Using rational screening and electron microscopy to optimize the crystallization of succinate:ubiquinone oxidoreductase from<i>Escherichia coli</i>

Horsefield, Rob; Yankovskaya, Victoria; Törnroth, Susanna; Luna-Chavez, C.; Stambouli, Elizabeth; Barber, James; Byrne, Bernadette; Cecchini, Gary; Iwata, So

doi:10.1107/s0907444903002075

Cited by 12 publications

(15 citation statements)

References 10 publications

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“…Isolation of the membrane fractions enriched with wild type and mutant SQR and QFR enzymes was as previously described (28,29). Protein extraction with the detergent Thesit (Anapoe C 12 E 9 (polyoxyethylene 9-dodecyl ether; Anatrace Inc., Maumee, OH) and purification on Q-Sepharose fast flow chromatography were carried out as previously described for wild type and mutant QFR and SQR (28,29).…”

Section: Enzyme Purificationmentioning

confidence: 99%

“…Protein extraction with the detergent Thesit (Anapoe C 12 E 9 (polyoxyethylene 9-dodecyl ether; Anatrace Inc., Maumee, OH) and purification on Q-Sepharose fast flow chromatography were carried out as previously described for wild type and mutant QFR and SQR (28,29). Purified enzyme fractions were pooled, concentrated under nitrogen using an Amicon cell and YM30 membrane, and stored at Ϫ80°C.…”

Section: Enzyme Purificationmentioning

confidence: 99%

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Fumarate Reductase and Succinate Oxidase Activity of Escherichia coli Complex II Homologs Are Perturbed Differently by Mutation of the Flavin Binding Domain

Maklashina

Iverson

Sher

et al. 2006

Journal of Biological Chemistry

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The Escherichia coli complex II homologues succinate:ubiquinone oxidoreductase (SQR, SdhCDAB) and menaquinol:fumarate oxidoreductase (QFR, FrdABCD) have remarkable structural homology at their dicarboxylate binding sites. Although both SQR and QFR can catalyze the interconversion of fumarate and succinate, QFR is a much better fumarate reductase, and SQR is a better succinate oxidase. An exception to the conservation of amino acids near the dicarboxylate binding sites of the two enzymes is that there is a Glu (FrdA Glu-49) near the covalently bound FAD cofactor in most QFRs, which is replaced with a Gln (SdhA Gln-50) in SQRs. The role of the amino acid side chain in enzymes with Glu/Gln/Ala substitutions at FrdA Glu-49 and SdhA Gln-50 has been investigated in this study. The data demonstrate that the mutant enzymes with Ala substitutions in either QFR or SQR remain functionally similar to their wild type counterparts. There were, however, dramatic changes in the catalytic properties when Glu and Gln were exchanged for each other in QFR and SQR. The data show that QFR and SQR enzymes are more efficient succinate oxidases when Gln is in the target position and a better fumarate reductase when Glu is present. Overall, structural and catalytic analyses of the FrdA E49Q and SdhA Q50E mutants suggest that coulombic effects and the electronic state of the FAD are critical in dictating the preferred directionality of the succinate/fumarate interconversions catalyzed by the complex II superfamily. . The membrane-extrinsic domain is bound to the membrane through interactions with the hydrophobic subunits of the complex. These subunits comprise two membrane anchor polypeptides, each containing three transmembrane helices and providing a binding site(s) for quinone (for reviews, see Refs. 5-8). In addition, the E. coli SQR hydrophobic peptides bind one b-type heme, whereas the E. coli QFR lacks heme.Comparison of the structures of complex II is possible due to the availability of x-ray crystallographic structures for both SQR and QFR of E. coli (9, 10), the porcine SQR (11), the QFR from Wolinella succinogenes (12), and soluble homologs of the flavoprotein subunit that function as periplasmically localized fumarate reductases (13-16). The flavoprotein subunits from the E. coli SQR and QFR are highly homologous, with 64% similarity and 44% identity of amino acid residues (3). The sequence similarity within the SQR/QFR superfamily is reflected in structural alignments of members whose structures are known (17)(18)(19). Within this group, the backbone C ␣ atoms can be superimposed with a maximal root mean square deviation of 1.5 Å (10). A detailed hydride transfer mechanism for fumarate reduction has been proposed based on structural data and enzyme assays of wild type and mutant enzymes (15, 18 -20). QFRs and SQRs all contain covalently bound FAD and are bidirectional (i.e. they will catalyze both succinate oxidation and fumarate reduction). There are, however, significant differences for the kinetics of fumarate reduction by...

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Section: Enzyme Purificationmentioning

confidence: 99%

Section: Enzyme Purificationmentioning

confidence: 99%

Fumarate Reductase and Succinate Oxidase Activity of Escherichia coli Complex II Homologs Are Perturbed Differently by Mutation of the Flavin Binding Domain

Maklashina

Iverson

Sher

et al. 2006

Journal of Biological Chemistry

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“…All data better than Ϫ2.0(I) were used for scaling. The crystals belong to the trigonal space group R32 with cell dimensions of a ϭ b ϭ 138.75 Å, c ϭ 521.87 Å and one molecule per asymmetric unit, very similar to those previously obtained (27). Phase information was obtained from the native SQR structure (Protein Data Bank (PDB) 1NEK).…”

Section: Methodsmentioning

confidence: 99%

“…Expression, Purification, and Crystallization-These methods are as previously described (26,27) with only minor modifications to the sample preparation following purification to introduce the inhibitor. Protein sample, containing ϳ60 mg ml Ϫ1 of purified SQR in Buffer C (20 mM Tris-HCl, 0.05% Thesit, pH 7.6) was diluted 1:10 with Buffer C plus 50 M Atpenin A5 (AA5) and incubated at 4°C for 14 h. Excess inhibitor was washed out by diluting (1:10) and re-concentrating the sample with Buffer C three times.…”

Section: Methodsmentioning

confidence: 99%

“…Protein sample, containing ϳ60 mg ml Ϫ1 of purified SQR in Buffer C (20 mM Tris-HCl, 0.05% Thesit, pH 7.6) was diluted 1:10 with Buffer C plus 50 M Atpenin A5 (AA5) and incubated at 4°C for 14 h. Excess inhibitor was washed out by diluting (1:10) and re-concentrating the sample with Buffer C three times. The enzyme was then re-concentrated to ϳ60 mg ml Ϫ1 and used to set up crystallization trials by the hanging-drop vapordiffusion method as previously described (27). Data Collection and Structure Determination-Crystals were frozen within 72 h of a trial set up for x-ray data collection.…”

Section: Methodsmentioning

confidence: 99%

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Structural and Computational Analysis of the Quinone-binding Site of Complex II (Succinate-Ubiquinone Oxidoreductase)

Horsefield

Yankovskaya

Sexton

et al. 2006

Journal of Biological Chemistry

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245

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The transfer of electrons and protons between membranebound respiratory complexes is facilitated by lipid-soluble redox-active quinone molecules (Q). This work presents a structural analysis of the quinone-binding site (Q-site) identified in succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli. SQR, often referred to as Complex II or succinate dehydrogenase, is a functional member of the Krebs cycle and the aerobic respiratory chain and couples the oxidation of succinate to fumarate with the reduction of quinone to quinol (QH 2 ). The interaction between ubiquinone and the Q-site of the protein appears to be mediated solely by hydrogen bonding between the O1 carbonyl group of the quinone and the side chain of a conserved tyrosine residue. In this work, SQR was co-crystallized with the ubiquinone binding-site inhibitor Atpenin A5 (AA5) to confirm the binding position of the inhibitor and reveal additional structural details of the Q-site. The electron density for AA5 was located within the same hydrophobic pocket as ubiquinone at, however, a different position within the pocket. AA5 was bound deeper into the site prompting further assessment using proteinligand docking experiments in silico. The initial interpretation of the Q-site was re-evaluated in the light of the new SQR-AA5 structure and protein-ligand docking data. Two binding positions, the Q 1 -site and Q 2 -site, are proposed for the E. coli SQR quinone-binding site to explain these data. At the Q 2 -site, the side chains of a serine and histidine residue are suitably positioned to provide hydrogen bonding partners to the O4 carbonyl and methoxy groups of ubiquinone, respectively. This allows us to propose a mechanism for the reduction of ubiquinone during the catalytic turnover of the enzyme.The Complex II family is comprised of two homologous integral membrane proteins (1-4). Succinate:quinone oxidoreductase (SQR), 4 or succinate dehydrogenase (SDH), is a functional member of the Krebs cycle and the aerobic respiratory chain coupling the oxidation of succinate to fumarate with the reduction of quinone (Q) to quinol (QH 2 ). Quinol:fumarate oxidoreductase (QFR), or fumarate reductase, catalyzes the reverse reaction to SQR during anaerobic respiration. Both enzymes have a similar subunit and cofactor composition (1-4). The hydrophilic subunits, composed of a flavoprotein (SdhA) and an ironsulfur protein (SdhB), have a high degree of sequence homology across species. The SdhAB catalytic subunits are anchored to the membrane by the hydrophobic SdhCD subunits, which in the case of mammalian and Escherichia coli Complex II form a cytochrome b that contains one heme b. The two-electron oxidation of succinate at the substrate binding site in the SdhA subunit is coupled to the two-electron reduction of quinone in the membrane domain of Complex II. Electrons are sequentially transferred to ubiquinone (in the case of mammalian and E. coli Quinones can undergo 2e Ϫ /2H ϩ oxidation-reduction reactions, an essential feature of respiration, allowing the t...

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Expression, purification, and crystallisationof membrane proteins

Byrne¹

2007

Evolving Methods for Macromolecular Crystallography

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Using rational screening and electron microscopy to optimize the crystallization of succinate:ubiquinone oxidoreductase fromEscherichia coli

Cited by 12 publications

References 10 publications

Fumarate Reductase and Succinate Oxidase Activity of Escherichia coli Complex II Homologs Are Perturbed Differently by Mutation of the Flavin Binding Domain

Fumarate Reductase and Succinate Oxidase Activity of Escherichia coli Complex II Homologs Are Perturbed Differently by Mutation of the Flavin Binding Domain

Structural and Computational Analysis of the Quinone-binding Site of Complex II (Succinate-Ubiquinone Oxidoreductase)

Expression, purification, and crystallisationof membrane proteins

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