The influenza virus M-2 ion channel protein: Probing the structure of the transmembrane domain in intact cells by using engineered disulfide cross-linking

The influenza A virus M-2 integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of virions in end osomes, and it also modulates the pH of the trans-Golgi network in virus-in fected cells. M-2 protein is a homo-oligomer of 97 residues with a single t ransmembrane (TM) domain whose residues encompass the pore region of the ch annel and the biologically active form of the channel is a homotetramer. To understand the structural arrangement of the TM domains, each residue of t he TM domain was changed in turn to cysteine, and oxidative disulfide cross -linking used to identify residues in close proximity. Oxidative treatment of M-2 protein in membranes using iodine resulted in maximum cross-linking at TM domain residues 27, 34, and 41. Oxidation of M-2 protein in membranes using the catalyst Cu(II)(1,10-phenanthroline)(3) resulted in cross-linkin g of many TM domain residues when the reaction was allowed to proceed at 37 degrees C, suggesting that rotational movements of the TM domains in the m embrane can occur. However, analysis of the kinetics of disulfide-linked di mer formation showed that TM domain residues 27 30, 34, 37 and 41 formed mo st rapidly. Furthermore, when oxidation was performed at 4 degrees C, maxim um cross-linking occurred at TM domain residues 27 30, 34, 37 and 41. These positions correspond to the a and d positions of a heptad repeat Thus thes e biochemical data are consistent with the TM domain region of the M-2 tetr amer forming a four-helix bundle. Analysis of the disulfide bonds that form ed when oxidation of M-2 protein in membranes was performed at pH 5.2 showe d greatly reduced cross-linking at TM domain residues 40, 42, and 43 than t hat found at pH 74. This pH-dependent change in cross-linking of residues t oward the cytoplasmic side of the TM domain parallels with the activation o f the M-2 ion channel at low pH. (C) 1999 Academic Press.