Abstract
Summary: Nicotinamide nucleotide transhydrogenase couples hydride transfer between NAD(H) and NADP(H) to proton translocation, supporting cellular NADPH production. Here we present cryo-EM structures of full-length Escherichia coli transhydrogenase in apo, substrate-bound, and inhibitor-bound states. In the apo state, both copies of the NADP(H)-binding domain (dIII) adopt a face-down configuration, with the binding pocket partially embedded in transmembrane domain dII, plugging the cytoplasmic side of the proton channel. NADP + or NADPH binding to dIII is accompanied by detachment of one or both dIII copies from dII and increased orientational dynamics. Detachment of dIII from dII allows a conformation compatible with hydride transfer between NADP(H) in dIII and NAD(H) in dI, and is accompanied by conformational changes in the dII proton channel that close periplasmic access and open cytoplasmic access. Palmitoyl-CoA stabilizes an apo-like conformation, trapping dIII in a face-down configuration. These results define the structural basis for transhydrogenase regulation through interdomain conformational coupling.