Solid-State NMR Investigations of Vpu Structural Domains in Oriented Phospholipid Bilayers: Interactions and Alignment

Abstract

The accessory 81-residue protein viral protein U (Vpu) of human immunodeficiency virus-1 (HIV-1) fulfills two important functions during the viral life cycle. First, its intact N-terminus is required during viral release, a function that is correlated to its capability to form channels in planar lipid bilayers. Second, its cytoplasmic domain is involved in the binding and degradation of viral receptors including CD4 and major histocompatibility complex-I (MHC-I). In order to develop a structural model of Vpu in phospholipid bilayers, various Vpu polypeptides have been prepared by chemical synthesis and labeled with 15N at selected backbone amide sites. After reconstitution of the peptides into oriented phospholipid bilayers, protondecoupled 15N solid-state nuclear magnetic resonance (NMR) spectra were recorded. The measured 15N chemical shifts are indicative of the interactions and alignment of structural domains of Vpu within the lipid membrane. Whereas the hydrophobic helix 1, which is located at the N-terminus, adopts a transmembrane tilt angle of approximately 20°, the amphipathic helix 2 in the center of the polypeptide is oriented 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 the Vpu cytoplasmic domain. The alanine-62 position of Vpu51–81 exhibits a pronounced 15N chemical shift anisotropy suggesting that interactions with the lipid bilayer of the C-terminal part of the protein are weak.