NMR structure and ion channel activity of the p7 protein from hepatitis C virus.
Related Articles NMR structure and ion channel activity of the p7 protein from hepatitis C virus.
J Biol Chem. 2010 Jul 28;
Authors: Montserret R, Saint N, Vanbelle C, Salvay AG, Simorre JP, Ebel C, Sapay N, Renisio JG, Bockmann A, Steinmann E, Pietschmann T, Dubuisson J, Chipot C, Penin F
The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch-clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride, but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly alpha-helical, irrespective of the membrane mimetic medium, e.g. lysolipids, detergents, organic solvent-water mixtures. The secondary structure elements of the monomeric form of p7 were determined by 1H and 13C NMR in trifluoroethanol-water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated to the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal alpha-helix to the first transmembrane helix TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion-channel gating mechanism.
PMID: 20667830 [PubMed - as supplied by publisher]
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PubMed