NMR structural studies of the integral membrane protein Vpu from HIV-1

Vpu is an 81-residue membrane protein that enhances the release of new virus particles from cells infected with HIV-1 and induces the degradation of the CD4 receptor protein. The ion channel activity of Vpu may be related to an increased virus budding rate, and the phosphorylation of two highly conserved serines is necessary for CD4 degradation. Uniformly isotopically labeled samples of several constructs of Vpu, which represent its functional domains, prepared by expression of a fusion protein in E. coli, were studied in the model membrane environments of dihexanoyl phophatidylcholine (DHPC) micelles and phospholipid bilayers by multidimensional solution and solid-state NMR spectroscopy, respectively. The biological activities of the constructs of Vpu were evaluated. Complete backbone resonance assignments and the secondary structure were obtained for the transmembrane and the cytoplasmic constructs of Vpu by solution NMR spectroscopy. Both short-range distance constraints and angular measurements from weakly oriented samples are being used to describe its tertiary structure in micelles. The solution NMR results are complementary to those based on angular constraints from highly oriented, immobile samples in lipid bilayers measured in solid-state NMR experiments. Methodology developments toward structure determination of membrane proteins are also discussed