The high-resolution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from human heart mitochondria

Background: Transhydrogenase, located in the inner membranes of animal mito chondria and the cytoplasmic membranes of bacteria, couples the transfer of reducing equivalents between NAD(H) and NADP(H) to proton pumping. The pro tein comprises three subunits termed dl, dll and dill. The dll component sp ans the membrane. The dl component, which contains the binding site for NAD (+)/NADH, and the dill component, which has the binding site for NADP(+)/NA DPH, protrude from the membrane. Proton pumping is probably coupled to chan ges in the binding affinities of dill for NADP(+) and NADPH. Results: The first X-ray structure of the NADP(H)-binding component, dill, of human heart transhydrogenase is described here at 2.0 Angstrom resolutio n. It comprises a single domain resembling the classical Rossmann fold, but NADP(+) binds to dill with a reversed orientation. The first beta alpha be ta alpha beta motif of dill contains a Gly-X-Gly-X-X-Ala/Val 'fingerprint', but it has a different function to that in the classical Rossmann structur e. The nicotinamide ring of NADP(+) is located on a ridge where it is expos ed to interaction with NADH on the dr subunit. Two distinctive features of the dill structure are helix D/loop D, which projects from the beta sheet, and loop E, which forms a 'lid' over the bound NADP(+). Conclusions: Helix D/loop D interacts with the bound nucleotide and loop E, and probably interacts with the membrane-spanning dll. Changes in ionisati on and conformation in helix D/loop D, resulting from proton translocation through dll, are thought to be responsible for the changes in affinity of d ill for NADP(+) and NADPH that drive the reaction.