Expression, purification, and activities of full‐length and truncated versions of the integral membrane protein Vpu from HIV‐1

Abstract

Vpu is an 81-residue accessory protein of HIV-1. Because it is a membrane protein, it presents substantial technical challenges for the characterization of its structure and function, which are of considerable interest because the protein enhances the release of new virus particles from cells infected with HIV-1 and induces the intracellular degradation of the CD4 receptor protein. The Vpu-mediated enhancement of the virus release rate from HIV-1-infected cells is correlated with the expression of an ion channel activity associated with the transmembrane hydrophobic helical domain. Vpu-induced CD4 degradation and, to a lesser extent, enhancement of particle release are both dependent on the phosphorylation of two highly conserved serine residues in the cytoplasmic domain of Vpu. To define the minimal folding units of Vpu and to identify their activities, we prepared three truncated forms of Vpu and compared their structural and functional properties to those of full-length Vpu (residues 2-81). Vpu 2-37 encompasses the N-terminal transmembrane ␣-helix; Vpu 2-51 spans the N-terminal transmembrane helix and the first cytoplasmic ␣-helix; Vpu 28-81 includes the entire cytoplasmic domain containing the two C-terminal amphipathic ␣-helices without the transmembrane helix. Uniformly isotopically labeled samples of the polypeptides derived from Vpu were prepared by expression of fusion proteins in E. coli and were studied in the model membrane environments of lipid micelles by solution NMR spectroscopy and oriented lipid bilayers by solid-state NMR spectroscopy. The assignment of backbone resonances enabled the secondary structure of the constructs corresponding to the transmembrane and the cytoplasmic domains of Vpu to be defined in micelle samples by solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in oriented lipid bilayers demonstrated that the topology of the domains is retained in the truncated polypeptides. The biological activities of the constructs of Vpu were evaluated. The ion channel activity is confined to the transmembrane ␣-helix. The C-terminal ␣-helices modulate or promote the oligomerization of Vpu in the membrane and stabilize the conductive state of the channel, in addition to their involvement in CD4 degradation.

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

“…However, the fact that 2D 13 C-13 C spectra of Vpu in bilayers show a single set of 13 C chemical shifts 17 suggests that peptide conformations and intermolecular interactions in the predominant oligomers are similar. Moreover, the sharp crosspeak signals in solid-state NMR spectra of Vpu TM peptides in uniaxially aligned bilayers reported by Opella and coworkers 1,11,[18][19][20][21][22] suggest that peptide orientations relative to the bilayer are similar, as orientational variations greater than $5 would produce significant line-broadening in their spectra. Therefore, although it is generally not valid to use data obtained from a structural ensemble to generate a single structural model, in the case of Vpu 1-40 oligomers it is reasonable to assume that oligomers of different size share common helix orientations and intermolecular contacts and to apply the constraints from our solid-state NMR measurements to oligomers with specific sizes.…”

“…This lipid composition was chosen for consistency with previous solid-state NMR studies. 11,17 PICUP has the advantages of high efficiency and short reaction times, so that crosslinking between different oligomers that encounter one another through translational diffusion can be minimized. In addition, PICUP does not require chemical modifications to the peptide.…”

“…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.…”

“…The precise number of Vpu molecules that form active ion channels and the precise molecular structures of these ion channels have not yet been established unequivocally by experiments, although considerable experimental information is available about the structure of monomeric Vpu and its orientation relative to bilayer membranes from nuclear magnetic resonance (NMR) and other measurements. 1,11,[16][17][18][19][20][21][22] Gel filtration chromatography suggests a pentameric structure, 23 whereas gel electrophoresis suggests oligomers in the tetramer-tohexamer range. 17 Computational modeling of oligomers of Vpu TM segments has been carried out by several groups, resulting in detailed models for tetramers, pentamers, and hexamers.…”

Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2271–326)

“…The recombinant expression of hydrophobic peptides or transmembrane proteins in IBs as a fusion protein coupled with a leading partner (such as GST, KSI or TrpLE) has been shown to be an effective strategy to achieve high yield [9,12,[24][25][26][27]]. We previously showed high level expression of fusion proteins when TrpLE was fused to the N terminal of other CB2 fragments [12,14,15].…”