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HIV-1 Vpu protein forms stable oligomers in aqueous solution via its transmembrane domain self-association.

ABSTRACT: We report our findings on the assembly of the HIV-1 protein Vpu into soluble oligomers. Vpu is a key to HIV-1 protein. It has been considered exclusively a single-pass membrane protein. However, we revealed that this protein forms stable oligomers in aqueous solution, which is an interesting and rather unique observation, as the number of proteins transitioning between soluble and membrane embedded states is limited. Therefore, we undertook a study to characterize these oligomers by utilizing protein engineering, size exclusion chromatography, cryoEM and electron paramagnetic resonance (EPR) spectroscopy. We found that Vpu oligomerizes via its N-terminal transmembrane domain (TM). CryoEM analyses suggest that the oligomeric state most likely is a hexamer or hexamer-to-heptamer equilibrium. Both cryoEM and EPR suggest that, within the oligomer, the distant C-terminal region of Vpu is highly flexible. To the best of our knowledge, this is the first comprehensive study on soluble Vpu. We propose that these oligomers are stabilized via possibly hydrophobic interactions between Vpu TMs. Our findings contribute valuable information about this protein properties and about protein supramolecular complexes formation. The acquired knowledge could be further used in protein engineering, and could also help to uncover possible physiological function of these Vpu oligomers.

SUBMITTER: Majeed S

PROVIDER: S-EPMC10197565 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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HIV-1 Vpu protein forms stable oligomers in aqueous solution via its transmembrane domain self-association.

Majeed Saman S Dang Lan L Islam Md Majharul MM Ishola Olamide O Borbat Peter P PP Ludtke Steven J SJ Georgieva Elka R ER

bioRxiv : the preprint server for biology 20230705

We report our findings on the assembly of the HIV-1 protein Vpu into soluble oligomers. Vpu is a key to HIV-1 protein. It has been considered exclusively a single-pass membrane protein. However, we revealed that this protein forms stable oligomers in aqueous solution, which is an interesting and rather unique observation, as the number of proteins transitioning between soluble and membrane embedded states is limited. Therefore, we undertook a study to characterize these oligomers by utilizing pr ...[more]

PMID: 37214796

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Project description:BackgroundThe acquisition of effective Vpu-mediated anti-tetherin activity to promote virion release following transmission of SIVcpzPtt from central chimpanzees (Pan troglodytes troglodytes) to humans distinguishes pandemic HIV-1 group M strains from non-pandemic group N, O and P viruses and may have been a prerequisite for their global spread. Some functional motifs in the cytoplasmic region of HIV-1 M Vpus proposed to be important for anti-tetherin activity are more frequently found in the Vpu proteins of SIVcpzPtt than in those of SIVcpzPts infecting eastern chimpanzees (P. t. schweinfurthii), that have not been detected in humans, and SIVgor from gorillas, which is closely related to HIV-1 O and P. Thus, SIVcpzPtt strains may require fewer adaptive changes in Vpu than SIVcpzPts or SIVgor strains to counteract human tetherin.ResultsTo examine whether SIVcpzPtt may only need changes in the transmembrane domain (TMD) of Vpu to acquire anti-tetherin activity, whereas SIVcpzPts and SIVgor may also require changes in the cytoplasmic region, we analyzed chimeras between the TMD of an HIV-1 M Vpu and the cytoplasmic domains of SIVcpzPtt (n = 2), SIVcpzPts (n = 2) and SIVgor (n = 2) Vpu proteins. Unexpectedly, all of these chimeras were capable of counteracting human tetherin to enhance virion release, irrespective of the presence or absence of the putative adaptor protein binding sites and the DSGxxS β-TrCP binding motif reported to be critical for effective anti-tetherin activity of M Vpus. It was also surprising that in three of the six chimeras the gain of anti-tetherin function was associated with a loss of the CD4 degradation activity since this function was conserved among all parental HIV-1, SIVcpz and SIVgor Vpu proteins.ConclusionsOur results show that changes in the TMD of SIVcpzPtt, SIVcpzPts and SIVgor Vpus are sufficient to render them active against human tetherin. Thus, several previously described domains in the extracellular region of Vpu are not absolutely essential for tetherin antagonism but may be required for other Vpu functions.

Project description:The three-dimensional backbone structure of the transmembrane domain of Vpu from HIV-1 was determined by solid-state NMR spectroscopy in two magnetically-aligned phospholipid bilayer environments (bicelles) that differed in their hydrophobic thickness. Isotopically labeled samples of Vpu(2-30+), a 36-residue polypeptide containing residues 2-30 from the N-terminus of Vpu, were incorporated into large (q = 3.2 or 3.0) phospholipid bicelles composed of long-chain ether-linked lipids (14-O-PC or 16-O-PC) and short-chain lipids (6-O-PC). The protein-containing bicelles are aligned in the static magnetic field of the NMR spectrometer. Wheel-like patterns of resonances characteristic of tilted transmembrane helices were observed in two-dimensional (1)H/(15)N PISEMA spectra of uniformly (15)N-labeled Vpu(2-30+) obtained on bicelle samples with their bilayer normals aligned perpendicular or parallel to the direction of the magnetic field. The NMR experiments were performed at a (1)H resonance frequency of 900 MHz, and this resulted in improved data compared to lower-resonance frequencies. Analysis of the polarity-index slant-angle wheels and dipolar waves demonstrates the presence of a transmembrane alpha-helix spanning residues 8-25 in both 14-O-PC and 16-O-PC bicelles, which is consistent with results obtained previously in micelles by solution NMR and mechanically aligned lipid bilayers by solid-state NMR. The three-dimensional backbone structures were obtained by structural fitting to the orientation-dependent (15)N chemical shift and (1)H-(15)N dipolar coupling frequencies. Tilt angles of 30 degrees and 21 degrees are observed in 14-O-PC and 16-O-PC bicelles, respectively, which are consistent with the values previously determined for the same polypeptide in mechanically-aligned DMPC and DOPC bilayers. The difference in tilt angle in C14 and C16 bilayer environments is also consistent with previous results indicating that the transmembrane helix of Vpu responds to hydrophobic mismatch by changing its tilt angle. The kink found in the middle of the helix in the longer-chain C18 bilayers aligned on glass plates was not found in either of these shorter-chain (C14 or C16) bilayers.

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HIV-1 Vpu protein forms stable oligomers in aqueous solution via its transmembrane domain self-association.

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HIV-1 Vpu protein forms stable oligomers in aqueous solution via its transmembrane domain self-association.

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