Abstract

The three-dimensional structure of the trans-membrane domain of virus protein "u" (Vpu) of HIV-1 was determined by NMR spectroscopy. Vpu_2-30+, a polypeptide consists of residues 2-30 from Vpu and a 6-residue "solubility tag" to enable its isolation, purification, and sample preparation of this hydrophobic minimal function domain of the protein. The vast majority of resonances in the ¹H-¹⁵N HSQC spectrum of uniformly ¹⁵N labeled Vpu_2-30+ in micelles are superimposable on those from the corresponding residues in the spectrum of the full-length Vpu, suggesting that the trans-membrane domain is not strongly influenced by the presence of the cytoplasmic domain. The two-dimensional PISEMA spectrum of the uniformly ¹⁵N labeled Vpu_2-30+ polypeptide in the bilayers has been fully resolved and assigned. The "wheel-like" pattern of resonances observed is characteristic of a tilted membrane-spanning helix. The analysis of PISA wheels and Dipolar waves obtained from both weakly and completely aligned samples show that Vpu_2-30+ has a trans-membrane α-helix spanning residues 8-25 with a slant angle of 13° that is slightly kinked at isoleucine 17 in the middle of the helix. A structural fit to the experimental solid-state NMR data results in a structure determination with an sccuracy equivalent to an RMSD of 0.4 Å that is consistent with the spectral analysis based on ideal helical segment. Vpu_2-30+ exists mainly as a pentamer on PFO-PAGE and forms ion-channels of most frequent conductance of 96 +/- 6pS in lipid bilayers, the structural features of the helix are likely to be determinants of the ion-channel activity thought to be associated with the protein activity that facilitates the budding of new virus particles from infected cells.

Schematic diagram of the topology of Vpu in bilayers that compares the polypeptide Vpu_2-30+ and full-length Vpu_2-30+

(a)SDS-PAGE: 1 & 2, supematant and precipitate of cell extract; 3, purified fusion protein; 4, mixture dissolved in HPLC injection solvent after CNBr cleavage; 5, purified Vpu_2-30+ by RP-HPLC
(b)RP-HPLC profile of Vpu_2-30+ after CNBr cleavage of fusion peptide
(c)PFO-PAGE:1, lysozyme (14.4 kDa); 2, Vpu_28-81 (6.3 kDa); 3, 100 µg of Vpu_2-30+; 4, 50 µg of Vpu_2-30+; 5, 25 µg of Vpu_2-30+; 6, 13 µg of Vpu_2-30+; 7, 7 µg of Vpu_2-30+

Amide regiona of ¹H-¹⁵N HSQC spectra of uniformly and selectively labeled Vpu_2-30+ in 100 mM DHPC at pH 4.0, 323K. The assignments of the amide resonances are indicated by residue number.