Myrta Oblatt-Montal

Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy

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 α-helices with no kinks. The AChR M2 peptide inserts in the lipid bilayer at an angle of 12° 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.

Identification of an ion channel activity of the Vpu transmembrane domain and its involvement in the regulation of virus release from HIV‐1‐infected cells

HIV‐1 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 full‐length 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.

Expression, purification, and activities of full‐length and truncated versions of the integral membrane protein Vpu from HIV‐1

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). Vpu2–37 encompasses the N‐terminal transmembrane α‐helix; Vpu2–51 spans the N‐terminal transmembrane helix and the first cytoplasmic α‐helix; Vpu28–81 includes the entire cytoplasmic domain containing the two C‐terminal amphipathic α‐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 α‐helix. The C‐terminal α‐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.

Dilute spin-exchange assignment of solid-state NMR spectra of oriented proteins: Acetylcholine M2 in bilayers

The assignment of amide resonances in the two-dimensional PISEMA (Polarization Inversion with Spin Exchange at the Magic Angle) spectrum of uniformly 15N labeled M2 peptide corresponding to the channel-lining segment of the acetylcholine receptor in oriented phospholipid bilayers is described. The majority of the resonances were assigned through comparisons with spectra from selectively 15N labeled recombinant peptides and specifically 15N labeled synthetic peptides. Some resonances were assigned to specific amino acid residues by means of homonuclear 15N spin-exchange spectroscopy. A modification to the conventional spin-exchange pulse sequence that significantly shortens the length of the experiments by combining the intervals for 15 N spin-exchange and 1H magnetization recovery is described.

Structure and Function of Vpu from HIV-1

Virus protein “u” (Vpu) contributes to the virulence of HIV-1 infections of humans by enhancing the production and release of progeny virus particles. Its biological activities are associated with the two distinct structural domains of the protein. Since the entire polypeptide consists of only 81 amino acid residues, each of the biological activities is associated with a relatively small and well-defined structural entity. This suggests that the three-dimensional structure of the protein will lead to a detailed understanding of its biological functions, and potentially to the identification of small molecules that act as drugs by interfering with its functions (Miller and Sarver, 1997) as has been done for other HIV-1 encoded proteins (Turner and Summers, 1999; Wlodawer, 2002). The many structure determinations of HIV protease alone and complexed with inhibitors led to the development of the highly effective drugs that are a mainstay of current therapy for AIDS (Erickson and Burt, 1996; Vondrasek et al., 1997). Even though the protease is about 20% larger than Vpu, its structure was determined very soon after its discovery (Navia et al., 1989; Wlodawer et al., 1989), while the structure of Vpu is yet to be determined. The reasons that Vpu has not followed quickly in the path of protease have their roots in the most fundamental aspects of experimental structural biology and biochemistry. Vpu is a helical membrane protein, and it requires the presence of lipids and water to adopt its native functional structure. The lipids interfere with the formation of crystals for X-ray diffraction as well as the preparation of samples suitable for multidimensional solution NMR spectroscopy. In contrast, protease is a globular, soluble protein well suited for experimental structure determination by both X-ray crystallography and