Proton-translocating transhydrogenase (also known as nicotinamide nucleotide transhydrogenase (NNT)) is found in the plasma membranes of bacteria and the inner mitochondrial membranes of eukaryotes. NNT catalyses the transfer of a hydride between NADH and NADP+, coupled to the translocation of one proton across the membrane. Its main physiological function is the generation of NADPH, which is a substrate in anabolic reactions and a regulator of oxidative status; however, NNT may also fine-tune the Krebs cycle1,2. NNT deficiency causes familial glucocorticoid deficiency in humans and metabolic abnormalities in mice, similar to those observed in type II diabetes3,4. The catalytic mechanism of NNT has been proposed to involve a rotation of around 180° of the entire NADP(H)-binding domain that alternately participates in hydride transfer and proton-channel gating. However, owing to the lack of high-resolution structures of intact NNT, the details of this process remain unclear5,6. Here we present the cryo-electron microscopy structure of intact mammalian NNT in different conformational states. We show how the NADP(H)-binding domain opens the proton channel to the opposite sides of the membrane, and we provide structures of these two states. We also describe the catalytically important interfaces and linkers between the membrane and the soluble domains and their roles in nucleotide exchange. These structures enable us to propose a revised mechanism for a coupling process in NNT that is consistent with a large body of previous biochemical work. Our results are relevant to the development of currently unavailable NNT inhibitors, which may have therapeutic potential in ischaemia reperfusion injury, metabolic syndrome and some cancers7,8,9.
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Structures of ‘double face-down’ and ‘single face-down’ NADP+ states and apo-NNT have been deposited in the Protein Data Bank with accession numbers 6QTI, 6QUE and 6S59, respectively. The corresponding cryo-EM density maps in have been deposited in the Electron Microscopy Data Bank with accession numbers EMD-4635, EMD-4637 and EMD-10099.
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We thank R. Thompson, G. Effantin and V.-V. Hodirnau for their assistance with collecting NADP+, NADPH and apo datasets, respectively. Data processing was performed at the IST high-performance computing cluster. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement no. 665385.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks Amandine Marechal and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, The domain split and organization of transhydrogenase in the four model species: T. thermophilus, Rhodospirillum rubrum, E. coli and Ovis aries. The dII–dIII linker is conserved in all transhydrogenases, whereas the dI–dII linker and TM1 are limited to NNTs with an α-β polypeptide split (for example, E. coli) and single-polypeptide NNTs (metazoans, including mammals). TM5 is present only in metazoans. b, Residue conservation scores (calculated in Consurf, coloured cyan to magenta from low to high conservation) mapped on the structure of a single monomer of transhydrogenase. The most highly conserved regions are the proton translocation pathway, the nucleotide binding sites and the dI–dIII, dII–dIII and dI–dI interfaces. c, The architecture of mammalian dII. Supernumerary helices TM1 and TM5 are coloured in a darker shade of blue. Residues of the proton transfer pathway are labelled in the helices. The 310-helix stretch within TM3 is depicted as a triangular helix. d, Alignment of conserved residues on TM3, TM9, TM13, TM14 and CL7 and CL8 important for proton translocation and reaction coupling in T. thermophilus (Tt), R. rubrum (Rr), E. coli (Ec), O. aries (Oa) and Homo sapiens (Hs). T. thermophilus and a few other species with an NGXGG motif on TM9 have the protonatable histidine on TM3 (α2H42 in T. thermophilus), whereas most other species that share an HSXXG motif on TM9 have the protonatable histidine on TM9 (βH91 in E. coli and H664 in O. aries).
a, Ovine NNT was purified chromatographically. The last step of purification—size-exclusion chromatography—is shown in two different detergents, FOM and LMNG. b, SDS–PAGE shows the presence of an approximately 110 kDa polypeptide of NNT. The highest purity fractions (which eluted at around 10.5 ml) were pooled and concentrated for cryo-EM sample preparation. c, NNT is highly active and stable when purified in LMNG as shown by undiminished activity over several days when stored at 4 °C. Error bars represent standard deviations based on n = 3 independent measurements. d, Reconstitution of purified NNT into DOPC liposomes shows that the reverse transhydrogenation reaction is tightly coupled to proton transfer, as it is stimulated around tenfold by the proton-gradient uncoupler CCCP and by solubilisation in CHAPS detergent. Error bars represent standard deviations based on n = 3 independent measurements. e, Trypsinolysis of NNT at different pH values in the presence of substrates. The full NNT (110 kDa) and previously identified15 fragments at 66 and 43 kDa are labelled with asterisks. As the pH decreases from 8 to 6, NADPH-induced proteolysis diminishes relative to that induced by NADP+. At pH 5 trypsin produces different fragments, but stabilization of the intact NNT by NADPH is evident. Trypsinolysis was performed independently three times with similar results. The purification of NNT was repeated independently ten times with similar results as shown in a, b. For gel source data, see Supplementary Fig. 1.
Thorough classification of particles resulted in six distinct classes with different domain orientations and resolutions. The best-resolution class (double face-down NNT) is almost symmetric with both dIIIs bound in the face-down position, whereas the other five have only one dIII bound in the face-down position, with the other dIII detached and the dI2 tilted at different angles. Simultaneous tilting of dI2 and dissociation of dIIIb is probably necessary to permit full dIIIb rotation during the catalytic cycle. Monomers with dIII detached from dII also show little or no density for the dI–dII linker, which suggests that it detaches from dIII to enable dI2 to open.
a, NNT bound to NADPH exhibited a large degree of conformational flexibility, which prevented a high-resolution (beyond 8 Å) refinement of any class of particles. Classification into 20 classes revealed that only a small proportion (around 5%) of particles have one dIII bound to dII in the face-down orientation (class C). About another 10% have partially detached dIII (classes A and B). The majority, however, have both dIIIs dissociated from the other domains, leading to a large degree of freedom of movement of dI2 and of both dIIIs independent of each other (see classes D, E and F). b, Apo-NNT exhibited a single conformational class, similar to the double face-down NNT class in the presence of NADP+.
All examples are from the double face-down class in the presence of NADP+, unless otherwise stated. a, Density of the transmembrane helices that line the proton channel (TM3 and TM13). b, Comparison of density of the TM2 between the double face-down and the single face-down (dIII-detached monomer) NADP+ classes, as well as the apo class, which remains in the same conformation upon dII opening while TM9 changes (Extended Data Fig. 6c). c, Beta-sheet density of dI. d, Beta-sheet density of dIII. e, α-helical segment density of dI. f, α-helical segment density of dIII. g, Two phosphatidyl cholines bound in the cavity enclosed by TM1, TM2 and TM6. h, Density of dIIIa–NADP+. i, Density of dIIIb–NADP+. j, Partial NAD+ density in dIa. k, Partial NAD+ density in dIb. The density for lipids and putative NAD+ is discussed in Methods.
Extended Data Fig. 6 Comparison of double face-down and single face-down NNT–NADP+ conformations and apo-NNT.
a, Local resolution and Fourier shell correlation (FSC) curves for the double face-down NNT–NADP+ structure. b, Local resolution and FSC curves for the single face-down NNT–NADP+ structure. c, Local resolution and FSC curves for the apo-NNT structure. d, Overall comparison of double face-down (cyan) and single face-down (green) classes of NNT. An increased dI tilt, dIIIb detachment and dIIb conformation change are visible. e, Comparison of the two monomers in the closed NADP+ class, as viewed from the dimerization interface reveals a tilt of dI2 and asymmetry in dI-dIIIa/b contacts. f, Overall comparison of double face-down NNT–NADP+ (cyan) and apo-NNT. dII and dIII are in the same conformation and dI2 is slightly more tilted in apo-NNT. g, Electrostatic surface potential of the proton entry cavities on the matrix (bottom) and IMS sides (top) as well as that of the membrane-facing side (right). h, Hydrophobicity of residues on the surface of dII2 (coloured white to red from hydrophobic to hydrophilic). Surface-exposed tyrosine and tryptophan residues, which often delineate the surface of the lipid membrane, are highlighted in green and the lipid-binding pocket is circled.
Extended Data Fig. 7 Changes in the proton translocation channel upon dIII detachment and comparison between ovine and T. thermophilus dII.
a, Comparison of T. thermophilus dII (salmon, PDB: 5UNI) and double face-down (dIII attached) ovine dII (cyan, supernumerary TM1 and TM5 in blue). Residues in the N side cavity display markedly different conformations. b, Comparison of T. thermophilus dII (salmon, PDB: 5UNI) and single face-down (dIII-detached monomer) ovine dII (blue). Residues in the N side cavity match more closely as both of these dII structures are detached from dIII. c, Comparison of the TM9 density in the double face-down, single face-down (dIII-detached monomer) and apo dII clearly displaying a H664 flip in the dIII-detached dII. d, Comparison of Dowser-predicted waters in the dIII-attached (cyan) and dIII-detached (green) dII. The dIII-attached structure has two water molecules—one below and one above the N side proton gate—whereas the dIII-detached structure only has one water molecule above the gate, which is consistent with the proposal that the channel is open to the P side when dIII is attached. e, Density for a water molecule coordinated between H664 and S492, consistent with Dowser-predicted water, is beginning to show in our cryo-EM density. f, Proton pathway profile (calculated in Mole 2.5) in dIII-attached dII reveals a diameter constriction between the N side and H664, a hydrophobic stretch between H664 and E806 and a negatively charged P side proton-entry site. g, Proton pathway profile in the dIII-detached dII. Additional constriction appearing between H664 and the P side upon dIII detachment is indicated.
a, A homology model of the asymmetric ovine NNT based on the T. thermophilus dI2dIII heterotrimer structure (PDB: 4J16). Note the putative interacting residues at the dIIIup–dIIIdown interface. The dI–dII linker contacts the loop D of dIII in the face-down conformation. D942 and Y941 from dIII form hydrogen bonds with R544 on the CL2 loop, which stabilizes the dII–dIII interface. Helix 4 and loop D also contribute towards formation of the dI–dIII interface, but Y941 is too far away to interact with dI. b, Comparison of ovine dIII (red) with dIII isolated from R. rubrum (PDB: 1PNO; chain A in blue and chain B in green). Helix 4, loop D and loop E are all more open around the nucleotide in the ovine structure, but the nucleotide is occluded by the interactions from the dII residues (not shown) in the ovine face-down structure. c, Comparison of the NADP(H) binding site in double face-down NNT (left) and apo-NNT (right). Loop E, K999 and R1000 are disordered in apo-NNT and R925 flips into an outward-facing orientation, opening the site to the solvent.
a, Summary of the biochemical evidence and mutagenesis data supporting the proposed mechanism. A full description of mutants in E. coli, R. rubrum and human patients are provided in Supplementary Tables 1–3. b, Our proposal for the reverse reaction. The reverse transhydrogenation reaction consumes NADPH and NAD+ and results in proton pumping, supporting the proton motive force. The driving forces for this reaction are the nucleotide ratios and low proton motive force, which promotes protonation of H664 from the matrix side.