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 ␣-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 ␣-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 ␣-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.

Hydration-optimized oriented phospholipid bilayer samples for solid-state NMR structural studies of membrane proteins

“…The problems associated with rf heating can be further mitigated by performing the NMR experiments at low temperature. Indeed, even moderately low temperatures of 0-5 °C result in improved sample stability and reduced losses of rf power [27]. Our initial experiments with hydration-optimized samples at n w ≈ 2, indicate that lipid bilayer orientation is not lost upon lowering the temperature below the onset of headgroup rotations, suggesting that it might be possible to use these samples for experiments at very low temperature.…”

“…Finally, while the samples described in this report were prepared by organic solvent deposition of mixed lipids and protein on glass slides, oriented lipid bilayer samples for solid-state NMR structural studies can also be prepared by depositing aqueous suspensions of protein/lipid vesicles, obtained by detergent dialysis [9,27]. The method of choice for reconstitution is protein-dependent, and both methods yield highly oriented lipid bilayer samples that are ready for hydration optimization in reduced humidity atmosphere.…”

Application of solid-state NMR restraint potentials in membrane protein modeling

“…There is a hydrophobic TM helix in the N-terminal domain and amphipathic helices in the C-terminal domain [44]. Vpu affects the budding of new virus particles, and the hydrophobic TM helix (residues 7-25) may play an important role as an ion channel that is selective for monovalent cations such as Na + and K + [45].…”

Oligomerization state and supramolecular structure of the HIV‐1 Vpu protein transmembrane segment in phospholipid bilayers

“…Vpu is an 81-residue integral membrane protein encoded by the HIV-1 genome for which two distinct functions have been identified [1][2][3] : (i) downregulation of cell-surface CD4 expression, produced by mediating the interaction of newly synthesized CD4 with bTrCP, a component of a ubiquitin ligase complex 4-8 ; (ii) enhancement of the release of new virions from host cell surfaces. 9 The second function depends principally on the single transmembrane TM segment of Vpu, contained within residues 1-30, as sequence alterations in the TM segment have been shown to interfere with this function.…”

“…9 The second function depends principally on the single transmembrane TM segment of Vpu, contained within residues 1-30, as sequence alterations in the TM segment have been shown to interfere with this function. 2 Full-length Vpu and various N-terminal peptides containing the TM segment have been shown to form cation-selective channels in model membranes 1,[10][11][12][13][14][15] and in cells. 12 These ion channels presumably form by association of a-helical TM segments into homo-oligomeric bundles, with ions passing through the central pore of the helix bundle.…”