Solution structure and orientation of the transmembrane anchor domain of the HTV-1-encoded virus protein U by high-resolution and solid-state NMR spectroscopy

The structure of the membrane anchor domain (Vpu(MA)) of the HIV-1-specific accessory protein Vpu has been investigated in solution and in lipid bilay ers by homonuclear two-dimensional and solid-state nuclear magnetic resonan ce spectroscopy, respectively. Simulated annealing calculations, using the nuclear Overhauser enhancement data for the soluble synthetic peptide Vpu(1 -39) (positions Met-1-Asp-39) in an aqueous 2,2,2-trifluoroethanol (TFE) so lution, afford a compact well-defined U-shaped structure comprised of an in itial turn (residues 1-6) followed by a linker (7-9) and a short helix on t he N-terminal side (10-16) and a further longer helix on the C-terminal sid e (22-36). The side chains of the two aromatic residues (Trp-22 and Tyr-29) in the longer helix are directed toward the center of the molecule around which the hydrophobic core of the folded Vpu(MA) is positioned. As the obse rved solution structure is inconsistent with the formation of ion-conductiv e membrane pores defined previously for Vpu(MA) in planar lipid bilayers, t he isolated Vpu(MA) domain as peptide Vpu(1-27) was investigated in oriente d phospholipid bilayers by proton-decoupled N-15 cross polarization solid-s tare NMR spectroscopy. The Line widths and chemical shift data of three sel ectively N-15-labeled peptides are consistent with a transmembrane alignmen t of a helical polypeptide. Chemical shift tensor calculations imply that t he data sets are compatible with a model in which the nascent helices of th e folded solution structure reassemble to form a more regular linear alpha- helix that lies parallel to the bilayer normal with a tilt angle of less th an or equal to 30 degrees. The arrangement of the membrane-associated struc tures described previously for the cytoplasmic domain and for the anchor do main of Vpu identified in this work is discussed.