The Crystal Structure of an Asymmetric Complex of the Two Nucleotide Binding Components of Proton-Translocating Transhydrogenase

Cotton, Nick P.J.; White, Scott A.; Peake, Sarah J.; McSweeney, Seán; Jackson, John L.

doi:10.1016/s0969-2126(01)00571-8

Cited by 60 publications

(161 citation statements)

References 54 publications

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“…The bovine domain III with NADP bound was superimposed onto domain III of the R. rubrum heterotrimeric complex (rms deviation 0.89 Å for 174 CR atoms). As expected, the resulting model shows overall shape complementarity of domains I and III and conserved residues in close proximity (Figure 7), as observed for the all R. rubrum heterotrimeric complex (10,11).…”

Section: Discussionsupporting

confidence: 79%

“…However, the C4 atoms of the nicotinamide rings of NADH and NADP are far apart (6.4 Å) and not coplanar ( Figure 7). A similar result was obtained when NAD was modeled into the 'B' subunit of domain I in the heterotrimeric complex, based on consideration of the NAD binding site in the 'A' subunit and rotation of flexible torsion angles to place NAD(H) adjacent to NADP in domain III (10,11). In this case, the C4 atoms are 6.5 Å apart and the nicotinamide rings are also significantly nonparallel.…”

Section: Discussionsupporting

confidence: 61%

“…The open pocket is formed by a 1.5-turn 3 10 -helix following the f 1 strand, and residues at the N-terminal end of the R-helix (residues 126-138) ( Figure 4). As noted for the NAD complex (9), six of these residues are conserved in TH domain I sequences, i.e., Pro126, Arg127, Ala131, Gln132, Asp135, and Ser138.…”

Section: Nadh-containing Crystalsmentioning

confidence: 99%

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Crystal Structures of Transhydrogenase Domain I with and without Bound NADH^,

et al. 2002

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Transhydrogenase (TH) is a dimeric integral membrane enzyme in mitochondria and prokaryotes that couples proton translocation across a membrane with hydride transfer between NAD(H) and NADP(H) in soluble domains. Crystal structures of the NAD(H) binding R1 subunit (domain I) of Rhodospirillum rubrum TH have been determined at 1.8 Å resolution in the absence of dinucleotide and at 1.9 Å resolution with NADH bound. Each structure contains two domain I dimers in the asymmetric unit (AB and CD); the dimers are intimately associated and related by noncrystallographic 2-fold axes. NADH binds to subunits A and D, consistent with the half-of-the-sites reactivity of the enzyme. The conformation of NADH in subunits A and D is very similar; the nicotinamide is in the anti conformation, the A-face is exposed to solvent, and both N7 and O7 participate in hydrogen bonds. Comparison of subunits A and D to six independent copies of the subunit without bound NADH reveals multiple conformations for residues and loops surrounding the NADH site, indicating flexibility for binding and release of the substrate (product). The NADH-bound structure is also compared to the structures of R. rubrum domain I with NAD bound (PDB code 1F8G) and with NAD bound in complex with domain III of TH (PDB code 1HZZ). The NADH-vs NAD-bound domain I structures reveal conformational differences in conserved residues in the NAD(H) binding site and in dinucleotide conformation that are correlated with the net charge, i.e., oxidation state, of the nicotinamides. The comparisons illustrate how nicotinamide oxidation state can affect the domain I conformation, which is relevant to the hydride transfer step of the overall reaction.The energy-transducing nicotinamide nucleotide transhydrogenases of eukaryotic mitochondria and bacteria are homodimeric integral membrane proteins of monomer molecular mass of about 110 kDa. They catalyze the direct and stereospecific transfer of a hydride ion between the 4A position of NAD(H) 1 and the 4B position of NADP(H) in a reaction that is coupled to transmembrane proton translocation with a H + /H -stoichiometry of n ) 1 (eq 1) (1-4).In bovine submitochondrial particles, the proton motive force (pmf) accelerates the forward reaction 10-12-fold, and shifts the equilibrium toward product formation. In the reverse direction, transhydrogenation from NADPH to NAD results in outward proton translocation and creation of a pmf. Because there is essentially no difference in the reduction potential of the nicotinamide cofactors, the driving force for proton translocation coupled to the reverse reaction is the difference in binding affinities for substrates (NADPH, NAD) and products (NADH, NADP). In mammalian mitochondria, a function of TH is to produce NADPH for reduction of toxic H 2 O 2 by glutathione reductase and glutathione peroxidase.TH monomers are composed of three domains: a 400-430-residue hydrophilic domain I, a 360-400-residue hydrophobic domain II, and a 200-residue hydrophilic domain III. In mammalian TH, the ...

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Section: Discussionsupporting

confidence: 79%

Section: Discussionsupporting

confidence: 61%

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Crystal Structures of Transhydrogenase Domain I with and without Bound NADH^,

et al. 2002

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“…1), and that there is strong evidence that isolated dI-dIII complexes from R. rubrum have their dIII fixed in the face-up orientation, are occluded and are capable of rapid hydride transfer between bound nucleotides [19,57,58]. The events taking place during operation of the dimer then fall into place (Fig.…”

Section: Alternation Of Sitesmentioning

confidence: 99%

“…High-resolution structures of isolated dI [13][14][15] and dIII [16][17][18], and that of a dI-dIII complex [19][20][21], have been published during the last decade, and have afforded insights particularly into nucleotide binding and the hydride transfer step. The recent structure of the membrane-spanning dII at 2.8 Å A 0 resolution, and of the holo-enzyme at 6.9 Å A 0 from Thermus thermophilus [22], provide clues as to how hydride transfer from NADH to NADP + at the interface of dI and dIII is coupled to proton translocation through dII.…”

Section: The DI Dii and Diii Components Of Transhydrogenasementioning

confidence: 99%

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

et al. 2015

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Edited by Peter BrzezinskiKeywords: Transhydrogenase Membrane-protein structure Nicotinamide nucleotide Proton-pump Proton-gating a b s t r a c tThe membrane protein transhydrogenase in animal mitochondria and bacteria couples reduction of NADP + by NADH to proton translocation. Recent X-ray data on Thermus thermophilus transhydrogenase indicate a significant difference in the orientations of the two dIII components of the enzyme dimer (Leung et al., 2015). The character of the orientation change, and a review of information on the kinetics and thermodynamics of transhydrogenase, indicate that dIII swivelling might assist in the control of proton gating by the redox state of bound NADP + /NADPH during enzyme turnover.

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Three‐Dimensional Model of Human Nicotinamide Nucleotide Transhydrogenase (NNT) and Sequence‐Structure Analysis of its Disease‐Causing Variations

et al. 2016

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Defective mitochondrial proteins are emerging as major contributors to human disease. Nicotinamide nucleotide transhydrogenase (NNT), a widely expressed mitochondrial protein, has a crucial role in the defence against oxidative stress. NNT variations have recently been reported in patients with familial glucocorticoid deficiency (FGD) and in patients with heart failure. Moreover, knockout animal models suggest that NNT has a major role in diabetes mellitus and obesity. In this study, we used experimental structures of bacterial transhydrogenases to generate a structural model of human NNT (H‐NNT). Structure‐based analysis allowed the identification of H‐NNT residues forming the NAD binding site, the proton canal and the large interaction site on the H‐NNT dimer. In addition, we were able to identify key motifs that allow conformational changes adopted by domain III in relation to its functional status, such as the flexible linker between domains II and III and the salt bridge formed by H‐NNT Arg882 and Asp830. Moreover, integration of sequence and structure data allowed us to study the structural and functional effect of deleterious amino acid substitutions causing FGD and left ventricular non‐compaction cardiomyopathy. In conclusion, interpretation of the function–structure relationship of H‐NNT contributes to our understanding of mitochondrial disorders.

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The Crystal Structure of an Asymmetric Complex of the Two Nucleotide Binding Components of Proton-Translocating Transhydrogenase

Cited by 60 publications

References 54 publications

Crystal Structures of Transhydrogenase Domain I with and without Bound NADH^,

Crystal Structures of Transhydrogenase Domain I with and without Bound NADH^,

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

Three‐Dimensional Model of Human Nicotinamide Nucleotide Transhydrogenase (NNT) and Sequence‐Structure Analysis of its Disease‐Causing Variations

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The Crystal Structure of an Asymmetric Complex of the Two Nucleotide Binding Components of Proton-Translocating Transhydrogenase

Cited by 60 publications

References 54 publications

Crystal Structures of Transhydrogenase Domain I with and without Bound NADH,

Crystal Structures of Transhydrogenase Domain I with and without Bound NADH,

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

Three‐Dimensional Model of Human Nicotinamide Nucleotide Transhydrogenase (NNT) and Sequence‐Structure Analysis of its Disease‐Causing Variations

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Product

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Crystal Structures of Transhydrogenase Domain I with and without Bound NADH^,

Crystal Structures of Transhydrogenase Domain I with and without Bound NADH^,