SOLUTION STRUCTURE OF THE HYDROPHILIC REGION OF HIV-1 ENCODED VIRUS PROTEIN-U (VPU) BY CD AND H-1-NMR SPECTROSCOPY

The HIV-1 specific Vpu is a class I oligomeric membrane phosphoprotein of unknown structure and mechanism, The first experimental evidence f or the position of secondary structural elements present in the hydrop hilic C-terminal region of Vpu under various solution regimes is repor ted. CD data for nine overlapping 15 amino-acid fragments and 3 longer fragments indicate the presence of only transitory amounts of stable structure in aqueous solution alone, while with increasing trifluoroet hanol content limiting structures were found indicating two helical se gments in the hydrophilic region of Vpu. These limiting structures wer e more precisely defined from a detailed study of Vpu(41-58), Vpu(52-7 4) and Vpu(63-81), by a combination of 2D H-1 NMR spectroscopy, distan ce geometry, and restrained molecular dynamics and energy minimization calculations. Sets of low-energy conformations compatible with the qu antitative NOE data indicate that Vpu(41-58) has an a-helix from resid ues 42 to 50 while a second helix is found for Vpu(52-74) from residue s 57 to 69. Vpu(63-81) shows only the presence of a single reverse tur n at residues 74 to 77, without any evidence of helix, under the same conditions, From CD measurements the first helix extends back to resid ue 30 and is connected to the N-terminal anchor of Vpu, Thus the hydro philic region of Vpu consists of two alpha-helices joined by a flexibl e region of 6 or 7 residues, which contains the phosphoacceptor sites of Vpu at positions 52 and 56. The second helix is followed by a singl e reverse turn and a flexible C-terminus. (C) Munksgaard 1995.