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

Abstract

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α and a β 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 α-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 Å), and visualized by SDS-PAGE.

In the α₂β₂ tetramer, αβ cross-links were obtained with the αG476C-βS2C, αG476C-βT54C and αG476C-βS183C double mutants. Significant αα cross-links were obtained with the αG476C single mutant in the loop connecting helix 3 and 4, whereas ββ cross-links were obtained with the βS2C, βT54C and βS183C 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 α and β subunits in the dimer is lined along an axis formed by helices 3 and 4 from the α subunit and helices 6, 7 and 8 from the β subunit. In addition, helices 2 and 4 in the α subunit together with helices 6 and 12 in the β subunit interact with their counterparts in the α₂β₂ tetramer. Each β subunit in the α₂β₂ tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.