biostudies-literature00600Padayatti PSXSEDENHLBI NIH HHSDOE Office of Biological and Environmental ResearchArgonne National LaboratoryNational Cancer InstituteNIHNational Institute of General Medical SciencesNIH HHSNIGMS NIH HHSNational Science Foundation1111-1119.e3https://www.ebi.ac.uk/biostudies/studies/S-EPMC5524145biostudies-literatureUnknown25(7)The nicotinamide nucleotide transhydrogenase (TH) is an integral membrane enzyme that uses the proton-motive force to drive hydride transfer from NADH to NADP⁺ in bacteria and eukaryotes. Here we solved a 2.2-Å crystal structure of the TH transmembrane domain (Thermus thermophilus) at pH 6.5. This structure exhibits conformational changes of helix positions from a previous structure solved at pH 8.5, and reveals internal water molecules interacting with residues implicated in proton translocation. Together with molecular dynamics simulations, we show that transient water flows across a narrow pore and a hydrophobic "dry" region in the middle of the membrane channel, with key residues His42^α2 (chain A) being protonated and Thr214^β (chain B) displaying a conformational change, respectively, to gate the channel access to both cytoplasmic and periplasmic chambers. Mutation of Thr214^β to Ala deactivated the enzyme. These data provide new insights into the gating mechanism of proton translocation in TH.Structure (London, England : 1993)Critical Role of Water Molecules in Proton Translocation by the Membrane-Bound Transhydrogenase.PMC5524145TG-MCB150029P41GM103393R01HL16101AGM-120061616874R01 HL016101S10 OD012289R01 GM103838P41 GM103393DE-AC02-06CH113575R01GM103838P41 GM104601ACB-120021S10OD012289-01A1Stout CDTajkhorshid ECherezov VMahinthichaichan PJackson JBGennis RBPadayatti PSIshchenko ALeung JHSoltis SMZhang Q60falseCritical Role of Water Molecules in Proton Translocation by the Membrane-Bound Transhydrogenase.The nicotinamide nucleotide transhydrogenase (TH) is an integral membrane enzyme that uses the proton-motive force to drive hydride transfer from NADH to NADP⁺ in bacteria and eukaryotes. Here we solved a 2.2-Å crystal structure of the TH transmembrane domain (Thermus thermophilus) at pH 6.5. This structure exhibits conformational changes of helix positions from a previous structure solved at pH 8.5, and reveals internal water molecules interacting with residues implicated in proton translocation. Together with molecular dynamics simulations, we show that transient water flows across a narrow pore and a hydrophobic "dry" region in the middle of the membrane channel, with key residues His42^α2 (chain A) being protonated and Thr214^β (chain B) displaying a conformational change, respectively, to gate the channel access to both cytoplasmic and periplasmic chambers. Mutation of Thr214^β to Ala deactivated the enzyme. These data provide new insights into the gating mechanism of proton translocation in TH.2017-01-01T00:00:00Z2017 Jul2024-11-14T05:11:46.629Z2019-03-26T23:44:05ZS-EPMC55241452864860910.1016/j.str.2017.05.022