Membrane interactions and alignment of structures within the HIV-1 Vpu cytoplasmic domain: effect of phosphorylation of serines 52 and 56

The cytoplasmic domain of the HIV-1 accessory protein Vpu is involved in the binding and degradation of the viral receptor CD4. In order to analyze previous structural models in the context of membrane environments, regions of Vpu CYTO incorporating particular conformational features have been synthesized and labelled with 15N at selected backbone amides. Well-oriented proton-decoupled 15N solid-state NMR spectra with 15N chemical shifts at the most upfield position indicate that the amphipathic helix within [ 15N-Leu 45]-Vpu 27–57 strongly interacts with mechanically aligned POPC bilayers and adopts an orientation parallel to the membrane surface. No major changes in the topology of this membrane-associated amphipathic helix were observed upon phosphorylation of serine residues 52 and 56, although this modification regulates biological function of Vpu. In contrast, [ 15N-Ala 62]-Vpu 51–81 exhibits a pronounced 15N chemical shift anisotropy.