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1
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Zöller J, Hong S, Eisinger ML, Anderson M, Radloff M, Desch K, Gennis R, Langer JD. Ligand binding and conformational dynamics of the E. coli nicotinamide nucleotide transhydrogenase revealed by hydrogen/deuterium exchange mass spectrometry. Comput Struct Biotechnol J 2022; 20:5430-5439. [PMID: 36212541 PMCID: PMC9529548 DOI: 10.1016/j.csbj.2022.09.036] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2022] [Revised: 09/22/2022] [Accepted: 09/22/2022] [Indexed: 11/17/2022] Open
Abstract
Nicotinamide nucleotide transhydrogenases are integral membrane proteins that utilizes the proton motive force to reduce NADP+ to NADPH while converting NADH to NAD+. Atomic structures of various transhydrogenases in different ligand-bound states have become available, and it is clear that the molecular mechanism involves major conformational changes. Here we utilized hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map ligand binding sites and analyzed the structural dynamics of E. coli transhydrogenase. We found different allosteric effects on the protein depending on the bound ligand (NAD+, NADH, NADP+, NADPH). The binding of either NADP+ or NADPH to domain III had pronounced effects on the transmembrane helices comprising the proton-conducting channel in domain II. We also made use of cyclic ion mobility separation mass spectrometry (cyclic IMS-MS) to maximize coverage and sensitivity in the transmembrane domain, showing for the first time that this technique can be used for HDX-MS studies. Using cyclic IMS-MS, we increased sequence coverage from 68 % to 73 % in the transmembrane segments. Taken together, our results provide important new insights into the transhydrogenase reaction cycle and demonstrate the benefit of this new technique for HDX-MS to study ligand binding and conformational dynamics in membrane proteins.
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2
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Energy transfer between the nicotinamide nucleotide transhydrogenase and ATP synthase of Escherichia coli. Sci Rep 2021; 11:21234. [PMID: 34707181 PMCID: PMC8551311 DOI: 10.1038/s41598-021-00651-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2021] [Accepted: 10/15/2021] [Indexed: 11/09/2022] Open
Abstract
Membrane bound nicotinamide nucleotide transhydrogenase (TH) catalyses the hydride transfer from NADH to NADP+. Under physiological conditions, this reaction is endergonic and must be energized by the pmf, coupled to transmembrane proton transport. Recent structures of transhydrogenase holoenzymes suggest new mechanistic details, how the long-distance coupling between hydride transfer in the peripheral nucleotide binding sites and the membrane-localized proton transfer occurs that now must be tested experimentally. Here, we provide protocols for the efficient expression and purification of the Escherichia coli transhydrogenase and its reconstitution into liposomes, alone or together with the Escherichia coli F1F0 ATP synthase. We show that E. coli transhydrogenase is a reversible enzyme that can also work as a NADPH-driven proton pump. In liposomes containing both enzymes, NADPH driven H+-transport by TH is sufficient to instantly fuel ATP synthesis, which adds TH to the pool of pmf generating enzymes. If the same liposomes are energized with ATP, NADPH production by TH is stimulated > sixfold both by a pH gradient or a membrane potential. The presented protocols and results reinforce the tight coupling between hydride transfer in the peripheral nucleotide binding sites and transmembrane proton transport and provide powerful tools to investigate their coupling mechanism.
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3
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Calisto F, Sousa FM, Sena FV, Refojo PN, Pereira MM. Mechanisms of Energy Transduction by Charge Translocating Membrane Proteins. Chem Rev 2021; 121:1804-1844. [PMID: 33398986 DOI: 10.1021/acs.chemrev.0c00830] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Life relies on the constant exchange of different forms of energy, i.e., on energy transduction. Therefore, organisms have evolved in a way to be able to harvest the energy made available by external sources (such as light or chemical compounds) and convert these into biological useable energy forms, such as the transmembrane difference of electrochemical potential (Δμ̃). Membrane proteins contribute to the establishment of Δμ̃ by coupling exergonic catalytic reactions to the translocation of charges (electrons/ions) across the membrane. Irrespectively of the energy source and consequent type of reaction, all charge-translocating proteins follow two molecular coupling mechanisms: direct- or indirect-coupling, depending on whether the translocated charge is involved in the driving reaction. In this review, we explore these two coupling mechanisms by thoroughly examining the different types of charge-translocating membrane proteins. For each protein, we analyze the respective reaction thermodynamics, electron transfer/catalytic processes, charge-translocating pathways, and ion/substrate stoichiometries.
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Affiliation(s)
- Filipa Calisto
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157, Oeiras, Portugal.,BioISI-Biosystems & Integrative Sciences Institute, University of Lisboa, Faculty of Sciences, Campo Grande, 1749-016 Lisboa, Portugal
| | - Filipe M Sousa
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157, Oeiras, Portugal.,BioISI-Biosystems & Integrative Sciences Institute, University of Lisboa, Faculty of Sciences, Campo Grande, 1749-016 Lisboa, Portugal
| | - Filipa V Sena
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157, Oeiras, Portugal.,BioISI-Biosystems & Integrative Sciences Institute, University of Lisboa, Faculty of Sciences, Campo Grande, 1749-016 Lisboa, Portugal
| | - Patricia N Refojo
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157, Oeiras, Portugal
| | - Manuela M Pereira
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157, Oeiras, Portugal.,BioISI-Biosystems & Integrative Sciences Institute, University of Lisboa, Faculty of Sciences, Campo Grande, 1749-016 Lisboa, Portugal
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4
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Nesci S, Trombetti F, Pagliarani A. Nicotinamide Nucleotide Transhydrogenase as a Sensor of Mitochondrial Biology. Trends Cell Biol 2020; 30:1-3. [PMID: 31753532 DOI: 10.1016/j.tcb.2019.11.001] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2019] [Accepted: 11/06/2019] [Indexed: 01/27/2023]
Abstract
The enzyme nicotinamide nucleotide transhydrogenase (NNT) transfers hydride from NADH to NADP+ coupled to H+ translocation across the inner mitochondrial membrane. In a recent study, Kampjut and Sazanov reveal that the bifunctional NNT mechanism rules the NAD(P)+/NAD(P)H interconversion ratio, which in turn regulates antioxidant defense and sirtuin actions.
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Affiliation(s)
- Salvatore Nesci
- Department of Veterinary Medical Sciences, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy.
| | - Fabiana Trombetti
- Department of Veterinary Medical Sciences, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy
| | - Alessandra Pagliarani
- Department of Veterinary Medical Sciences, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy
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5
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Kampjut D, Sazanov LA. Structure and mechanism of mitochondrial proton-translocating transhydrogenase. Nature 2019; 573:291-295. [PMID: 31462775 DOI: 10.1038/s41586-019-1519-2] [Citation(s) in RCA: 58] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2019] [Accepted: 07/31/2019] [Indexed: 11/09/2022]
Abstract
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 cancers7-9.
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Affiliation(s)
- Domen Kampjut
- Institute of Science and Technology Austria, Klosterneuburg, Austria
| | - Leonid A Sazanov
- Institute of Science and Technology Austria, Klosterneuburg, Austria.
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6
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Ghirotto B, Terra FF, Câmara NOS, Basso PJ. Sirtuins in B lymphocytes metabolism and function. World J Exp Med 2019; 9:1-13. [PMID: 30705866 PMCID: PMC6354076 DOI: 10.5493/wjem.v9.i1.1] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/07/2018] [Revised: 10/29/2018] [Accepted: 01/05/2019] [Indexed: 02/06/2023] Open
Abstract
Sirtuins (SIRTs) are NAD+-dependent histone deacetylases and play a role in virtually all cell biological processes. As SIRTs functions vary according to their subtypes, they can either activate or inhibit signaling pathways upon different conditions or tissues. Recent studies have focused on metabolic effects performed by SIRTs in several cell types since specific metabolic pathways (e.g., aerobic glycolysis, oxidative phosphorylation, β-oxidation, glutaminolysis) are used to determine the cell fate. However, few efforts have been made to understand the role of SIRTs on B lymphocytes metabolism and function. These cells are associated with humoral immune responses by secreting larger amounts of antibodies after differentiating into antibody-secreting cells. Besides, both the SIRTs and B lymphocytes are potential targets to treat several immune-mediated disorders, including cancer. Here, we provide an outlook of recent studies regarding the role of SIRTs in general cellular metabolism and B lymphocytes functions, pointing out the future perspectives of this field.
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Affiliation(s)
- Bruno Ghirotto
- Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo 05508-000, Brazil
| | - Fernanda Fernandes Terra
- Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo 05508-000, Brazil
| | - Niels Olsen Saraiva Câmara
- Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo 05508-000, Brazil
- Division of Nephrology, School of Medicine, Federal University of São Paulo, São Paulo 04023-062, Brazil
- Laboratory of Renal Physiology (LIM 16), School of Medicine, University of São Paulo, São Paulo 01246-903, Brazil
| | - Paulo José Basso
- Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo 05508-000, Brazil
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7
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Zhang Q, Padayatti PS, Leung JH. Proton-Translocating Nicotinamide Nucleotide Transhydrogenase: A Structural Perspective. Front Physiol 2017; 8:1089. [PMID: 29312000 PMCID: PMC5742237 DOI: 10.3389/fphys.2017.01089] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2017] [Accepted: 12/11/2017] [Indexed: 01/07/2023] Open
Abstract
Nicotinamide nucleotide transhydrogenase (TH) is an enzyme complex in animal mitochondria and bacteria that utilizes the electrochemical proton gradient across membranes to drive the production of NADPH. The enzyme plays an important role in maintaining the redox balance of cells with implications in aging and a number of human diseases. TH exists as a homodimer with each protomer containing a proton-translocating transmembrane domain and two soluble nucleotide binding domains that mediate hydride transfer between NAD(H) and NADP(H). The three-domain architecture of TH is conserved across species but polypeptide composition differs substantially. The complex domain coupling mechanism of TH is not fully understood despite extensive biochemical and structural characterizations. Herein the progress is reviewed, focusing mainly on structural findings from 3D crystallization of isolated soluble domains and more recently of the transmembrane domain and the holo-enzyme from Thermus thermophilus. A structural perspective and impeding challenges in further elucidating the mechanism of TH are discussed.
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Affiliation(s)
- Qinghai Zhang
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, United States
| | - Pius S Padayatti
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, United States
| | - Josephine H Leung
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, United States
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8
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Padayatti PS, Leung JH, Mahinthichaichan P, Tajkhorshid E, Ishchenko A, Cherezov V, Soltis SM, Jackson JB, Stout CD, Gennis RB, Zhang Q. Critical Role of Water Molecules in Proton Translocation by the Membrane-Bound Transhydrogenase. Structure 2017. [PMID: 28648609 DOI: 10.1016/j.str.2017.05.022] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
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 His42α2 (chain A) being protonated and Thr214β (chain B) displaying a conformational change, respectively, to gate the channel access to both cytoplasmic and periplasmic chambers. Mutation of Thr214β to Ala deactivated the enzyme. These data provide new insights into the gating mechanism of proton translocation in TH.
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Affiliation(s)
- Pius S Padayatti
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
| | - Josephine H Leung
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Paween Mahinthichaichan
- Department of Biochemistry, University of Illinois Urbana-Champaign, Champaign, IL 61801, USA
| | - Emad Tajkhorshid
- Department of Biochemistry, University of Illinois Urbana-Champaign, Champaign, IL 61801, USA
| | - Andrii Ishchenko
- Department of Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA
| | - Vadim Cherezov
- Department of Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA; Laboratory for Structural Biology of GPCRs, Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia
| | - S Michael Soltis
- Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA
| | - J Baz Jackson
- School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK
| | - C David Stout
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Robert B Gennis
- Department of Biochemistry, University of Illinois Urbana-Champaign, Champaign, IL 61801, USA
| | - Qinghai Zhang
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
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9
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Metherell LA, Guerra-Assunção JA, Sternberg MJ, David A. Three-Dimensional Model of Human Nicotinamide Nucleotide Transhydrogenase (NNT) and Sequence-Structure Analysis of its Disease-Causing Variations. Hum Mutat 2016; 37:1074-84. [PMID: 27459240 PMCID: PMC5026163 DOI: 10.1002/humu.23046] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2016] [Revised: 06/23/2016] [Accepted: 06/28/2016] [Indexed: 12/22/2022]
Abstract
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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Affiliation(s)
- Louise A Metherell
- Centre for Endocrinology, William Harvey Research Institute, Queen Mary University of London, Charterhouse Square, London, UK
| | - José Afonso Guerra-Assunção
- Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London, UK
| | - Michael J Sternberg
- Centre for Integrative System Biology and Bioinformatics, Imperial College London, London, UK
| | - Alessia David
- Centre for Integrative System Biology and Bioinformatics, Imperial College London, London, UK.
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10
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Marreiros BC, Calisto F, Castro PJ, Duarte AM, Sena FV, Silva AF, Sousa FM, Teixeira M, Refojo PN, Pereira MM. Exploring membrane respiratory chains. BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 2016; 1857:1039-1067. [PMID: 27044012 DOI: 10.1016/j.bbabio.2016.03.028] [Citation(s) in RCA: 54] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/15/2016] [Revised: 03/16/2016] [Accepted: 03/18/2016] [Indexed: 01/20/2023]
Abstract
Acquisition of energy is central to life. In addition to the synthesis of ATP, organisms need energy for the establishment and maintenance of a transmembrane difference in electrochemical potential, in order to import and export metabolites or to their motility. The membrane potential is established by a variety of membrane bound respiratory complexes. In this work we explored the diversity of membrane respiratory chains and the presence of the different enzyme complexes in the several phyla of life. We performed taxonomic profiles of the several membrane bound respiratory proteins and complexes evaluating the presence of their respective coding genes in all species deposited in KEGG database. We evaluated 26 quinone reductases, 5 quinol:electron carriers oxidoreductases and 18 terminal electron acceptor reductases. We further included in the analyses enzymes performing redox or decarboxylation driven ion translocation, ATP synthase and transhydrogenase and we also investigated the electron carriers that perform functional connection between the membrane complexes, quinones or soluble proteins. Our results bring a novel, broad and integrated perspective of membrane bound respiratory complexes and thus of the several energetic metabolisms of living systems. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
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Affiliation(s)
- Bruno C Marreiros
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157 Oeiras, Portugal
| | - Filipa Calisto
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157 Oeiras, Portugal
| | - Paulo J Castro
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157 Oeiras, Portugal
| | - Afonso M Duarte
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157 Oeiras, Portugal
| | - Filipa V Sena
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157 Oeiras, Portugal
| | - Andreia F Silva
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157 Oeiras, Portugal
| | - Filipe M Sousa
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157 Oeiras, Portugal
| | - Miguel Teixeira
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157 Oeiras, Portugal
| | - Patrícia N Refojo
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157 Oeiras, Portugal
| | - Manuela M Pereira
- Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157 Oeiras, Portugal.
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11
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Jackson JB, Leung JH, Stout CD, Schurig-Briccio LA, Gennis RB. Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel. FEBS Lett 2015; 589:2027-33. [PMID: 26143375 DOI: 10.1016/j.febslet.2015.06.027] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2015] [Revised: 06/17/2015] [Accepted: 06/17/2015] [Indexed: 11/26/2022]
Abstract
The 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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Affiliation(s)
- J Baz Jackson
- School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
| | - Josephine H Leung
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92307, USA
| | - Charles D Stout
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92307, USA
| | | | - Robert B Gennis
- Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA
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12
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Leung JH, Schurig-Briccio LA, Yamaguchi M, Moeller A, Speir JA, Gennis RB, Stout CD. Structural biology. Division of labor in transhydrogenase by alternating proton translocation and hydride transfer. Science 2015; 347:178-81. [PMID: 25574024 DOI: 10.1126/science.1260451] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
NADPH/NADP(+) (the reduced form of NADP(+)/nicotinamide adenine dinucleotide phosphate) homeostasis is critical for countering oxidative stress in cells. Nicotinamide nucleotide transhydrogenase (TH), a membrane enzyme present in both bacteria and mitochondria, couples the proton motive force to the generation of NADPH. We present the 2.8 Å crystal structure of the transmembrane proton channel domain of TH from Thermus thermophilus and the 6.9 Å crystal structure of the entire enzyme (holo-TH). The membrane domain crystallized as a symmetric dimer, with each protomer containing a putative proton channel. The holo-TH is a highly asymmetric dimer with the NADP(H)-binding domain (dIII) in two different orientations. This unusual arrangement suggests a catalytic mechanism in which the two copies of dIII alternatively function in proton translocation and hydride transfer.
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Affiliation(s)
- Josephine H Leung
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | | | - Mutsuo Yamaguchi
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Arne Moeller
- National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Jeffrey A Speir
- National Resource for Automated Molecular Microscopy, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Robert B Gennis
- Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA
| | - Charles D Stout
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
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13
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Parenti MD, Bruzzone S, Nencioni A, Del Rio A. Selectivity hot-spots of sirtuin catalytic cores. MOLECULAR BIOSYSTEMS 2015; 11:2263-72. [DOI: 10.1039/c5mb00205b] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
We report a comprehensive study aimed to classify and identify the selectivity hot-spots for targeting the catalytic cores of human sirtuins using small molecule modulators.
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Affiliation(s)
- Marco Daniele Parenti
- Department of Experimental, Diagnostic and Specialty Medicine (DIMES)
- University of Bologna
- 40126 Bologna
- Italy
| | - Santina Bruzzone
- Department of Experimental Medicine
- Section of Biochemistry
- and CEBR
- University of Genoa
- 16132 Genoa
| | - Alessio Nencioni
- Department of Internal Medicine
- University of Genoa
- 16132 Genoa
- Italy
| | - Alberto Del Rio
- Department of Experimental, Diagnostic and Specialty Medicine (DIMES)
- University of Bologna
- 40126 Bologna
- Italy
- Institute of Organic Synthesis and Photoreactivity (ISOF)
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14
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Abstract
Sir2 proteins, or sirtuins, are a family of enzymes that catalyze NAD(+)-dependent deacetylation reactions and can also process ribosyltransferase, demalonylase, and desuccinylase activities. More than 40 crystal structures of sirtuins have been determined, alone or in various liganded forms. These high-resolution architectural details lay the foundation for understanding the molecular mechanisms of catalysis, regulation, substrate specificity, and inhibition of sirtuins. In this minireview, we summarize these structural features and discuss their implications for understanding sirtuin function.
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Affiliation(s)
- Hua Yuan
- Program in Gene Expression and Regulation, The Wistar Institute, University of Pennsylvania,Philadelphia, Pennsylvania 19104, USA
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15
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Jackson JB. A review of the binding-change mechanism for proton-translocating transhydrogenase. BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 2012; 1817:1839-46. [PMID: 22538293 DOI: 10.1016/j.bbabio.2012.04.006] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/24/2012] [Revised: 04/04/2012] [Accepted: 04/10/2012] [Indexed: 11/17/2022]
Abstract
Proton-translocating transhydrogenase is found in the inner membranes of animal mitochondria, and in the cytoplasmic membranes of many bacteria. It catalyses hydride transfer from NADH to NADP(+) coupled to inward proton translocation. Evidence is reviewed suggesting the enzyme operates by a "binding-change" mechanism. Experiments with Escherichia coli transhydrogenase indicate the enzyme is driven between "open" and "occluded" states by protonation and deprotonation reactions associated with proton translocation. In the open states NADP(+)/NADPH can rapidly associate with, or dissociate from, the enzyme, and hydride transfer is prevented. In the occluded states bound NADP(+)/NADPH cannot dissociate, and hydride transfer is allowed. Crystal structures of a complex of the nucleotide-binding components of Rhodospirillum rubrum transhydrogenase show how hydride transfer is enabled and disabled at appropriate steps in catalysis, and how release of NADP(+)/NADPH is restricted in the occluded state. Thermodynamic and kinetic studies indicate that the equilibrium constant for hydride transfer on the enzyme is elevated as a consequence of the tight binding of NADPH relative to NADP(+). The protonation site in the translocation pathway must face the outside if NADP(+) is bound, the inside if NADPH is bound. Chemical shift changes detected by NMR may show where alterations in protein conformation resulting from NADP(+) reduction are initiated. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
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16
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Huxley L, Quirk PG, Cotton NPJ, White SA, Jackson JB. The specificity of proton-translocating transhydrogenase for nicotinamide nucleotides. BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 2010; 1807:85-94. [PMID: 20732298 DOI: 10.1016/j.bbabio.2010.08.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/06/2010] [Accepted: 08/10/2010] [Indexed: 11/29/2022]
Abstract
In its forward direction, transhydrogenase couples the reduction of NADP(+) by NADH to the outward translocation of protons across the membrane of bacteria and animal mitochondria. The enzyme has three components: dI and dIII protrude from the membrane and dII spans the membrane. Hydride transfer takes place between nucleotides bound to dI and dIII. Studies on the kinetics of a lag phase at the onset of a "cyclic reaction" catalysed by complexes of the dI and dIII components of transhydrogenase from Rhodospirillum rubrum, and on the kinetics of fluorescence changes associated with nucleotide binding, reveal two features. Firstly, the binding of NADP(+) and NADPH to dIII is extremely slow, and is probably limited by the conversion of the occluded to the open state of the complex. Secondly, dIII can also bind NAD(+) and NADH. Extrapolating to the intact enzyme this binding to the "wrong" site could lead to slip: proton translocation without change in the nucleotide redox state, which would have important consequences for bacterial and mitochondrial metabolism.
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Affiliation(s)
- Lucinda Huxley
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
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17
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Veronesi G, Whitehead SJ, Francia F, Giachini L, Boscherini F, Venturoli G, Cotton NP, Jackson JB. X-ray absorption studies of Zn2+-binding sites in Escherichia coli transhydrogenase and its βH91K mutant. BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 2010; 1797:494-500. [DOI: 10.1016/j.bbabio.2010.01.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/09/2009] [Revised: 01/08/2010] [Accepted: 01/11/2010] [Indexed: 12/01/2022]
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18
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Whitehead SJ, Iwaki M, Cotton NPJ, Rich PR, Jackson JB. Inhibition of proton-transfer steps in transhydrogenase by transition metal ions. BIOCHIMICA ET BIOPHYSICA ACTA 2009; 1787:1276-88. [PMID: 19505432 DOI: 10.1016/j.bbabio.2009.06.001] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/01/2009] [Revised: 06/02/2009] [Accepted: 06/02/2009] [Indexed: 11/20/2022]
Abstract
Transhydrogenase couples proton translocation across a bacterial or mitochondrial membrane to the redox reaction between NAD(H) and NADP(H). Purified intact transhydrogenase from Escherichia coli was prepared, and its His tag removed. The forward and reverse transhydrogenation reactions catalysed by the enzyme were inhibited by certain metal ions but a "cyclic reaction" was stimulated. Of metal ions tested they were effective in the order Pb(2+)>Cu(2+)>Zn(2+)=Cd(2+)>Ni(2+)>Co(2+). The results suggest that the metal ions affect transhydrogenase by binding to a site in the proton-transfer pathway. Attenuated total-reflectance Fourier-transform infrared difference spectroscopy indicated the involvement of His and Asp/Glu residues in the Zn(2+)-binding site(s). A mutant in which betaHis91 in the membrane-spanning domain of transhydrogenase was replaced by Lys had enzyme activities resembling those of wild-type enzyme treated with Zn(2+). Effects of the metal ion on the mutant were much diminished but still evident. Signals in Zn(2+)-induced FTIR difference spectra of the betaHis91Lys mutant were also attributable to changes in His and Asp/Glu residues but were much smaller than those in wild-type spectra. The results support the view that betaHis91 and nearby Asp or Glu residues participate in the proton-transfer pathway of transhydrogenase.
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19
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Sanders BD, Jackson B, Marmorstein R. Structural basis for sirtuin function: what we know and what we don't. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2009; 1804:1604-16. [PMID: 19766737 DOI: 10.1016/j.bbapap.2009.09.009] [Citation(s) in RCA: 173] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Received: 06/12/2009] [Revised: 08/26/2009] [Accepted: 09/10/2009] [Indexed: 12/18/2022]
Abstract
The sirtuin (silent information regulator 2) proteins are NAD(+)-dependent deacetylases that are implicated in diverse biological processes including DNA regulation, metabolism, and longevity. Homologues of the prototypic yeast Sir2p have been identified in all three kingdoms of life, and while bacteria and archaea typically contain one to two sirtuins, eukaryotic organisms contain multiple members. Sirtuins are regulated in part by the cellular concentrations of the noncompetitive inhibitor, nicotinamide, and several synthetic modulators of these enzymes have been identified. The x-ray crystal structures of several sirtuin proteins in various liganded forms have been determined. This wealth of structural information, together with related biochemical studies, have provided important insights into the catalytic mechanism, substrate specificity, and inhibitory mechanism of sirtuin proteins. Implications for future structural studies to address outstanding questions in the field are also discussed.
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Affiliation(s)
- Brandi D Sanders
- The Wistar Institute and Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA
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20
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Proton-translocating transhydrogenase: an update of unsolved and controversial issues. J Bioenerg Biomembr 2008; 40:463-73. [PMID: 18972197 DOI: 10.1007/s10863-008-9170-x] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2008] [Accepted: 08/11/2008] [Indexed: 10/21/2022]
Abstract
Proton-translocating transhydrogenases, reducing NADP(+) by NADH through hydride transfer, are membrane proteins utilizing the electrochemical proton gradient for NADPH generation. The enzymes have important physiological roles in the maintenance of e.g. reduced glutathione, relevant for essentially all cell types. Following X-ray crystallography and structural resolution of the soluble substrate-binding domains, mechanistic aspects of the hydride transfer are beginning to be resolved. However, the structure of the intact enzyme is unknown. Key questions regarding the coupling mechanism, i.e., the mechanism of proton translocation, are addressed using the separately expressed substrate-binding domains. Important aspects are therefore which functions and properties of mainly the soluble NADP(H)-binding domain, but also the NAD(H)-binding domain, are relevant for proton translocation, how the soluble domains communicate with the membrane domain, and the mechanism of proton translocation through the membrane domain.
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21
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Brondijk THC, van Boxel GI, Mather OC, Quirk PG, White SA, Jackson JB. The role of invariant amino acid residues at the hydride transfer site of proton-translocating transhydrogenase. J Biol Chem 2006; 281:13345-13354. [PMID: 16533815 DOI: 10.1074/jbc.m513230200] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
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.
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Affiliation(s)
- T Harma C Brondijk
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
| | - Gijs I van Boxel
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
| | - Owen C Mather
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
| | - Philip G Quirk
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
| | - Scott A White
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
| | - J Baz Jackson
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
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22
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Johansson T, Oswald C, Pedersen A, Törnroth S, Okvist M, Karlsson BG, Rydström J, Krengel U. X-ray structure of domain I of the proton-pumping membrane protein transhydrogenase from Escherichia coli. J Mol Biol 2005; 352:299-312. [PMID: 16083909 DOI: 10.1016/j.jmb.2005.07.022] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2005] [Revised: 07/05/2005] [Accepted: 07/07/2005] [Indexed: 11/30/2022]
Abstract
The dimeric integral membrane protein nicotinamide nucleotide transhydrogenase is required for cellular regeneration of NADPH in mitochondria and prokaryotes, for detoxification and biosynthesis purposes. Under physiological conditions, transhydrogenase couples the reversible reduction of NADP+ by NADH to an inward proton translocation across the membrane. Here, we present crystal structures of the NAD(H)-binding domain I of transhydrogenase from Escherichia coli, in the absence as well as in the presence of oxidized and reduced substrate. The structures were determined at 1.9-2.0 A resolution. Overall, the structures are highly similar to the crystal structure of a previously published NAD(H)-binding domain, from Rhodospirillum rubrum transhydrogenase. However, this particular domain is unique, since it is covalently connected to the integral-membrane part of transhydrogenase. Comparative studies between the structures of the two species reveal extensively differing surface properties and point to the possible importance of a rigid peptide (PAPP) in the connecting linker for conformational coupling. Further, the kinetic analysis of a deletion mutant, from which the protruding beta-hairpin was removed, indicates that this structural element is important for catalytic activity, but not for domain I:domain III interaction or dimer formation. Taken together, these results have important implications for the enzyme mechanism of the large group of transhydrogenases, including mammalian enzymes, which contain a connecting linker between domains I and II.
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Affiliation(s)
- Tomas Johansson
- Department of Chemistry and Bioscience, Chalmers University of Technology, P.O. Box 462, SE-405 30 Göteborg, Sweden.
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23
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Bizouarn T, van Boxel GI, Bhakta T, Jackson JB. Nucleotide binding affinities of the intact proton-translocating transhydrogenase from Escherichia coli. BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 2005; 1708:404-10. [PMID: 15935988 DOI: 10.1016/j.bbabio.2005.04.004] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/18/2005] [Revised: 04/28/2005] [Accepted: 04/29/2005] [Indexed: 12/01/2022]
Abstract
Transhydrogenase (E.C. 1.6.1.1) couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane. The enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane. We have estimated dissociation constants (K(d) values) for NADPH (0.87 microM), NADP(+) (16 microM), NADH (50 microM), and NAD(+) (100-500 microM) for intact, detergent-dispersed transhydrogenase from Escherichia coli using micro-calorimetry. This is the first complete set of dissociation constants of the physiological nucleotides for any intact transhydrogenase. The K(d) values for NAD(+) and NADH are similar to those previously reported with isolated dI, but the K(d) values for NADP(+) and NADPH are much larger than those previously reported with isolated dIII. There is negative co-operativity between the binding sites of the intact, detergent-dispersed transhydrogenase when both nucleotides are reduced or both are oxidized.
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Affiliation(s)
- Tania Bizouarn
- Laboratoire de Chimie Physique, Bat 350, Université Paris XI-Orsay, 91405 Orsay, France
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24
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Sundaresan V, Chartron J, Yamaguchi M, Stout CD. Conformational diversity in NAD(H) and interacting transhydrogenase nicotinamide nucleotide binding domains. J Mol Biol 2004; 346:617-29. [PMID: 15670609 DOI: 10.1016/j.jmb.2004.11.070] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2004] [Revised: 11/17/2004] [Accepted: 11/27/2004] [Indexed: 11/26/2022]
Abstract
Transhydrogenase (TH) couples direct and stereospecific hydride transfer between NAD(H) and NADP(H), bound within soluble domains I and III, respectively, to proton translocation across membrane bound domain II. The cocrystal structure of Rhodospirillum rubrum TH domains I and III has been determined in the presence of limiting NADH, under conditions in which the subunits reach equilibrium during crystallization. The crystals contain three heterotrimeric complexes, dI(2)dIII, in the asymmetric unit. Multiple conformations of loops and side-chains, and NAD(H) cofactors, are observed in domain I pertaining to substrate/product exchange, and highlighting electrostatic interactions during the hydride transfer. Two interacting NAD(H)-NADPH pairs are observed where alternate conformations of the NAD(H) phosphodiester and conserved arginine side-chains are correlated. In addition, the stereochemistry of one NAD(H)-NADPH pair approaches that expected for nicotinamide hydride transfer reactions. The cocrystal structure exhibits non-crystallographic symmetry that implies another orientation for domain III, which could occur in dimeric TH. Superposition of the "closed" form of domain III (PDB 1PNO, chain A) onto the dI(2)dIII complex reveals a severe steric conflict of highly conserved loops in domains I and III. This overlap, and the overlap with a 2-fold related domain III, suggests that motions of loop D within domain III and of the entire domain are correlated during turnover. The results support the concept that proton pumping in TH is driven by the difference in binding affinity for oxidized and reduced nicotinamide cofactors, and in the absence of a difference in redox potential, must occur through conformational effects.
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Affiliation(s)
- Vidyasankar Sundaresan
- Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
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25
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Pedersen A, Johansson T, Rydström J, Göran Karlsson B. Titration of E. coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range. BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 2004; 1707:254-8. [PMID: 15863102 DOI: 10.1016/j.bbabio.2004.12.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/13/2004] [Revised: 12/14/2004] [Accepted: 12/15/2004] [Indexed: 10/26/2022]
Abstract
A pH-titration 2D NMR study of Escherichia coli transhydrogenase domain III with bound NADP(+) or NADPH has been carried out, in which the pH was varied between 5.4 and 12. In this analysis, individual amide protons served as reporter groups. The apparent pK(a) values of the amide protons, determined from the pH-dependent chemical shift changes, were attributed to actual pK(a) values for several titrating residues in the protein. The essential Asp392 is shown to be protonated at neutral pH in both the NADP(+) and NADPH forms of domain III, but with a marked difference in pK(a) not only attributable to the charge difference between the substrates. Titrating residues found in loop D/alpha5 point to a conformational difference of these structural elements that is redox-dependent, but not pH dependent. The observed apparent pK(a) values of these residues are discussed in relation to the crystal structure of Rhodospirillum rubrum domain III, the solution structure of E. coli domain III and the mechanism of intact proton-translocating transhydrogenase.
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Affiliation(s)
- Anders Pedersen
- Department of Chemistry, Göteborg University, P.O. Box 462, SE-405 30 Göteborg, Sweden
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26
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Broos J, Gabellieri E, van Boxel GI, Jackson JB, Strambini GB. Tryptophan phosphorescence spectroscopy reveals that a domain in the NAD(H)-binding component (dI) of transhydrogenase from Rhodospirillum rubrum has an extremely rigid and conformationally homogeneous protein core. J Biol Chem 2003; 278:47578-84. [PMID: 12972415 DOI: 10.1074/jbc.m309287200] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The characteristics of tryptophan phosphorescence from the NAD(H)-binding component (dI) component of Rhodospirillum rubrum transhydrogenase are described. This enzyme couples hydride transfer between NAD(H) and NADP(H) to proton translocation across a membrane and is only active as a dimer. Tryptophan phosphorescence spectroscopy is a sensitive technique for the detection of protein conformational changes and was used here to characterize dI under mechanistically relevant conditions. Our results indicate that the single tryptophan in dI, Trp-72, is embedded in a rigid, compact, and homogeneous protein matrix that efficiently suppresses collisional quenching processes and results in the longest triplet lifetime for Trp ever reported in a protein at ambient temperature (2.9 s). The protein matrix surrounding Trp-72 is extraordinarily rigid up to 50 degrees C. In all previous studies on Trp-containing proteins, changes in structure were reflected in a different triplet lifetime. In dI, the lifetime of Trp-72 phosphorescence was barely affected by protein dimerization, cofactor binding, complexation with the NADP(H)-binding component (dIII), or by the introduction of two amino acid substitutions at the hydride-transfer site. It is suggested that the rigidity and structural invariance of the protein domain (dI.1) housing this Trp residue are important to the mechanism of transhydrogenase: movement of dI.1 affects the width of a cleft which, in turn, regulates the positioning of bound nucleotides ready for hydride transfer. The unique protein core in dI may be a paradigm for the design of compact and stable de novo proteins.
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Affiliation(s)
- Jaap Broos
- Department of Biochemistry and Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands
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27
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Yamaguchi M, Stout CD. Essential glycine in the proton channel of Escherichia coli transhydrogenase. J Biol Chem 2003; 278:45333-9. [PMID: 12952962 DOI: 10.1074/jbc.m308236200] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III, to proton translocation by the membrane-intercalated domain II. Previous experiments have established the involvement of three conserved domain II residues in the proton pumping function of the enzyme: His91, Ser139, and Asn222, located on helices 9, 10, and 13, respectively. Eight highly conserved domain II glycines in helices 9, 10, 13, and 14 were mutated to alanine, and the mutant enzymes were assayed for hydride transfer between domains I and III and for proton translocation by domain II. One of the glycines on helix 14, Gly252, was further mutated to Cys, Ser, Thr, and Val, expression levels of the mutant enzymes were evaluated, and each was purified and assayed. The results show that Gly252 is essential for function and support a model for the proton channel composed of helices 9, 10, 13, and 14. Gly252 would allow spatial proximity of His91, Ser139, and Asn222 for proton conductance within the channel. Gly252 mutants are distinguished by high levels of cyclic transhydrogenation activity in the absence of added NADP(H) and by complete loss of proton pumping activity. The purified G252A mutant has <1% proton translocation and reverse transhydrogenation activity, retains 0.9 mol of NADP(H) per domain III, and has 96% intrinsic cyclic transhydrogenation activity, which does not exceed 100% upon the addition of NADP(H). These properties imply that Gly252 mutants exhibit a native-like domain II conformation while blocking proton translocation and coupled exchange of NADP(H) in domain III.
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Affiliation(s)
- Mutsuo Yamaguchi
- Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA
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28
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Singh A, Venning JD, Quirk PG, van Boxel GI, Rodrigues DJ, White SA, Jackson JB. 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. J Biol Chem 2003; 278:33208-16. [PMID: 12791694 DOI: 10.1074/jbc.m303061200] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
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.
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Affiliation(s)
- Avtar Singh
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
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29
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Abstract
Transhydrogenase, in animal mitochondria and bacteria, couples hydride transfer between NADH and NADP(+) to proton translocation across a membrane. Within the protein, the redox reaction occurs at some distance from the proton translocation pathway and coupling is achieved through conformational changes. In an 'open' conformation of transhydrogenase, in which substrate nucleotides bind and product nucleotides dissociate, the dihydronicotinamide and nicotinamide rings are held apart to block hydride transfer; in an 'occluded' conformation, they are moved into apposition to permit the redox chemistry. In the two monomers of transhydrogenase, there is a reciprocating, out-of-phase alternation of these conformations during turnover.
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Affiliation(s)
- J Baz Jackson
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
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30
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Pedersen A, Karlsson J, Althage M, Rydström J. Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme. BIOCHIMICA ET BIOPHYSICA ACTA 2003; 1604:55-9. [PMID: 12765762 DOI: 10.1016/s0005-2728(03)00028-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called "occluded" intermediary state of the reaction cycle of the intact enzyme. Despite a K(d) in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP(+) is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2'-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD(+) by NADH, indicating that 3-acetylpyridine-NAD(+) and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2'-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.
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Affiliation(s)
- Anders Pedersen
- Department of Biochemistry and Biophysics, Göteborg University, Box 462, 405 30, Göteborg, Sweden
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31
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Yamaguchi M, Stout CD, Hatefi Y. The proton channel of the energy-transducing nicotinamide nucleotide transhydrogenase of Escherichia coli. J Biol Chem 2002; 277:33670-5. [PMID: 12087099 DOI: 10.1074/jbc.m204170200] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple direct hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III to proton translocation by the membrane-intercalated domain II. To delineate the proton channel of the enzyme, 25 conserved and semiconserved prototropic amino acid residues of domain II of the Escherichia coli transhydrogenase were mutated, and the mutant enzymes were assayed for transhydrogenation from NADPH to an NAD analogue and for the coupled outward proton translocation. The results confirmed the previous findings of others and ourselves on the essential roles of three amino acid residues and identified another essential residue. Three of these amino acids, His-91, Ser-139, and Asn-222, occur in three separate membrane-spanning alpha helices of domain II of the beta subunit of the enzyme. Another residue, Asp-213, is probably located in a cytosolic-side loop that connects to the alpha helix bearing Asn-222. It is proposed that the three helices bearing His-91, Ser-139, and Asn-222 come together, possibly with another highly conserved alpha helix to form a four-helix bundle proton channel and that Asp-213 serves to conduct protons between the channel and domain III where NADPH binding energy is used via protein conformation change to initiate outward proton translocation.
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Affiliation(s)
- Mutsuo Yamaguchi
- Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA
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Rodrigues DJ, Jackson JB. A conformational change in the isolated NADP(H)-binding component (dIII) of transhydrogenase induced by low pH: a reflection of events during proton translocation by the complete enzyme? BIOCHIMICA ET BIOPHYSICA ACTA 2002; 1555:8-13. [PMID: 12206884 DOI: 10.1016/s0005-2728(02)00247-5] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Transhydrogenase couples the reduction of NADP(+) by NADH to inward proton translocation across the bacterial (or mitochondrial) membrane. Conformational changes in the NADP(H)-binding component of the enzyme (dIII) are central to the coupling mechanism. In the "open" state, NADP(H) bound to dIII can readily exchange with nucleotides in the solvent but hydride transfer [to/from NAD(H) bound to dI] is prevented. In the "occluded" state, bound NADP(H) cannot exchange with solvent nucleotides but the hydride transfer reaction is permitted. It was previously found that the conformational state of isolated, recombinant dIII is pH dependent. At neutral pH, the protein adopts a conformation resembling the occluded state, and at low pH, it adopts a conformation resembling the open state. The crystal structure of dIII indicates that the loop E "lid" might be largely responsible for the very high affinity of the protein for NADP(H). In this paper we show, using fluorescence resonance energy transfer, that the distance between the apex of loop E of isolated dIII, and the core of the protein, increases when the solution pH is lowered. This is consistent with the view that the lid is retracted to permit NADPH release during turnover of the complete enzyme.
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33
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Bizouarn T, Althage M, Pedersen A, Tigerström A, Karlsson J, Johansson C, Rydström J. The organization of the membrane domain and its interaction with the NADP(H)-binding site in proton-translocating transhydrogenase from E. coli. BIOCHIMICA ET BIOPHYSICA ACTA 2002; 1555:122-7. [PMID: 12206903 DOI: 10.1016/s0005-2728(02)00266-9] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Proton-translocating nicotinamide nucleotide transhydrogenase is a conformationally driven pump which catalyzes the reversibel reduction of NADP(+) by NADH. Transhydrogenases contain three domains, i.e., the hydrophilic NAD(H)-binding domain I and the NADP(H)-binding domain III, and the hydrophobic domain II containing the proton channel. Domains I and III have been separately expressed and characterized structurally by, e.g. X-ray crystallography and NMR. These domains catalyze transhydrogenation in the absence of domain II. However, due to the absence of the latter domain, the reactions catalyzed by domains I and III differ significantly from those catalyzed by the intact enzyme. Mutagenesis of residues in domain II markedly affects the activity of the intact enzyme. In order to resolve the structure-function relationships of the intact enzyme, and the molecular mechanism of proton translocation, it is therefore essential to establish the structure and function of domain II and its interactions with domains I and III. This review describes some relevant recent results in this field of research.
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34
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Johansson C, Pedersen A, Karlsson BG, Rydström J. Redox-sensitive loops D and E regulate NADP(H) binding in domain III and domain I-domain III interactions in proton-translocating Escherichia coli transhydrogenase. EUROPEAN JOURNAL OF BIOCHEMISTRY 2002; 269:4505-15. [PMID: 12230562 DOI: 10.1046/j.1432-1033.2002.03144.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Membrane-bound transhydrogenases are conformationally driven proton-pumps which couple an inward proton translocation to the reversible reduction of NADP+ by NADH (forward reaction). This reaction is stimulated by an electrochemical proton gradient, Delta p, presumably through an increased release of NADPH. The enzymes have three domains: domain II spans the membrane, while domain I and III are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively. Separately expressed domain I and III together catalyze a very slow forward reaction due to tightly bound NADP(H) in domain III. With the aim of examining the mechanistic role(s) of loop D and E in domain III and intact cysteine-free Escherichia coli transhydrogenase by cysteine mutagenesis, the conserved residues beta A398, beta S404, beta I406, beta G408, beta M409 and beta V411 in loop D, and residue beta Y431 in loop E were selected. In addition, the previously made mutants betaD392C and betaT393C in loop D, and beta G430C and beta A432C in loop E, were included. All loop D and E mutants, especially beta I406C and beta G430C, showed increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild-type enzyme. Determination of values indicated that the former increase was due to a strongly increased dissociation of NADPH caused by an altered conformation of loops D and E. In contrast, the cysteine-free G430C mutant of the intact enzyme showed the same inhibition of both forward and reverse rates. Most domain III mutants also showed a decreased affinity for domain I. The results support an important and regulatory role of loops D and E in the binding of NADP(H) as well as in the interaction between domain I and domain III.
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35
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Bottoms CA, Smith PE, Tanner JJ. A structurally conserved water molecule in Rossmann dinucleotide-binding domains. Protein Sci 2002; 11:2125-37. [PMID: 12192068 PMCID: PMC2373605 DOI: 10.1110/ps.0213502] [Citation(s) in RCA: 108] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
A computational comparison of 102 high-resolution (</=1.90 A) enzyme-dinucleotide (NAD, NADP, FAD) complexes was performed to investigate the role of solvent in dinucleotide recognition by Rossmann fold domains. The typical binding site contains about 9-12 water molecules, and about 30% of the hydrogen bonds between the protein and the dinucleotide are water mediated. Detailed inspection of the structures reveals a structurally conserved water molecule bridging dinucleotides with the well-known glycine-rich phosphate-binding loop. This water molecule displays a conserved hydrogen-bonding pattern. It forms hydrogen bonds to the dinucleotide pyrophosphate, two of the three conserved glycine residues of the phosphate-binding loop, and a residue at the C-terminus of strand four of the Rossmann fold. The conserved water molecule is also present in high-resolution structures of apo enzymes. However, the water molecule is not present in structures displaying significant deviations from the classic Rossmann fold motif, such as having nonstandard topology, containing a very short phosphate-binding loop, or having alpha-helix "A" oriented perpendicular to the beta-sheet. Thus, the conserved water molecule appears to be an inherent structural feature of the classic Rossmann dinucleotide-binding domain.
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36
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Klon AE, Héroux A, Ross LJ, Pathak V, Johnson CA, Piper JR, Borhani DW. Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution. J Mol Biol 2002; 320:677-93. [PMID: 12096917 DOI: 10.1016/s0022-2836(02)00469-2] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The crystal structures of two human dihydrofolate reductase (hDHFR) ternary complexes, each with bound NADPH cofactor and a lipophilic antifolate inhibitor, have been determined at atomic resolution. The potent inhibitors 6-([5-quinolylamino]methyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9439) and (Z)-6-(2-[2,5-dimethoxyphenyl]ethen-1-yl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9662) were developed at Southern Research Institute against Toxoplasma gondii DHFR-thymidylate synthase. The 5-deazapteridine ring of each inhibitor adopts an unusual puckered conformation that enables the formation of identical contacts in the active site. Conversely, the quinoline and dimethoxybenzene moieties exhibit distinct binding characteristics that account for the differences in inhibitory activity. In both structures, a salt-bridge is formed between Arg70 in the active site and Glu44 from a symmetry-related molecule in the crystal lattice that mimics the binding of methotrexate to DHFR.
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Affiliation(s)
- Anthony E Klon
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 35294, USA
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37
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Martinez-Cruz LA, Dreyer MK, Boisvert DC, Yokota H, Martinez-Chantar ML, Kim R, Kim SH. Crystal structure of MJ1247 protein from M. jannaschii at 2.0 A resolution infers a molecular function of 3-hexulose-6-phosphate isomerase. Structure 2002; 10:195-204. [PMID: 11839305 DOI: 10.1016/s0969-2126(02)00701-3] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The crystal structure of the hypothetical protein MJ1247 from Methanococccus jannaschii at 2 A resolution, a detailed sequence analysis, and biochemical assays infer its molecular function to be 3-hexulose-6-phosphate isomerase (PHI). In the dissimilatory ribulose monophosphate (RuMP) cycle, ribulose-5-phosphate is coupled to formaldehyde by the 3-hexulose-6-phosphate synthase (HPS), yielding hexulose-6-phosphate, which is then isomerized to fructose-6-phosphate by the enzyme 3-hexulose-6-phosphate isomerase. MJ1247 is an alpha/beta structure consisting of a five-stranded parallel beta sheet flanked on both sides by alpha helices, forming a three-layered alpha-beta-alpha sandwich. The fold represents the nucleotide binding motif of a flavodoxin type. MJ1247 is a tetramer in the crystal and in solution and each monomer has a folding similar to the isomerase domain of glucosamine-6-phosphate synthase (GlmS).
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Affiliation(s)
- Luis Alfonso Martinez-Cruz
- Physical Biosciences Division, Lawrence Berkeley National Laboratory and Department of Chemistry, University of California, Berkeley, 94720, USA.
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38
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Pinheiro TJ, Venning JD, Jackson JB. Fast hydride transfer in proton-translocating transhydrogenase revealed in a rapid mixing continuous flow device. J Biol Chem 2001; 276:44757-61. [PMID: 11577115 DOI: 10.1074/jbc.m109227200] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Transhydrogenase couples the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Coupling is achieved through changes in protein conformation. Upon mixing, the isolated nucleotide-binding components of transhydrogenase (dI, which binds NAD(H), and dIII, which binds NADP(H)) form a catalytic dI(2).dIII(1) complex, the structure of which was recently solved by x-ray crystallography. The fluorescence from an engineered Trp in dIII changes when bound NADP(+) is reduced. Using a continuous flow device, we have measured the Trp fluorescence change when dI(2).dIII(1) complexes catalyze reduction of NADP(+) by NADH on a sub-millisecond scale. At elevated NADH concentrations, the first-order rate constant of the reaction approaches 21,200 s(-1), which is larger than that measured for redox reactions of nicotinamide nucleotides in other, soluble enzymes. Rather high concentrations of NADH are required to saturate the reaction. The deuterium isotope effect is small. Comparison with the rate of the reverse reaction (oxidation of NADPH by NAD(+)) reveals that the equilibrium constant for the redox reaction on the complex is >36. This high value might be important in ensuring high turnover rates in the intact enzyme.
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Affiliation(s)
- T J Pinheiro
- Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom
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Venning JD, Rodrigues DJ, Weston CJ, Cotton NP, Quirk PG, Errington N, Finet S, White SA, Jackson JB. The heterotrimer of the membrane-peripheral components of transhydrogenase and the alternating-site mechanism of proton translocation. J Biol Chem 2001; 276:30678-85. [PMID: 11399770 DOI: 10.1074/jbc.m104429200] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
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 nucleotide-binding 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.
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Affiliation(s)
- J D Venning
- School of Biosciences, University of Birmingham, Edgbaston, United Kingdom
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40
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Bragg PD, Hou C. Characterization of mutants of beta histidine91, beta aspartate213, and beta asparagine222, possible components of the energy transduction pathway of the proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli. Arch Biochem Biophys 2001; 388:299-307. [PMID: 11368169 DOI: 10.1006/abbi.2001.2298] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The roles of three residues (betaHis91, betaAsp213, and betaAsn222) implicated in energy transduction in the membrane-spanning domain II of the proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli have been examined using site-directed mutagenesis. All mutations affected transhydrogenation and proton pumping activities, although to various extents. Replacing betaHis91 or betaAsn222 of domain II by the basic residues lysine or arginine resulted in occlusion of NADP(H) at the NADP(H)-binding site of domain III. This was not seen with betaD213K or betaD213R mutants. It is suggested that betaHis91 and betaAsn222 interact with betaAsp392, a residue probably involved in initiating conformational changes at the NADP(H)-binding site in the normal catalytic cycle of the enzyme (M. Jeeves et al. (2000) Biochim. Biophys. Acta 1459, 248-257). The introduced positive charges in the betaHis91 and betaAsn222 mutants might stabilize the carboxyl group of betaAsp392 in its anionic form, thus locking the NADP(H)-binding site in the occluded conformation. In comparison with the nonmutant enzyme, and those of mutants of betaAsp213, most mutant enzymes at betaHis91 and betaAsn222 bound NADP(H) more slowly at the NADP(H)-binding site. This is consistent with the effect of these two residues on the binding site. We could not demonstrate by mutation or crosslinking or through the formation of eximers with pyrene maleimide that betaHis91 and betaAsn222 were in proximity in domain II.
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Affiliation(s)
- P D Bragg
- Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
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41
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Abstract
The SIR2 protein family comprises a novel class of nicotinamide-adenine dinucleotide (NAD)-dependent protein deacetylases that function in transcriptional silencing, DNA repair, and life-span extension in Saccharomyces cerevisiae. Two crystal structures of a SIR2 homolog from Archaeoglobus fulgidus complexed with NAD have been determined at 2.1 A and 2.4 A resolutions. The structures reveal that the protein consists of a large domain having a Rossmann fold and a small domain containing a three-stranded zinc ribbon motif. NAD is bound in a pocket between the two domains. A distinct mode of NAD binding and an unusual configuration of the zinc ribbon motif are observed. The structures also provide important insights into the catalytic mechanism of NAD-dependent protein deacetylation by this family of enzymes.
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Affiliation(s)
- J Min
- W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
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42
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Rodrigues DJ, Venning JD, Quirk PG, Jackson JB. A change in ionization of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase regulates both hydride transfer and nucleotide release. EUROPEAN JOURNAL OF BIOCHEMISTRY 2001; 268:1430-8. [PMID: 11231296 DOI: 10.1046/j.1432-1327.2001.02008.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Transhydrogenase couples the transfer of hydride-ion equivalents between NAD(H) and NADP(H) to proton translocation across a membrane. The enzyme has three components: dI binds NAD(H), dIII binds NADP(H) and dII spans the membrane. Coupling between transhydrogenation and proton translocation involves changes in the binding of NADP(H). Mixtures of isolated dI and dIII from Rhodospirillum rubrum transhydrogenase catalyse a rapid, single-turnover burst of hydride transfer between bound nucleotides; subsequent turnover is limited by NADP(H) release. Stopped-flow experiments showed that the rate of the hydride transfer step is decreased at low pH. Single Trp residues were introduced into dIII by site-directed mutagenesis. Two mutants with similar catalytic properties to those of the wild-type protein were selected for a study of nucleotide release. The way in which Trp fluorescence was affected by nucleotide occupancy of dIII was different in the two mutants, and hence two different procedures for determining the rate of nucleotide release were developed. The apparent first-order rate constants for NADP(+) release and NADPH release from isolated dIII increased dramatically at low pH. It is concluded that a single ionisable group in dIII controls both the rate of hydride transfer and the rate of nucleotide release. The properties of the protonated and unprotonated forms of dIII are consistent with those expected of intermediates in the NADP(H)-binding-change mechanism. The ionisable group might be a component of the proton-translocation pathway in the complete enzyme.
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Affiliation(s)
- D J Rodrigues
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK
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43
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Cotton NP, White SA, Peake SJ, McSweeney S, Jackson JB. The crystal structure of an asymmetric complex of the two nucleotide binding components of proton-translocating transhydrogenase. Structure 2001; 9:165-76. [PMID: 11250201 DOI: 10.1016/s0969-2126(01)00571-8] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Membrane-bound ion translocators have important functions in biology, but their mechanisms of action are often poorly understood. Transhydrogenase, found in animal mitochondria and bacteria, links the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Linkage is achieved through changes in protein conformation at the nucleotide binding sites. The redox reaction takes place between two protein components located on the membrane surface: dI, which binds NAD(H), and dIII, which binds NADP(H). A third component, dII, provides a proton channel through the membrane. Intact membrane-located transhydrogenase is probably a dimer (two copies each of dI, dII, and dIII). RESULTS We have solved the high-resolution crystal structure of a dI:dIII complex of transhydrogenase from Rhodospirillum rubrum-the first from a transhydrogenase of any species. It is a heterotrimer, having two polypeptides of dI and one of dIII. The dI polypeptides fold into a dimer. The loop on dIII, which binds the nicotinamide ring of NADP(H), is inserted into the NAD(H) binding cleft of one of the dI polypeptides. The cleft of the other dI is not occupied by a corresponding dIII component. CONCLUSIONS The redox step in the transhydrogenase reaction is readily visualized; the NC4 atoms of the nicotinamide rings of the bound nucleotides are brought together to facilitate direct hydride transfer with A-B stereochemistry. The asymmetry of the dI:dIII complex suggests that in the intact enzyme there is an alternation of conformation at the catalytic sites associated with changes in nucleotide binding during proton translocation.
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Affiliation(s)
- N P Cotton
- School of Biosciences, University of Birmingham, Edgbaston, B15 2TT, Birmingham, United Kingdom
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44
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Hou C, Bragg PD. Intersubunit crosslinking of the heterotetrameric proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli defines intersubunit contacts between transmembrane helices of the beta subunits. Biochem Biophys Res Commun 2001; 280:466-70. [PMID: 11162540 DOI: 10.1006/bbrc.2000.4142] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta, organized as an alpha(2)beta(2) tetramer. The protein contains three recognizable domains, of which domain II is the transmembrane region of the molecule containing the pathway for proton translocation. Domain II is composed of four transmembrane helices at the carboxyl-terminus of the alpha subunit and nine transmembrane helices at the amino-terminal region of the beta subunit. We have introduced pairs of cysteine residues into all of the loops connecting the transmembrane helices of domain II of the beta subunit. Crosslinking between the two beta subunits of the tetramer was induced spontaneously, or by treatment with cupric 1,10-phenanthrolinate or o-phenylenedimaleimide. Crosslinks between pairs of betaA114C, betaS183C, and betaA262C residues were observed, suggesting that pairs of domain II transmembrane helices 11, 12, and 14 were in proximity. These results, together with previous data (Bragg and Hou (2000) Biochem. Biophys. Res. Commun. 273, 955-959) suggest that the transhydrogenase tetramer is formed by apposition of alpha(2) and beta(2) dimers. Crosslinking between pairs of cysteine residues in the same beta subunit was not observed, possibly because the interhelical loops of the domain II region of the beta subunit were too short to allow correct orientation of the sulfhydryl groups for crosslinking.
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Affiliation(s)
- C Hou
- Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3
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45
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Abstract
We have expressed and purified a protein fragment from Entamoeba histolytica. It catalyses transhydrogenation between analogues of NAD(H) and NADP(H). The characteristics of this reaction resemble those of the reaction catalysed by a complex of the NAD(H)- and NADP(H)-binding subunits of proton-translocating transhydrogenases from bacteria and mammals. It is concluded that the complete En. histolytica protein, which, along with similar proteins from other protozoan parasites, has an unusual subunit organisation, is also a proton-translocating transhydrogenase. The function of the transhydrogenase, thought to be located in organelles which do not have the enzymes of oxidative phosphorylation, is not clear.
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Affiliation(s)
- C J Weston
- School of Biosciences, University of Birmingham, P.O. Box 363, Edgbaston, B15 2TT, Birmingham, UK
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46
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Jeeves M, Smith KJ, Quirk PG, Cotton NP, Jackson JB. Solution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from Rhodospirillum rubrum. BIOCHIMICA ET BIOPHYSICA ACTA 2000; 1459:248-57. [PMID: 11004437 DOI: 10.1016/s0005-2728(00)00159-6] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Abstract
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.
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Affiliation(s)
- M Jeeves
- School of Biosciences, University of Birmingham, Edgbaston, B15 2TT, UK
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47
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Bizouarn T, Meuller J, Axelsson M, Rydström J. The transmembrane domain and the proton channel in proton-pumping transhydrogenases. BIOCHIMICA ET BIOPHYSICA ACTA 2000; 1459:284-90. [PMID: 11004441 DOI: 10.1016/s0005-2728(00)00163-8] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
Proton-pumping nicotinamide nucleotide transhydrogenases are composed of three main domains, the NAD(H)-binding and NADP(H)-binding hydrophilic domains I (dI) and III (dIII), respectively, and the hydrophobic domain II (dII) containing the assumed proton channel. dII in the Escherichia coli enzyme has recently been characterised with regard to topology and a packing model of the helix bundle in dII is proposed. Extensive mutagenesis of conserved charged residues of this domain showed that important residues are betaHis91 and betaAsn222. The pH dependence of betaH91D, as well as betaH91C (unpublished), when compared to that of wild type shows that reduction of 3-acetylpyridine-NAD(+) by NADPH, i.e., the reverse reaction, is optimal at a pH essentially coinciding with the pK(a) of the residue in the beta91 position. It is therefore concluded that the wild-type transhydrogenase is regulated by the degree of protonation of betaHis91. The mechanisms of the interactions between dI+dIII and dII are suggested to involve pronounced conformational changes in a 'hinge' region around betaR265.
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Affiliation(s)
- T Bizouarn
- Department of Biochemistry and Biophysics, Göteborg University, Sweden
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Buckley PA, Baz Jackson J, Schneider T, White SA, Rice DW, Baker PJ. Protein-protein recognition, hydride transfer and proton pumping in the transhydrogenase complex. Structure 2000; 8:809-15. [PMID: 10997900 DOI: 10.1016/s0969-2126(00)00171-4] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
BACKGROUND Membrane-bound ion pumps are involved in metabolic regulation, osmoregulation, cell signalling, nerve transmission and energy transduction. How the ion electrochemical gradient interacts with the scalar chemistry and how the catalytic machinery is gated to ensure high coupling efficiency are fundamental to the mechanism of action of such pumps. Transhydrogenase is a conformationally coupled proton pump linking a proton gradient to the redox reaction between NAD(H) and NADP(H). The enzyme has three components; dI binds NAD(H), dII spans the membrane and dIII binds NADP(H). RESULTS The first crystal structure of a transhydrogenase dI component (from Rhodospirillum rubrum) has been determined at 2.0 A resolution. The monomer comprises two domains. Both are involved in dimer formation, and one has a Rossmann fold that binds NAD+ in a novel mode. The two domains can adopt different conformations. In the most closed conformation, the nicotinamide ring is expelled from the cleft between the two domains and is exposed on the outside of the protein. In this conformation it is possible to dock the structure of dI/NAD+ with that of a dIII/NADP+ complex to provide the first insights into the molecular basis of the hydride-transfer step. CONCLUSIONS Analysis of the model of the dI/dIII complex identifies residues potentially involved in dI/dIII interaction and shows how domain motion in dI results in a shift in position of the nicotinamide ring of NAD+. We propose that this movement is responsible for switching between the forbidden and allowed states for hydride transfer during proton pumping.
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Affiliation(s)
- P A Buckley
- Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, UK
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Bragg PD, Hou C. The presence of an aqueous cavity in the proton-pumping pathway of the pyridine nucleotide transhydrogenase of Escherichia coli is suggested by the reaction of the enzyme with sulfhydryl inhibitors. Arch Biochem Biophys 2000; 380:141-50. [PMID: 10900143 DOI: 10.1006/abbi.2000.1923] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The pyridine nucleotide transhydrogenase of Escherichia coli carries out transmembrane proton translocation coupled to transfer of a hydride ion equivalent between NAD(+) and NADP(+). The membrane domain (domain II) of the enzyme is composed of 13 transmembrane helices. Previous studies (N. A. Glavas et al., Biochemistry 34, 7694-7702, 1995) have suggested that betaHis91 in transmembrane helix 9 is involved in the translocation pathway of protons across the membrane. In this study we have replaced amino acid residues on the same face of helix 9 as betaHis91 by single cysteine residues. We then examined the effect of the sulfhydryl inhibitors N-ethylmaleimide (NEM) and p-chloromercuriphenylsulfonate (pCMPS) on enzyme activity and, in the case of [(14)C]NEM, as an enzyme label. The pattern of enzyme inhibition and labelling is consistent with the presence of an aqueous cavity through domain II from the cytosolic surface to the region of betaHis91. Residue betaAsn222 in helix 13, which appears also to be involved in the proton pathway across domain II, may interface with this aqueous cavity. A further series of mutants of betaGlu124 on helix 10 confirms the proposal (P. D. Bragg and C. Hou, Arch. Biochem. Biophys. 363, 182-190, 1999) that this residue is involved in passive permeation of protons across domain II.
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Affiliation(s)
- P D Bragg
- Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3, Canada.
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Bragg PD, Hou C. Crosslinking between alpha and beta subunits defines the orientation and spatial relationship of some of the transmembrane helices of the proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli. Biochem Biophys Res Commun 2000; 273:955-9. [PMID: 10891354 DOI: 10.1006/bbrc.2000.3037] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta, organized as an alpha(2)beta(2) tetramer. The protein contains three recognizable domains, of which domain II is the transmembrane region of the molecule containing the pathway for proton translocation. Domain II is composed of four transmembrane helices at the carboxyl-terminus of the alpha subunit and either eight or nine transmembrane helices at the amino-terminal region of the beta subunit. We have introduced pairs of cysteine residues into a cysteine-free transhydrogenase by site-directed mutagenesis. Disulfide bond formation between some of these cysteine residues occurred spontaneously or on treatment with cupric 1, 10-phenanthrolinate. Analysis of crosslinked products confirmed that there are nine transmembrane helices in the domain II region of the beta subunit. The proximity to one another of several of the transmembrane helices was determined. Thus, helices 2 and 4 are close to helix 6 (nomenclature of Meuller and Rydström, J. Biol. Chem. 274, 19072-19080, 1999), and helix 3 and the carboxyl-terminal eight residues of the alpha subunit are close to helix 7. In the alpha(2)beta(2) tetramer, helices 2 and 4 of one alpha subunit are close to the same pair of transmembrane helices of the other alpha subunit, and helix 6 of one beta subunit is close to helix 6 of the other beta subunit.
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Affiliation(s)
- P D Bragg
- Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3, Canada.
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