vpu Transmembrane Peptide Structure Obtained by Site-Specific Fourier Transform Infrared Dichroism and Global Molecular Dynamics Searching

Abstract

The recently developed method of site-directed Fourier transform infrared dichroism for obtaining orientational constraints of oriented polymers is applied here to the transmembrane domain of the vpu protein from the human immunodeficiency virus type 1 (HIV-1). The infrared spectra of the 31-residue-long vpu peptide reconstituted in lipid vesicles reveal a predominantly alpha-helical structure. The infrared dichroism data of the (13)C-labeled peptide yielded a helix tilt beta = (6.5 +/- 1.7) degrees from the membrane normal. The rotational pitch angle omega, defined as zero for a residue located in the direction of the helix tilt, is omega = (283 +/- 11) degrees for the (13)C labels Val(13)/Val(20) and omega = (23 +/- 11) degrees for the (13)C labels Ala(14)/Val(21). A global molecular dynamics search protocol restraining the helix tilt to the experimental value was performed for oligomers of four, five, and six subunits. From 288 structures for each oligomer, a left-handed pentameric coiled coil was obtained, which best fits the experimental data. The structure reveals a pore occluded by Trp residues at the intracellular end of the transmembrane domain.

Application of solid-state NMR restraint potentials in membrane protein modeling

“…They also argued that the resulting pore lining of Ile17 in the right-handed conformation can be supported by the fact that the highly conserved Ile17 may have an essential role in its ion channel activity. However, modeling based on the experimental data from site-directed Fourier transform infrared dichroism supports the left-handed pentamer conformation and reveals a pore occluded by Trp residues at the end of the TM domain [50]. Our assembly calculations with flexible sidechains show that the left-handed pentamer conformation can have a slightly lower energy structure than the right-handed conformation without clashes of the Trp22 sidechains inside the pore.…”

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

“…10,26,[30][31][32] Our approach to modeling and data analysis is similar to the approach used by Kukol and Arkin to interpret Fourier-transform infrared (FTIR) measurements on Vpu TM oligomers. 16 In our modeling procedure, oligomers with a right-handed twist about the symmetry axis have positive s, whereas those with left-handed twist have negative s. With q ¼ 0 , C a sites of I8, I15, and W22 are in the central pore of the oligomer, whereas C a sites of V13, V20, and I27 are on the outside, nearly diametrically opposite to the symmetry axis. Increasingly positive values of q correspond to increasing right-handed rotations of each helix about its axis, running from C-terminal end to N-terminal end.…”

“…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.…”

Viroporins: structure, function and potential as antiviral targets

“…The majority of studies favour the formation of a pentameric TMD helical bundle, with a lumen lined by both ionizable (e.g. Ser23) and hydrophobic aromatic side chains, including Trp22, which could act as a molecular gate (Cordes et al, 2001;Kukol & Arkin, 1999;Lu et al, 2010;Park et al, 2003Park et al, , 2006Sharpe et al, 2006). In vitro, Vpu TMD peptides display relatively weak channel-like properties, adopting more of a pore-like character with Michaelis-Menten characteristics in the presence of increasing salt concentration (Mehnert et al, 2008).…”