Structural Studies of the HIV-1 Accessory Protein Vpu in Langmuir Monolayers: Synchrotron X-ray Reflectivity

Abstract

Vpu is an 81 amino acid integral membrane protein encoded by the HIV-1 genome with a N-terminal hydrophobic domain and a C-terminal hydrophilic domain. It enhances the release of virus from the infected cell and triggers degradation of the virus receptor CD4. Langmuir monolayers of mixtures of Vpu and the phospholipid 1,2-dilignoceroyl-sn-glycero-3-phosphocholine (DLgPC) at the water-air interface were studied by synchrotron radiation-based x-ray reflectivity over a range of mole ratios at constant surface pressure and for several surface pressures at a maximal mole ratio of Vpu/DLgPC. Analysis of the x-ray reflectivity data by both slab model-refinement and model-independent box-refinement methods firmly establish the monolayer electron density profiles. The electron density profiles as a function of increasing Vpu/DLgPC mole ratio at a constant, relatively high surface pressure indicated that the amphipathic helices of the cytoplasmic domain lie on the surface of the phospholipid headgroups and the hydrophobic transmembrane helix is oriented approximately normal to the plane of monolayer within the phospholipid hydrocarbon chain layer. At maximal Vpu/DLgPC mole ratio, the tilt of the transmembrane helix with respect to the monolayer normal decreases with increasing surface pressure and the conformation of the cytoplasmic domain varies substantially with surface pressure.

Characterization of a Novel Simian Immunodeficiency Virus (SIVmonNG1) Genome Sequence from a Mona Monkey (Cercopithecus mona)

“…The SIVgsn Vpu was also predicted to have only one alpha-helix. In comparison, by using the same prediction algorithms, HIV-1 and SIVcpz Vpu proteins had two alpha-helices, one on either side of the phosphoserines, a finding consistent with the structural data (12,52,53). This could be interpreted as indicating a difference in biological activity of the cytoplasmic domain of the SIVmonNG1 Vpu compared to the equiv- Sequence comparisons of the SIVmonNG1 genome further illustrate the complexity of the relationships between the primate lentiviruses.…”

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.…”

“…1,11,[18][19][20][21][22] Solid-state NMR techniques employed in their studies are qualitatively different from the techniques described above, as they do not involve MAS and are applicable to uniaxially aligned bilayer systems. Specific models for Vpu TM tetramers and pentamers (Protein Data Bank files 1PI8 and 1PI7, respectively) have been proposed by Opella and coworkers.…”

“…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.…”

Bundles Consisting of Extended Transmembrane Segments of Vpu from HIV-1: Computer Simulations and Conductance Measurements

“…With this model we are able to focus not only on the residues within the TM region but also on particular residues such as glutamic acid, tyrosine, and arginine (EYR motif), which are suggested to be involved in the bend between the TM helix and the first extramembraneous helix (25). This helix lies with its helix long axis parallel to the membrane surface (20).…”