles show that both isotropic and orientationally dependent frequencies associated with the backbone amide of His18 are specifically perturbed by rimantadine binding. The isotropic chemical shifts of other residues, especially Ile15 and Trp22 were also affected by the channel blocker. It has been established that the highly conserved sequence motif His-X-X-X-Trp within the trans-membrane helix of the influenza M2 protein is essential for ion-channel activity and inhibition by amantadine, a similar channel blocker (Okada et al. 2001; Tang et al. 2002; Takeuchi et al. 2003; Hout et al. 2006a). Our results on A18H Vpu TM suggest that His18 is the key residue for rimantadine binding, although other residues located on the same face of the helix are also involved.

The comparisons of wild-type Vpu TM and A18H Vpu TM further demonstrate the structural plasticity of Vpu. Remarkably, a single amino acid substitution in the middle of the trans-membrane helix causes four additional residues to become helical, lengthening the helix and consequently causing a dramatic increase in the tilt angle of the helix in phospholipid bilayers. This would result in a significant change in the structure and topology of the oligomer. The specific changes observed in the spectra of A18H Vpu TM demonstrate that the histidine residue is likely to be involved in the binding of channel blockers. There is a direct correlation of structure to biological activity, because it has been shown that when this mutation is incorporated into the full-length protein, Vpu retains its biological functions and makes the in vivo HIV infection susceptible to channel blockers (Hout et al. 2006a). The nature of the changes in the spectra and the protein structure resulting from a single-site mutation and interactions with a drug are qualitatively different than those generally observed with globular proteins, demonstrating that structural studies of membrane proteins in phospholipid bilayers have the potential to reveal new aspects of their structural biology by preserving their ability to change in response to the phospholipids, protein sequence, and added small molecules.

Materials and Methods

Sample preparation

The expression and purification of wild-type Vpu TM have been described previously (Park et al. 2003). The mutant A18H Vpu TM was made using a QuikChange site-directed mutagenesis kit (Stratagene). Uniformly 15N-labeled polypeptides were obtained by growing bacteria in M9 minimal medium with (15NH4)2SO4 as the sole nitrogen source. Selectively, 15N-labeled samples were obtained by growing bacteria in minimal medium with unlabeled ammonium sulfate supplemented with each of the 19 amino acid residues and the chosen 15N-labeled amino acid. After purification of the inclusion bodies by immobilized metal ion affinity chromatography, followed by cleavage with cyanogen bromide, the peptide was purified by HPLC and lyophilized.

The preparation of samples in micelles for solution NMR and bicelles for solid-state NMR experiments have also been described previously (Park et al. 2003, 2006). Solution NMR samples were prepared by dissolving purified polypeptide in 400 μL of 100 mM deuterated DHPC (Cambridge Isotope Laboratories) micelles, 10% (v/v) 2H2O (pH 4.0). Weakly aligned samples were prepared by soaking the polypeptide-containing micelles into a dried 7% polyacrylamide gel overnight with a length of the gel restricted to 20 mm from an initial length of 27 mm. Samples for the solid-state NMR experiments were prepared by dissolving the polypeptide in an aqueous solution containing short-chain lipids, 6-O-PC, and then adding this solution to a dispersion of long-chain lipids, 14-O-PC, in water. The lipids were purchased from Avanti Polar Lipids. The bicelle sample for q = 3.2 and a lipid concentration of 28% (w/v), was ∼300 mM in 14-O-PC in a 180-μL volume at pH 4.0. A short, flat-bottomed NMR tube with 5 mm outer diameter (New Era Enterprises) was filled with 160 μL of the bicelle solution and the tube was sealed with a rubber cap to create a tight seal. In order to prepare 2H2O exchanged samples, the samples in water were lyophilized and redissolved in 99% 2H2O (Cambridge Isotope Laboratories). For the measurement of chemical shift and dipolar coupling perturbations by the addition of rimantadine, 0.2 M stock solution of rimantadine hydrochloride (Sigma-Aldrich) that was prepared immediately before use in water was added directly into the micelle and bicelle samples to a final concentration of 10 mM. After adding rimantadine to the peptide, the sample pH was adjusted.

Solution NMR spectroscopy

The solution NMR experiments were performed on a Bruker DRX 600 MHz NMR spectrometer equipped with a 5-mm triple resonance cryogenic probe and z-axis gradient at 50°C. The two-dimensional 1H/15N HSQC spectra were obtained on uniformly and selectively 15N-labeled samples with a protein concentration of ∼0.5 mM. To monitor the amide proton exchange, 1H/15N HSQC spectra were obtained 1 h after the addition of 2H2O to a lyophilized sample of uniformly 15N-labeled samples. Amide resonances of A18H Vpu TM were assigned by using a two-dimensional HMQC-NOESY spectrum with 200 ms mixing time (Shon and Opella 1989). IPAP-HSQC (Ding and Gronenborn 2004) spectra obtained on isotropic and weakly aligned samples were used to measure the 1H-15N residual dipolar couplings. The NMR data were processed using the programs NMRPipe/NMRDraw and NMRView (Delaglio et al. 1995). The chemical shift perturbations by addition of rimantadine were calculated using the equation

graphic file with name 2205equ1.jpg

where ΔδH is the change in the backbone amide proton chemical shift and ΔδN is the change in backbone amide nitrogen chemical shift.

Solid-state NMR spectroscopy

The solid-state NMR experiments were performed on a spectrometer with a 1H resonance frequency of 750 MHz, consisting of a Bruker Avance console and Magnex magnet and room temperature shims. Double-resonance (1H, 15N) home-built and triple-resonance (1H, 15N, 13C) Bruker low-E probes with 5-mm inner diameter solenoid coils were used. The two-dimensional SAMPI4 (Nevzorov and Opella 2007) spectra of uniformly 15N-labeled sample resulted from a total of 64 t1 increments and 512 t2 complex points with 128 scans for each t1 increment. The spectra for selectively 15N-labeled samples were obtained using between 32 and 64 t1 increments, depending on the sensitivity of each sample. The B1 radio-frequency strength of 50 kHz and 6 sec recycle delay with 5 ms acquisition time were used. All quantitative frequency measurements used in the structural analysis were obtained from the spectra of the uniformly 15N-labeled peptide to eliminate variability due to slight differences from sample to sample. The data were zero filled in both the t2 and t1 dimensions, yielding a 1024 × 1024 real matrix. A suitable phase-shifted sine bell multiplication followed by 50–100 Hz of exponential multiplication was applied in both dimensions prior to Fourier transformation. The NMR data were processed using the programs NMRPipe/NMRDraw (Delaglio et al. 1995). All chemical shifts were externally referenced to 15N-labeled solid ammonium sulfate, set to 26.8 ppm, corresponding to the signal from liquid ammonia at 0 ppm.

Dipolar waves and structure calculations

Dipolar waves (Mesleh et al. 2002, 2003; Mesleh and Opella 2003), the fitting of the 1H-15N dipolar couplings as a function of residue number to a sinusoid of period 3.6 corresponding to the pitch of an α-helix, were analyzed to characterize the length, orientation, and rotation of the α-helices. Regions for which the RMSD/point was within the experimental error (0.5 Hz for residual dipolar couplings and 0.4 kHz for full dipolar couplings) defined the N and C termini of the helix.

A set of structures was obtained from the orientationally dependent experimental 1H-15N dipolar coupling and 15N chemical shift frequencies in bicelles using the modified structural fitting algorithm (Nevzorov and Opella 2003). The order parameter S was set to 0.8 for the bicelle sample, which was determined previously (Park et al. 2006). The Ramachandran angles ϕ and ψ between consecutive residues were obtained by direct calculation from the dipolar and chemical shift frequencies, assuming constant peptide plane geometry. The 15N chemical shift tensor values were assigned constant values (σ11 = 64 ppm, σ22 = 77 ppm, σ33 = 222 ppm and σ11 = 41 ppm, σ22 = 64 ppm, σ33 = 215 ppm for glycines) and were allowed to vary within ±300 Hz in each frequency dimension for the spectral frequencies, in order to account for experimental reproducibility, site-to-site variations in the tensor values, and variatioin or uncercetainty of the scaling factor in the dipolar frequency dimension. For every calculation the torsion angles were allowed to vary within ±15° relative to the average values for an α-helix, ϕ = −61°, ψ = −45°. The precision of the structural fit was evaluated by comparing the multiple structural solutions from repeated calculations within these defined limits, which gives rise to a RMSD of ∼0.5 Å for the complete backbone structure. Side-chain atoms were added to the backbone structures, and their coordinates were optimized using the program SCWRL (side chain with a rotamer library) (Bower et al. 1997).

The coordinates have been deposited in the Protein Data Bank (PDB ID Code 2jpx), for release upon publication.

Acknowledgments

We thank C.H. Wu and C.V. Grant for assistance with the instrumentation. This research was supported by National Institutes of Health grant RO1GM066978 and it utilized the Biomedical Technology Resource for NMR Molecular Imaging of Proteins, supported by NIH grant P41EB002031.

Footnotes

Reprint requests to: Stanley J. Opella, Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0307, USA; e-mail: sopella@ucsd.edu; fax: (858) 822-4821.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.073041107.

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