Crystal structure of transhydrogenase domain III at 1.2 angstrom resolution

The nicotinamide nucleotide transhydrogenases (TH) of mitochondria and bact eria are membrane-intercalated proton pumps that transduce substrate bindin g energy and protonmotive force via protein conformational changes. In mito chondria, TH utilizes protonmotive force to promote direct hydride ion tran sfer from NADH to NADP, which are bound at the distinct extramembranous dom ains I and III, respectively, Domain II is the membrane-intercalated domain and contains the enzyme's proton channel, This paper describes the crystal structure of the NADP(H) binding domain III of bovine TR at 1.2 Angstrom r esolution, The structure reveals that NADP is bound in a manner inverted fr om that previously observed for nucleotide binding folds, The nonclassical binding mode exposes the NADP(H) nicotinamide ring for direct contact with NAD(H) in domain I, in accord with biochemical data. The surface of domain III surrounding the exposed nicotinamide is comprised of conserved residues presumed to form the interface with domain I during hydride ion transfer. Further, an adjacent region contains a number of acidic residues, forming a surface with negative electrostatic potential which may interact with extr amembranous loops of domain II. Together, the distinctive surface features allow mechanistic considerations regarding the NADP(H)-promoted conformatio n changes that are involved in the interactions of domain III with domains I and II for hydride ion transfer and proton translocation.