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

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 alpha-helix spanning residues 6-27 with an average tilt angle of 41 degrees in C14 phospholipid bicelles, indicating that the tilt angle is increased by 11 degrees 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.

Figure 1.

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

Figure 2.

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.

Figure 3.

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.

Figure 4.

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 5.

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.

Figure 6.

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.

Figure 7.

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.