Philip G. Quirk

Structural changes in the recombinant, NADP(H)-binding component of proton translocating transhydrogenase revealed by NMR spectroscopy

We have analysed 1H, 15N‐HSQC spectra of the recombinant, NADP(H)‐binding component of transhydrogenase in the context of the emerging three dimensional structure of the protein. Chemical shift perturbations of amino acid residues following replacement of NADP+ with NADPH were observed in both the adenosine and nicotinamide parts of the dinucleotide binding site and in a region which straddles the protein. These observations reflect the structural changes resulting from hydride transfer. The interactions between the recombinant, NADP(H)‐binding component and its partner, NAD(H)‐binding protein, are complicated. Helix B of the recombinant, NADP(H)‐binding component may play an important role in the binding process.

Solution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from Rhodospirillum rubrum

Transhydrogenase is a proton pump found in the membranes of bacteria and animal mitochondria. The solution structure of the expressed, 21.5 kDa, NADP(H)-binding component (dIII) of transhydrogenase from Rhodospirillum rubrum has been solved by NMR methods. This is the first description of the structure of dIII from a bacterial source. The protein adopts a Rossmann fold: an open, twisted, parallel beta-sheet, flanked by helices. However, the binding of NADP(+) to dIII is profoundly different to that seen in other Rossmann structures, in that its orientation is reversed: the adenosine moiety interacts with the first betaalphabetaalphabeta motif, and the nicotinamide with the second. Features in the structure that might be responsible for changes in nucleotide-binding affinity during catalysis, and for interaction with other components of the enzyme, are identified. The results are compared with the recently determined, high-resolution crystal structures of human and bovine dIII which also show the reversed nucleotide orientation.

Sequential phosphorylation of adjacent serine residues on the N-terminal region of cardiac troponin-I: structure-activity implications of ordered phosphorylation

We have used NMR spectroscopy to monitor the phosphorylation of a peptide corresponding to the N‐terminal region of human cardiac troponin‐I (residues 17–30), encompassing the two adjacent serine residues of the dual phosphorylation site. An ordered incorporation of phosphate catalysed by PKA was observed, with phosphorylation of Ser‐24 preceding that of Ser‐23. Diphosphorylation induced a conformational transition in this region, involving the specific association of the Arg‐22 and Ser‐24P side‐chains, and maximally stabilised when both phosphoserines were in the di‐anionic form. The results suggest that the second phosphorylation at Ser‐23 of cardiac troponin‐I is of particular significance in the mechanism by which adrenaline regulates the calcium sensitivity of the myofibrillar actomyosin Mg‐ATPase.

Interdomain hydride transfer in proton-translocating transhydrogenase.

We describe the use of the recombinant, nucleotide-binding domains (domains I and III) of transhydrogenase to study structural, functional and dynamic features of the protein that are important in hydride transfer and proton translocation. Experiments on the transient state kinetics of the reaction show that hydride transfer takes place extremely rapidly in the recombinant domain I:III complex, even in the absence of the membrane-spanning domain II. We develop the view that proton translocation through domain II is coupled to changes in the binding characteristics of NADP+ and NADPH in domain III. A mobile loop region which emanates from the surface of domain I, and which interacts with NAD+ and NADH during nucleotide binding has been studied by NMR spectroscopy and site-directed mutagenesis. An important role for the loop region in the process of hydride transfer is revealed.

The mobile loop region of the NAD(H) binding component (dI) of proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum: complete NMR assignment and effects of bound nucleotides

The dI component of transhydrogenase binds NAD+ and NADH. A mobile loop region of dI plays an important role in the nucleotide binding process, and mutations in this region result in impaired hydride transfer in the complete enzyme. We have previously employed one-dimensional 1H-NMR spectroscopy to study wild-type and mutant dI proteins of Rhodospirillum rubrum and the effects of nucleotide binding. Here, we utilise two- and three-dimensional NMR experiments to assign the signals from virtually all of the backbone and side-chain protons of the loop residues. The mobile loop region encompasses 17 residues: Asp223-Met239. The assignments also provide a much strengthened basis for interpreting the structural changes occurring upon nucleotide binding, when the loop closes down onto the surface of the protein and loses mobility. The role of the mobile loop region in catalysis is discussed with particular reference to a newly-developed model of the dI protein, based on its homology with alanine dehydrogenase.

Letter to the Editor: Sequence-specific resonance assignments for the NADP(H)-binding component (domain III) of proton- translocating transhydrogenase from Rhodospisrillum rubrum

Transhydrogenase is a proton pump, found in the inner membrane of animal mitochondria, and the cytoplasmic membrane of bacteria. It has a tripartite structure. Domains I and III protrude from the membrane (on the cytoplasmic side in bacteria, and on the matrix side in mitochondria). The domain II component spans the membrane, and serves as a channel for proton conduction. Transhydrogenase couples the transfer of reducing equivalents (hydride ion equivalents) between NAD(H) and NADP(H) to the translocation of protons across the membrane (reviewed by Jackson et al., 1998),

Interactions between Transhydrogenase and Thio-nicotinamide Analogues of NAD(H) and NADP(H) Underline the Importance of Nucleotide Conformational Changes in Coupling to Proton Translocation

Transhydrogenase couples the reduction of NADP+ by NADH to inward proton translocation across mitochondrial and bacterial membranes. The coupling reactions occur within the protein by long distance conformational changes. In intact transhydrogenase and in complexes formed from the isolated, nucleotide-binding components, thio-NADP(H) is a good analogue for NADP(H), but thio-NAD(H) is a poor analogue for NAD(H). Crystal structures of the nucleotide-binding components show that the twists of the 3-carbothiamide groups of thio-NADP+ and of thio-NAD+ (relative to the planes of the pyridine rings), which are defined by the dihedral, Xam, are altered relative to the twists of the 3-carboxamide groups of the physiological nucleotides. The finding that thio-NADP+ is a good substrate despite an increased Xam value shows that approach of the NADH prior to hydride transfer is not obstructed by the S atom in the analogue. That thio-NAD(H) is a poor substrate appears to be the result of failure in the conformational change that establishes the ground state for hydride transfer. This might be a consequence of restricted rotation of the 3-carbothiamide group during the conformational change.

The role of invariant amino acid residues at the hydride transfer site of proton-translocating transhydrogenase.

Transhydrogenase couples proton translocation across a membrane to hydride transfer between NADH and NADP+. Previous x-ray structures of complexes of the nucleotide-binding components of transhydrogenase (“dI2dIII1” complexes) indicate that the dihydronicotinamide ring of NADH can move from a distal position relative to the nicotinamide ring of NADP+ to a proximal position. The movement might be responsible for gating hydride transfer during proton translocation. We have mutated three invariant amino acids, Arg-127, Asp-135, and Ser-138, in the NAD(H)-binding site of Rhodospirillum rubrum transhydrogenase. In each mutant, turnover by the intact enzyme is strongly inhibited. Stopped-flow experiments using dI2dIII1 complexes show that inhibition results from a block in the steps associated with hydride transfer. Mutation of Asp-135 and Ser-138 had no effect on the binding affinity of either NAD+ or NADH, but mutation of Arg-127 led to much weaker binding of NADH and slightly weaker binding of NAD+. X-ray structures of dI2dIII1 complexes carrying the mutations showed that their effects were restricted to the locality of the bound NAD(H). The results are consistent with the suggestion that in wild-type protein movement of the Arg-127 side chain, and its hydrogen bonding to Asp-135 and Ser-138, stabilizes the dihydronicotinamide of NADH in the proximal position for hydride transfer.

Mutation of amino acid residues in the mobile loop region of the NAD(H)-binding domain of proton-translocating transhydrogenase

The effects of single amino acid substitutions in the mobile loop region of the recombinant NAD(H)-binding domain (dI) of transhydrogenase have been examined. The mutations lead to clear assignments of well-defined resonances in one-dimensional 1H-NMR spectra. As with the wild-type protein, addition of NADH, or higher concentrations of NAD+, led to broadening and some shifting of the well-defined resonances. With many of the mutant dI proteins more nucleotide was required for these effects than with wild-type protein. Binding constants of the mutant proteins for NADH were determined by equilibrium dialysis and, where possible, by NMR. Generally, amino acid changes in the mobile loop region gave rise to a 2-4-fold increase in the dI-nucleotide dissociation constants, but substitution of Ala236 for Gly had a 10-fold effect. The mutant dI proteins were reconstituted with dI-depleted bacterial membranes with apparent docking affinities that were indistinguishable from that of wild-type protein. In the reconstituted system, most of the mutants were more inhibited in their capacity to perform cyclic transhydrogenation (reduction of acetyl pyridine adenine dinucleotide, AcPdAD+, by NADH in the presence of NADP+) than in either the simple reduction of AcPdAD+ by NADPH, or the light-driven reduction of thio-NADP+ by NADH, which suggests that they are impaired at the hydride transfer step. A cross-peak in the 1H-1H nuclear Overhauser enhancement spectrum of a mixture of wild-type dI and NADH was assigned to an interaction between the A8 proton of the nucleotide and the betaCH3 protons of Ala236. It is proposed that, following nucleotide binding, the mobile loop folds down on to the surface of the dI protein, and that contacts, especially from Tyr235 in a Gly-Tyr-Ala motif with the adenosine moiety of the nucleotide, set the position of the nicotinamide ring of NADH close to that of NADP+ in dIII to effect direct hydride transfer.

Proton-Translocating Transhydrogenase From Rhodospirillum Rubrum

Transhydrogenase is found in the cytoplamic membranes of many bacteria. It catalyses the following reaction: Under physiological conditions the reaction probably proceeds from left to right, in favour of NADPH formation, driven by the protonmotive force generated by either respiratory or photosynthetic electron transport. Under experimental conditions the reaction can be measured in real time using nucleotide analogues. In this report we use acetyl pyridine adenine dinucleotide, AcPdAD+, an NAD+ analogue, whose reduced form has a characteristic ultra-violet absorbance.