NMR characterization of the NADP(H)‐binding domain of <i>Escherichia coli</i> transhydrogenase: sequential assignment and global fold

Johansson, Christina; Bergkvist, Anders; Fjellström, Ola; Rydström, Jan; Karlsson, Bengt

doi:10.1016/s0014-5793(99)01156-4

Cited by 24 publications

(39 citation statements)

References 22 publications

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“…NMR experiments with R. rubrum dIII also indicate that the protein is a monomer (25), although on size-exclusion chromatography it behaves anomalously; the apparent M r value is intermediate between those expected for the monomer and the dimer (13). E. coli dIII is monomeric on size-exclusion chromatography (16), but has an unexpectedly large correlation time in NMR experiments, perhaps indicating some tendency to aggregate (37). Fig.…”

Section: Resultsmentioning

confidence: 88%

The Heterotrimer of the Membrane-peripheral Components of Transhydrogenase and the Alternating-site Mechanism of Proton Translocation

Venning¹,

Rodrigues²,

Weston³

et al. 2001

Journal of Biological Chemistry

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Transhydrogenase undergoes conformational changes to couple the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The protein comprises three components: dI, which binds NAD(H); dIII, which binds NADP(H); and dII, which spans the membrane. Experiments using isothermal titration calorimetry, analytical ultracentrifugation, and small angle x-ray scattering show that, as in the crystalline state, a mixture of recombinant dI and dIII from Rhodospirillum rubrum transhydrogenase readily forms a dI 2 dIII 1 heterotrimer in solution, but we could find no evidence for the formation of a dI 2 dIII 2 tetramer using these techniques. The asymmetry of the complex suggests that there is an alternation of conformations at the nucleotidebinding sites during proton translocation by the complete enzyme. The characteristics of nucleotide interaction with the isolated dI and dIII components and with the dI 2 dIII 1 heterotrimer were investigated. (a) The rate of release of NADP ؉ from dIII was decreased 5-fold when the component was incorporated into the heterotrimer. (b) The binding affinity of one of the two nucleotide-binding sites for NADH on the dI dimer was decreased about 17-fold in the dI 2 dIII 1 complex; the other binding site was unaffected. These observations lend strong support to the alternating-site mechanism.Transhydrogenase, found in the cytoplasmic membranes of bacteria, and in the inner membranes of animal mitochondria, couples the redox reaction between NAD(H) and NADP(H) to the translocation of protons.Its function in energy metabolism, biosynthesis, and detoxification has been discussed at length (1, 2). In different organisms (and possibly in different tissues of the same organism) transhydrogenase can either utilize the proton electrochemical gradient (⌬p) to drive reduction of NADP ϩ by NADH, or it can use NADPH oxidation by NAD ϩ to augment ⌬p formation. Energy coupling in transhydrogenase is indirect. In the forward direction (Reaction 1), protein conformational changes accompanying proton translocation bring together the nicotinamide rings of the bound nucleotides to allow the redox reaction (3). This "binding-change mechanism" may share common features with energy coupling in some ion-translocating ATPases. More generally, transhydrogenase has a number of properties that make it an excellent model for understanding the principles of operation of conformationally linked pumps in biology.The polypeptide organization of transhydrogenases varies between species, but the arrangement of the three components, dI, dII, and dIII, is essentially the same in all (Fig. 1). NAD(H) binds to dI, and NADP(H) binds to dIII; these two components protrude from the membrane (into the bacterial cytoplasm or mitochondrial matrix). The dII component spans the membrane, probably in 13 or 14 transmembrane helices (reviewed in Ref. 4). There is cross-linking and hydrodynamic evidence that both the bovine (5, 6) and Escherichia coli (7) transhydrogenases have two copies each of dI, dII, and dIII...

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Section: Resultsmentioning

confidence: 88%

The Heterotrimer of the Membrane-peripheral Components of Transhydrogenase and the Alternating-site Mechanism of Proton Translocation

Venning¹,

Rodrigues²,

Weston³

et al. 2001

Journal of Biological Chemistry

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“…However, TH soluble domains have been expressed for biochemical and structural investigations, including human and bovine, Escherichia coli, and R. rubrum domains I and III. [21][22][23][24][25][26][27][28][29][30] Reconstituted mixtures of domain I and domain III catalyze reaction between NAD(H) and NADP(H) in the absence of membrane-bound domain II, and this activity has been studied to characterize the hydride transfer function of TH. [31][32][33][34][35] Regardless of their arrangement in intact TH, the soluble domains must allow the (dihydro)nicotinamide rings of NAD(H) and NADP(H) to come into direct contact.…”

Section: Introductionmentioning

confidence: 99%

Conformational Diversity in NAD(H) and Interacting Transhydrogenase Nicotinamide Nucleotide Binding Domains

Sundaresan

Chartron

Yamaguchi

et al. 2005

Journal of Molecular Biology

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“…The structural properties of domain I (dI) and domain III (dIII), from the three sources mentioned previously, have been characterized by expressing them separately. The determination of their structures has been realized by structural predictions [4–6], NMR [6–9] and X‐ray crystallography [10,11]. From these studies, it was found that dI was made of two similar domains (dIa and dIb) each of which comprise a parallel β sheet surrounded by mainly β helices.…”

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confidence: 99%

Interactions between the soluble domain I of nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and transhydrogenase from Escherichia coli

Bizouarn

Fjellström

Axelsson

et al. 2000

European Journal of Biochemistry

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Nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of two subunits, the a and the b subunits, each of which contains a hydrophilic domain, domain I and III, respectively, as well as several transmembrane helices, collectively denoted domain II. The interactions between domain I from Rhodospirillum rubrum (rrI) and the intact or the protease-treated enzyme from E. coli was investigated using the separately expressed and purified domain I from R. rubrum, and His-tagged intact and trypsin-treated E. coli transhydrogenase.Despite harsh treatments with, e.g. detergents and denaturing agents, the a and b subunits remained tightly associated. A monoclonal antibody directed towards the a subunit was strongly inhibitory, an effect that was relieved by added rrI. In addition, rrI also reactivated the trypsin-digested E. coli enzyme in which domain I had been partly removed. This suggests that the hydrophilic domains I and III are not in permanent contact but are mobile during catalysis while being anchored to domain II.Replacement of domain I of intact, as well as trypsin-digested, E. coli transhydrogenase with rrI resulted in a markedly different pH dependence of the cyclic reduction of 3-acetyl-pyridine-NAD 1 by NADH in the presence of NADP(H), suggesting that the protonation of one or more protonable groups in domain I is controlling this reaction. The reverse reaction and proton pumping showed a less pronounced change in pH dependence, demonstrating the regulatory role of domain II in these reactions.Keywords: transhydrogenase; NADP, NAD; membrane protein; proton pump. (Fig. 1). The active forms of the enzymes are dimers (220 kDa), either as a homodimer in the case of the mitochondrial H 1 -TH, as (a,b) 2 in the case of the Escherichia coli H 1 -TH or as (a1,a2,b) 2 in the case of Rhodospirillum rubrum H 1 -TH (1). As shown in Fig. 1, the a subunit (54 kDa) of E. coli H 1 -TH comprises domain I (40 kDa) and four transmembraneous helices, which form one component of dII. The b subunit (49 kDa) is composed of domain III (20 kDa) and nine helices spanning the membrane which constitute the second part of dII. The a1 subunit of R. rubrum corresponds only to domain I and is therefore purified as a soluble homodimer of 81 kDa [3]. The structural properties of domain I (dI) and domain III (dIII), from the three sources mentioned previously, have been characterized by expressing them separately. The determination of their structures has been realized by structural predictions [4±6], NMR [6±9] and X-ray crystallography [10,11]. From these studies, it was found that dI was made of two similar domains (dIa and dIb) each of which comprise a parallel b sheet surrounded by mainly b helices. The structure of domain III also exhibited a classical dinucleotide-binding fold [10,11].

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NMR characterization of the NADP(H)‐binding domain of Escherichia coli transhydrogenase: sequential assignment and global fold

Cited by 24 publications

References 22 publications

The Heterotrimer of the Membrane-peripheral Components of Transhydrogenase and the Alternating-site Mechanism of Proton Translocation

The Heterotrimer of the Membrane-peripheral Components of Transhydrogenase and the Alternating-site Mechanism of Proton Translocation

Conformational Diversity in NAD(H) and Interacting Transhydrogenase Nicotinamide Nucleotide Binding Domains

Interactions between the soluble domain I of nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and transhydrogenase from Escherichia coli

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