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

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.