Crystal structures of reduced and oxidized DsbA: investigation of domain motion and thiolate stabilization

Guddat, Luke W.; Bardwell, James C.A.; Martin, Jennifer L.

doi:10.1016/s0969-2126(98)00077-x

Cited by 155 publications

(170 citation statements)

References 55 publications

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“…Nevertheless, the fact that about 70% of all variants of DsbA obtained after complete randomization of six residues from the active-site helix ␣1 retained native tertiary structure and biological activity is remarkable and unexpected. This is because helix ␣1 is a well conserved element of regular secondary structure in the DsbA family, and because the dipole of helix ␣1 and hydrogen bonds within the 3 10 -helical, N-terminal part of ␣1 are assumed to cause the low pK a value of Cys 30 (7). The low pK a of Cys 30 is indeed considered the key factor underlying the enormous oxidative force of DsbA and the extreme reactivity of its activesite disulfide bond (14,24).…”

Section: Discussionmentioning

confidence: 99%

“…The three-dimensional structure of both oxidized and reduced DsbA has been solved by x-ray crystallography and NMR spectroscopy (5)(6)(7). The enzyme possesses a thioredoxin fold, which is common to most thiol-disulfide oxidoreductases and contains the active-site disulfide (8).…”

mentioning

confidence: 99%

“…In addition to the thioredoxin domain, DsbA possesses a second, purely ␣-helical domain, which is inserted into the thioredoxin domain (5). The structural differences between oxidized and reduced DsbA are locally restricted to the active-site region, and there is no major domain motion related to the redox state of DsbA (6,7).…”

mentioning

confidence: 99%

“…In addition, His 32 presumably contributes about one pK a unit to the lowered pK a of Cys 30 , possibly by formation of a hydrogen bond between the Cys 30 thiolate and N␦1 (7, 16 -18). In addition, possible hydrogen bonds with the thiol of Cys 33 , the amide hydrogen of His 32 , and the amide hydrogen of Cys 33 further stabilize the Cys 30 thiolate (7).…”

mentioning

confidence: 99%

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Randomization of the Entire Active-site Helix α1 of the Thiol-disulfide Oxidoreductase DsbA from Escherichia coli

Philipps¹,

Glockshuber²

2002

Journal of Biological Chemistry

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DsbA from Escherichia coli is the most oxidizing member of the thiol-disulfide oxidoreductase family (E o ‫؍‬ ؊122 mV) and is required for efficient disulfide bond formation in the periplasm. The reactivity of the catalytic disulfide bond (Cys 30 -Pro 31 -His 32 -Cys 33 ) is primarily due to an extremely low pK a value (3.4) of Cys 30 , which is stabilized by the partial positive dipole charge of the active-site helix ␣1 (residues 30 -37). We have randomized all non-cysteine residues of helix ␣1 (residues 31, 32, and 34 -37) and found that two-thirds of the resulting variants complement DsbA deficiency in a dsbA deletion strain. Sequencing of 98 variants revealed a large number of non-conservative replacements in active variants, even at well conserved positions. This indicates that tertiary structure context strongly determines ␣-helical secondary structure formation of the randomized sequence. A subset of active and inactive variants was further characterized. All these variants were more reducing than wild type DsbA, but the redox potentials of active variants did not drop below ؊210 mV. All inactive variants had redox potentials lower than ؊210 mV, although some of the inactive proteins were still re-oxidized by DsbB. This demonstrates that efficient oxidation of substrate polypeptides is the crucial property of DsbA in vivo.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Randomization of the Entire Active-site Helix α1 of the Thiol-disulfide Oxidoreductase DsbA from Escherichia coli

Philipps¹,

Glockshuber²

2002

Journal of Biological Chemistry

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“…These hydrogen bond donors are the N␦ atom of His-32, the backbone nitrogen of Cys-33, the S␥ of Cys-33, and the carbonyl oxygen of Val-150, respectively. No destabilizing effects have been described for reduced DsbA (64). The active-site interactions of DsbA thus serve to stabilize its reduced state, resulting in a relatively high midpoint redox potential.…”

Section: High-resolution Structures Of Oxidized and Reduced Resa-mentioning

confidence: 99%

Structural Basis of Redox-coupled Protein Substrate Selection by the Cytochrome c Biosynthesis Protein ResA

Crow

Acheson

Brun

et al. 2004

Journal of Biological Chemistry

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Post-translational maturation of cytochromes c involves the covalent attachment of heme to the Cys-XxxXxx-Cys-His motif of the apo-cytochrome. For this process, the two cysteines of the motif must be in the reduced state. In bacteria, this is achieved by dedicated, membrane-bound thiol-disulfide oxidoreductases with a high reducing power, which are essential components of cytochrome c maturation systems and are also linked to cellular disulfide-bond formation machineries. Here we report high-resolution structures of oxidized and reduced states of a soluble, functional domain of one such oxidoreductase, ResA, from Bacillus subtilis. The structures elucidate the structural basis of the protein's high reducing power and reveal the largest redox-coupled conformational changes observed to date in any thioredoxin-like protein. These redox-coupled changes alter the protein surface and illustrate how the redox state of ResA predetermines to which substrate it binds. Furthermore, a polar cavity, present only in the reduced state, may confer specificity to recognize apo-cytochrome c. The described features of ResA are likely to be general for bacterial cytochrome c maturation systems.

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Fluorescence quenching in the DsbA protein fromEscherichia coli: Complete picture of the excited-state energy pathway and evidence for the reshuffling dynamics of the microstates of tryptophan

Sillen

Hennecke²,

Roethlisberger³

et al. 1999

Proteins

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The disulfide oxidoreductase DsbA is a strong oxidant of protein thiols and is required for efficient disulfide bond formation in the bacterial periplasm. DsbA contains two tryptophans: W76 and W126. The fluorescence of W76 changes upon reduction of the disulfide bridge, as analyzed previously (Hennecke et al., Biochemistry 1997;36:6391-6400). The fluorescence of W126 is highly quenched. The only two potential side chain quenchers are Q74 and N127, and these were replaced by alanine, resulting in a threefold increase in fluorescence intensity. The fluorescence intensity increase is not due to the removal of dynamic quenchers but to an increase in the population with the longest lifetime. In this report, the possibility of a change in the conformation of W126 is investigated theoretically by using molecular mechanics and dynamic simulations and experimentally by using a reaction with N-bromosuccinimide. This reacts preferably with the most exposed microstate of tryptophan, which is responsible for the longest lifetime. The simulations and the experimental results reveal that the amino acid replacements allow W126 to increase the population of its antiperpendicular conformation. The selectivity of the N-bromosuccinimide reaction allows the visualization of the reshuffling kinetics at exhausting reagent concentration. To the authors' knowledge, this is the first time that the kinetics of Trp population reshuffling have been measured.

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Crystal structures of reduced and oxidized DsbA: investigation of domain motion and thiolate stabilization

Cited by 155 publications

References 55 publications

Randomization of the Entire Active-site Helix α1 of the Thiol-disulfide Oxidoreductase DsbA from Escherichia coli

Randomization of the Entire Active-site Helix α1 of the Thiol-disulfide Oxidoreductase DsbA from Escherichia coli

Structural Basis of Redox-coupled Protein Substrate Selection by the Cytochrome c Biosynthesis Protein ResA

Fluorescence quenching in the DsbA protein fromEscherichia coli: Complete picture of the excited-state energy pathway and evidence for the reshuffling dynamics of the microstates of tryptophan

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