Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain.

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an alpha and a beta subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane alpha-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix-helix distances were investigated by the introduction of one or two cysteines in helix-helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 A), and visualized by SDS-PAGE. In the alpha(2)beta(2) tetramer, alphabeta cross-links were obtained with the alphaG476C-betaS2C, alphaG476C-betaT54C and alphaG476C-betaS183C double mutants. Significant alphaalpha cross-links were obtained with the alphaG476C single mutant in the loop connecting helix 3 and 4, whereas betabeta cross-links were obtained with the betaS2C, betaT54C and betaS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the alpha and beta subunits in the dimer is lined along an axis formed by helices 3 and 4 from the alpha subunit and helices 6, 7 and 8 from the beta subunit. In addition, helices 2 and 4 in the alpha subunit together with helices 6 and 12 in the beta subunit interact with their counterparts in the alpha(2)beta(2) tetramer. Each beta subunit in the alpha(2)beta(2) tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.