Structure and Dynamics of the HIV-1 Vpu Transmembrane Domain Revealed by Solid-State NMR with Magic-Angle Spinning

Abstract

We report solid-state nuclear magnetic resonance (NMR) measurements on the peptide Vpu(1-40), comprising residues 1-40 of the 81-residue type 1 integral membrane protein Vpu encoded by the HIV-1 genome. On the basis of a combination of 13C and 15N NMR chemical shifts under magic-angle spinning (MAS), effects of local mobility on NMR signal intensities, site-specific MAS NMR line widths, and NMR-detected hydrogen-deuterium exchange, we develop a model for the structure and dynamics of the Vpu(1-40) monomer in phospholipid bilayer membranes. Our data are largely consistent with earlier structural studies of Vpu peptides by Opella and co-workers, in which solution NMR and solid-state NMR without MAS were used, but our data provide new information about local variations in the degree of mobility and structural order. In addition, our data indicate that the transmembrane alpha-helix of Vpu(1-40) extends beyond the hydrophobic core of the bilayer. We find no evidence for heterogeneity in the conformation and intermolecular contacts of the transmembrane alpha-helix, with the exception of two distinct chemical shifts observed for the C alpha and C beta atoms of A18 that may reflect distinct modes of helix-helix interaction. These results have possible implications for the supramolecular structure of Vpu oligomers that form cation-selective ion channels.

Oligomerization state and supramolecular structure of the HIV‐1 Vpu protein transmembrane segment in phospholipid bilayers

“…This lipid composition was chosen for consistency with previous solid-state NMR studies. 11,17 PICUP has the advantages of high efficiency and short reaction times, so that crosslinking between different oligomers that encounter one another through translational diffusion can be minimized. In addition, PICUP does not require chemical modifications to the peptide.…”

“…13 C chemical shift assignments for these spectra are given in Ref. 17 . In these experiments, each Vpu 1-40 molecule contained six uniformly labeled Val residues, eight uniformly labeled Ile residues, or four uniformly labeled Ala residues [see Figure 7(e,f), or in any of the other positions where intermolecular crosspeaks might be found (green rectangles).…”

“…Residues 7-27 were chosen because earlier solid-state NMR measurements 17 indicated that the N-terminal helix of Vpu includes residues 3-27, and because all data in Figures 7-10 pertain to sites in residues 7-27. Figure 11 shows a typical example of initial and energyminimized models, for a pentameric bundle with s ¼ À30 and q ¼ 0 .…”

“…The precise number of Vpu molecules that form active ion channels and the precise molecular structures of these ion channels have not yet been established unequivocally by experiments, although considerable experimental information is available about the structure of monomeric Vpu and its orientation relative to bilayer membranes from nuclear magnetic resonance (NMR) and other measurements. 1,11,[16][17][18][19][20][21][22] Gel filtration chromatography suggests a pentameric structure, 23 whereas gel electrophoresis suggests oligomers in the tetramer-tohexamer range. 17 Computational modeling of oligomers of Vpu TM segments has been carried out by several groups, resulting in detailed models for tetramers, pentamers, and hexamers.…”

“…1,11,[16][17][18][19][20][21][22] Gel filtration chromatography suggests a pentameric structure, 23 whereas gel electrophoresis suggests oligomers in the tetramer-tohexamer range. 17 Computational modeling of oligomers of Vpu TM segments has been carried out by several groups, resulting in detailed models for tetramers, pentamers, and hexamers. 10,[24][25][26][27][28][29] Predictions of channel conductance based on these models favor a pentamer or larger oligomer as the predominant channel.…”