The structures of functional peptides corresponding to the predicted channel-lining M2 segments of the nicotinic acetylcholine receptor (AChR) and of a glutamate receptor of the NMDA subtype (NMDAR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. Both M2 segments form straight transmembrane alpha-helices with no kinks. The AChR M2 peptide inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminal side of the membrane, which is assigned to be intracellular. A model built from these solid-state NMR data, and assuming a symmetric pentameric arrangement of M2 helices, results in a funnel-like architecture for the channel, with the wide opening on the N-terminal intracellular side.

HIV-I Vpu catalyzes two independent functions, degradation of the virus receptor CD4 in the endoplasmic reticulum and enhancement of virus release from the cell surface. These activities are confined to distinct structural domains of Vpu, the cytoplasmic tail and the transmembrane (TM) anchor, respectively. It was recently reported that Vpu forms cationselective ion channels in lipid bilayers. Here we report that this property of Vpu is a characteristic of its TM anchor. Expression of full-length Vpu in Xenopus oocytes increases membrane conductance. The Vpu-induced conductance is selective to monovalent cations over anions, does not discriminate Na + over K + and shows marginal permeability to divalent cations. Notably, introduction of the scrambled TM sequence into fulllength Vpu abrogates its capacity to increase membrane conductance in oocytes and to promote virus release from infected cells. Reconstitution of synthetic Vpu fragments in lipid bilayers identified an ion channel activity for a sequence corresponding to the TM domain of Vpu. In contrast, a peptide with the same amino acid composition but with a scrambled sequence does not form ion channels. Our findings therefore suggest that the ability of Vpu to increase virus release from infected cells may be correlated with an ion channel activity of the TM domain, thereby providing a potential target for drug intervention based on the development of Vpu-specific channel blockers.

No abstract

Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic ␣-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly 15 N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane ␣-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu 2-51 (residues 2-51), which contains the transmembrane ␣-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu 28 -81 (residues 28 -81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2-81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 ؎ 3 pS and 12 ؎ 3 pS in 0.5 M KCl and 29 ؎ 3 pS and 12 ؎ 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu 2-51, which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu 28 -81, which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.

Vpu is an 81-residue accessory protein of HIV-1. Because it is a membrane protein, it presents substantial technical challenges for the characterization of its structure and function, which are of considerable interest because the protein enhances the release of new virus particles from cells infected with HIV-1 and induces the intracellular degradation of the CD4 receptor protein. The Vpu-mediated enhancement of the virus release rate from HIV-1-infected cells is correlated with the expression of an ion channel activity associated with the transmembrane hydrophobic helical domain. Vpu-induced CD4 degradation and, to a lesser extent, enhancement of particle release are both dependent on the phosphorylation of two highly conserved serine residues in the cytoplasmic domain of Vpu. To define the minimal folding units of Vpu and to identify their activities, we prepared three truncated forms of Vpu and compared their structural and functional properties to those of full-length Vpu (residues 2-81). Vpu 2-37 encompasses the N-terminal transmembrane ␣-helix; Vpu 2-51 spans the N-terminal transmembrane helix and the first cytoplasmic ␣-helix; Vpu 28-81 includes the entire cytoplasmic domain containing the two C-terminal amphipathic ␣-helices without the transmembrane helix. Uniformly isotopically labeled samples of the polypeptides derived from Vpu were prepared by expression of fusion proteins in E. coli and were studied in the model membrane environments of lipid micelles by solution NMR spectroscopy and oriented lipid bilayers by solid-state NMR spectroscopy. The assignment of backbone resonances enabled the secondary structure of the constructs corresponding to the transmembrane and the cytoplasmic domains of Vpu to be defined in micelle samples by solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in oriented lipid bilayers demonstrated that the topology of the domains is retained in the truncated polypeptides. The biological activities of the constructs of Vpu were evaluated. The ion channel activity is confined to the transmembrane ␣-helix. The C-terminal ␣-helices modulate or promote the oligomerization of Vpu in the membrane and stabilize the conductive state of the channel, in addition to their involvement in CD4 degradation.

Synthetic peptides with sequences representing putative transmembrane (M) segments of CFTR (the cystic fibrosis transmembrane conductance regulator) were used as tools to identify the involvement of such segments in forming the ionic pore of the CFTR Cl channel. Peptides with sequences corresponding to M2 and M6 form anion-selective channels after reconstitution in lipid bilayers. In contrast, peptides with the sequences of Ml, M3, M4, and M5, or peptides of the same amino acid composition as M2 and M6 but with scrambled sequences, do not form channels. Conductive heterooligomers of M2 and M6 exhibit a single channel conductance of 8 pS (in 0.15 M KCI) and a 95% selectivity for anions over cations, properties that emulate both the conductance and the selectivity of the authentic CFTR channel. The identification of sequence-specific motifs that account for key functional attributes of the CFTR channel suggests that such modules may represent fundamental units of function and are plausible constituents of the pore-forming structure of the CF`TR Cl-channel.A defect in epithelial C1-transport is a major biochemical factor in the pathogenesis of cystic fibrosis (CF) (1). CFTR (the CF transmembrane conductance regulator), the protein product of the CF-susceptibility gene CFTR, is a phosphorylation-regulated Cl-channel (2-13). The primary sequence of CFTR reveals two hydrophobic repeats, each with six putative transmembrane segments and a nucleotide-binding fold (1, 2), joined by a charged central R domain that contains potential phosphorylation sites and is thought to regulate channel open probability (1,9,14). A deletion of a phenylalanine at amino acid 508 in CFTR (AF508CFTR) is the most common mutation in CF patients that affects primarily protein insertion into the apical epithelial membrane and reduces channel open probability, compromising its function (1, 2, 11, 15). Rarer missense mutations, associated with milder forms of the disease, generate channels that insert in the epithelial membrane but exhibit lower conductances (13). These mutations affect basic residues at positions 117, 334, and 347 in transmembrane segments M2 and M6, suggesting their participation in lining the pore. To identify sequence-specific motifs associated to functional modules in channel proteins, especially structural components of the ionic pore, synthetic peptides with sequences corresponding to each of the transmembrane segments have been assayed for channel activity after reconstitution in lipid bilayers (16). Here, we focus on one of the two homologous repeats of CFTR and show that peptides corresponding to the sequences of M2 and M6 form anion-selective channels in lipid bilayers, whereas peptides with the sequences of Ml, M3, M4, and M5 do not. Further, conductive heterooligomers of M2 and M6 peptides approximate the single-channel conductance and the anion selectivity characteristic of CFTR. We propose that a parallel heterotetramer of M2, M6, and the corresponding segments of the second repeat may form the pore-lining ...

Vpu is an 81-residue integral membrane protein encoded in the HIV-1 genome that is of considerable interest because it plays important roles in the release of virus particles from infected cells and in the degradation of the cellular receptor. We report here the total chemical synthesis of full-length Vpu(1-81) as well as a site-specifically (15)N-labeled analogue, Vpu(2-81), using native chemical ligation methodologies and also report a structural and functional comparison of these constructs with recombinant protein obtained via bacterial expression. The structures of the synthetic and expressed polypeptides were similar in lipid micelles using solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in aligned hydrated lipid bilayers indicated that their overall topologies were also very comparable. Further, the channel activity of the synthetic protein was found to be analogous to that previously characterized for the recombinant protein. We have thus demonstrated that using solid phase peptide synthesis and chemical ligation it is feasible to obtain large quantities of a purified and homogeneous membrane protein in a structurally and functionally relevant form for future structural and characterization studies.

No abstract