Transmembrane Peptide NB of Influenza B:  A Simulation, Structure, and Conductance Study

Abstract

The putative transmembrane segment of the ion channel forming peptide NB from influenza B was synthesized by standard solid-phase peptide synthesis. Insertion into the planar lipid bilayer revealed ion channel activity with conductance levels of 20, 61, 107, and 142 pS in a 0.5 M KCl buffer solution. In addition, levels at -100 mV show conductances of 251 and 413 pS. A linear current-voltage relation reveals a voltage-independent channel formation. In methanol and in vesicles the peptide appears to adopt an R-helical-like structure. Computational models of R-helix bundles using N ) 4, 5, and 6 NB peptides per bundle revealed water-filled pores after 1 ns of MD simulation in a solvated lipid bilayer. Calculated conductance values [using HOLE (Smart et al. (1997) Biophys. J. 72, 1109-1126] of ca. 20, 60, and 90 pS, respectively, suggested that the multiple conductance levels seen experimentally must correspond to different degrees of oligomerization of the peptide to form channels.In the genome of influenza B, RNA segment 6 encodes two integral membrane proteins, NB and NA. NB is a protein of ca. 100 amino acids. It has ca. 18 extramembraneous residues on its N-terminal end, a 22-residue membrane spanning segment, and a ca. 60 residue long C-terminal end (1, 2). NB is expressed at the surface of infected cells (3)(4)(5). In the virion the N-terminal site is located at the external surface. Experiments with gold immunolabeled freezefractured virions indicate that the C-terminus is located at the inside of the virion (4). By analogy with the M2 protein of influenza A, it was suggested (reviewed in ref 6) that NB might be a channel-forming protein.Conductance measurements with purified NB expressed in Escherichia coli and reconstituted in lipid bilayers reveal a conductance of 45 pS. However, when NB is incorporated in liposomes and then added to planar lipid bilayers, the conductance ranged from ca. 10 pS at very low NB concentration in the liposomes to several hundred picosiemens for higher concentrations (7). At neutral pH the channels are more permeable for sodium ions than chloride ions (P Na /P Cl ≈ 9 at pH 6.5). In asymmetrical NaCl solutions at pH 2.5 the permeability is higher for chloride ions (P Cl / P Na ≈ 4). A recent study on NB expressed in the plasmalemma of MEL 1 cells showed that the channel peptides induce a Na + -dependent proton current and a pH-activated Cl -current (8).One way in which single integral membrane proteins can form ion channels is via oligomerization to form a bundle of transmembrane helices. For the analogous channel peptide in influenza A, M2, it is believed that a tetrameric R-helix bundle is the minimal form of the proton-permeable ion channel (9-11). For NB the number of subunits in the bundle is not known. Purification of NB on gels using nonreducing conditions revealed the formation of disulfide-linked dimers and higher oligomers (2). Molecular modeling studies on bundles of the transmembrane parts of the peptide have been performed in vacuo with four, five, and si...

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

“…100 ps and does not exceed a value of 0.3 nm ( Figure 1B). This is in accordance with other successfully conducted simulations on other TM bundles (33,34). Figure 2 shows the structure of the bundles with a view down the pore from the N-to the C-terminus.…”

The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives

“…Other viral ion channels such as the tetrameric proton channel M2 from influenza A virus (31,32) exhibit conductance levels in the femtosiemen range (33,34). Channel-forming peptides show conductance levels as high as the ones we observed for HCV p7 when five or more transmembrane helices assemble to form a pore (26,27). We therefore conclude that (provided only one of p7's two transmembrane domains is involved in forming the actual pore) p7 needs to form at least a pentamer to account for the conductance levels observed.…”

“…NB and Vpu, ion channel-forming proteins of influenza B virus and HIV-1, respectively, generate similar conductances of Ϸ50 pS and less (23)(24)(25). Membrane-spanning domains inserted into lipid bilayers also give rise to similar conductance levels (24,(26)(27)(28). Vpr, another HIV-1 auxiliary protein, induces channel activity when inserted in lipid bilayers (29).…”

Vpu from HIV‐1 on an atomic scale: experiments and computer simulations

“…In comparison, calculations for NB, an ion channel‐forming protein of influenza B sharing approximately the same sequence length and topology as Vpu (see [45]), shows conductance of 22, 56, and 88 pS for the tetrameric (NB‐4), pentameric (NB‐5) and hexameric bundle (NB‐6), respectively [46] (Table 2). Compared with experimental findings [5,46] the values for the tetramer do not exclude this bundle from its possible contribution to channel activity of NB. Calculations of the conductivity of the bundles calculated in [40] support the overall trend.…”