Bundles Consisting of Extended Transmembrane Segments of Vpu from HIV-1:  Computer Simulations and Conductance Measurements

Abstract

Part of the genome of the human immunodeficiency virus type 1 (HIV-1) encodes for a short membrane protein Vpu, which has a length of 81 amino acids. It has two functional roles: (i) to downregulate CD4 and (ii) to support particle release. These roles are attributed to two distinct domains of the peptide, the cytoplasmic and transmembrane (TM) domains, respectively. It has been suggested that the enhanced particle release function is linked to the ion channel activity of Vpu, with a slight preference for cations over anions. To allow ion flux across the membrane Vpu would be required to assemble in homooligomers to form functional water-filled pores. In this study molecular dynamics simulations are used to address the role of particular amino acids in 4, 5, and 6 TM helix bundle structures. The helices (Vpu 6-33 ) are extended to include hydrophilic residues such as Glu, Tyr, and Arg (EYR motif). Our simulations indicate that this motif destabilizes the bundles at their C-terminal ends. The arginines point into the pore to form a positive charged ring that could act as a putative selectivity filter. The helices of the bundles adopt slightly higher average tilt angles with decreasing number of helices. We also suggest that the helices are kinked. Conductance measurements on a peptide (Vpu 1-32 ) reconstituted into lipid membranes show that the peptide forms ion channels with several conductance levels.

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

“…Comparable stability for structurally diverse oligomers in bilayers may explain experimental observations of multiple conductance levels for Vpu ion channels. 1,10,[13][14][15] Given that a variety of Vpu 1-40 oligomer structures coexist in DOPC/DOPG bilayers, we can not analyze our solid-state NMR data unequivocally in terms of a single structure. However, the fact that 2D 13 C-13 C spectra of Vpu in bilayers show a single set of 13 C chemical shifts 17 suggests that peptide conformations and intermolecular interactions in the predominant oligomers are similar.…”

“…Rather different pentamer models were used in simulations by Pertagias et al, 32 Grice et al, 25 and Cordes et al 10 In these simulations, helix orientations were chosen such that the side chain of S23 was located in the central pore, with the assumption that such an orientation would be thermodynamically preferred. Using our definition of the helix rotation angle, such models have q % 150 and do not fit our 2D CHHC and DIPSHIFT data.…”

“…9 The second function depends principally on the single transmembrane TM segment of Vpu, contained within residues 1-30, as sequence alterations in the TM segment have been shown to interfere with this function. 2 Full-length Vpu and various N-terminal peptides containing the TM segment have been shown to form cation-selective channels in model membranes 1,[10][11][12][13][14][15] and in cells. 12 These ion channels presumably form by association of a-helical TM segments into homo-oligomeric bundles, with ions passing through the central pore of the helix bundle.…”

“…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. 10,25 Computational studies include investigations of interactions of channel-blocking compounds with Vpu ion channels, 30 effects of site-specific mutations on secondary structure and supramolecular structure, 31 and channel selectivity.…”

Assembly of Viral Membrane Proteins

“…These structures suggest the lumen of the pore to be a widely hydrophobic stretch. Model 4 is similar to the one used so far in MD simulations with the TM part of Vpu, 11,49,50 whereas model 1 corresponds to a model which has been suggested earlier. 7,10 In the former model Ser-24 points into the pore, while in the latter it is Trp-23.…”