Correlation of the structural and functional domains in the membrane protein Vpu from HIV-1

Abstract

Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic alpha-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly (15)N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane alpha-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu(2-51) (residues 2-51), which contains the transmembrane alpha-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu(28-81) (residues 28-81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2-81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 +/- 3 pS and 12 +/- 3 pS in 0.5 M KCl and 29 +/- 3 pS and 12 +/- 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu(2-51), which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu(28-81), which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.

Figure 1

NMR spectra of three uniformly ¹⁵N-labeled recombinant Vpu constructs. Vpu has three helical segments. (Top) The overall architecture of the three constructs used in this study in the context of a membrane bilayer, with the hydrophobic helix in blue and both amphipathic helices in red. (Middle) Two-dimensional heteronuclear single quantum correlation spectra of the three Vpu constructs in dihexanoyl phosphatidylcholine micelles: Vpu (A), Vpu_2–51 (B), and Vpu_28–81 (C). Representative assigned resonances are highlighted in the boxes: amide resonances from Gly-53, Gly-58, Gly-67, and Gly-71 (red boxes); Ser-23 (blue boxes); and indole resonances from Trp-22 and Trp-76 (red/blue boxes). (Bottom) One-dimensional solid-state ¹⁵N NMR spectra of the three Vpu constructs obtained at 0°C in oriented lipid bilayers: Vpu (D), Vpu_2–51 (E), and Vpu_28–81 (F). The orientations of transmembrane (blue) and in-plane (red) amide NH bonds are indicated above the spectra.

Figure 2

Two-dimensional solid-state NMR ¹H–¹⁵N dipolar coupling/¹⁵N chemical shift correlation PISEMA planes from uniformly ¹⁵N-labeled full-length Vpu in oriented lipid bilayers. (A) Plane extracted from a three-dimensional ¹H chemical shift/¹⁵N dipolar coupling/¹⁵N chemical shift spectrum at the ¹H chemical shift frequency of 15.9 ppm. (B) Two-dimensional PISEMA spectrum. (C–G) Two-dimensional PISEMA spectra calculated for a 17-residue α-helix, with uniform dihedral angles (φ = −57°; ψ = −47°) for the transmembrane orientations 0° (C), 10° (D), 15° (E), 20° (F), and 30° (G) between the long axis of the transmembrane helix and the normal to the membrane plane. The blue box highlights the spectral region occupied by resonances in the two-dimensional PISEMA spectrum from residues in the single transmembrane helix of Vpu.

Figure 3

Single-channel recordings from three Vpu constructs: Vpu (Left), Vpu_2–51 (Center), and Vpu_28–81 (Right) in symmetric 0.5 M KCl (A) or 0.5 M NaCl (B) and 10 mM Hepes (pH 7.4). Short segments of continuous recordings were selected to illustrate the occurrence of the 22-pS (K⁺) and 29-pS (Na⁺) channel for full-length Vpu and the 26-pS (K⁺) and 23-pS (Na⁺) channel for truncated Vpu_2–51. The cytoplasmic domain Vpu_28–81 does not form ion channels. Note the occurrence of only stray, erratic fluctuations in membrane current produced by Vpu_28–81 in contrast with the square events produced by Vpu and Vpu_2–51. The currents of the closed (C) and open (O) states are indicated. Upward deflection indicates channel opening. Corresponding cumulative current histograms and Gaussian fits for the primary conductances generated from continuous segments of recordings lasting several minutes. (A) For Vpu, the probability of the channel being open (P_o) is 0.56 recorded at 85 mV. For Vpu_2–51, P_o = 0.76 recorded at 100 mV. (B) For Vpu, P_o = 0.75 recorded at 50 mV; for Vpu_2–51, P_o = 0.56 recorded at 50 mV.