Crystal structures of reduced, oxidized, and mutated human thioredoxins: evidence for a regulatory homodimer

Weichsel, A.; Gasdaska, John R.; Powis, Garth; Montfort, W.R.

doi:10.1016/s0969-2126(96)00079-2

Cited by 362 publications

(391 citation statements)

References 71 publications

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“…12 C73S mutant thioredoxin yields the same dimer interface as the wild type protein but without disulfide linkage. 12,18 The C69S/C73S double mutant described herein displays this same dimer interface despite crystallizing in two new crystal forms and, for the oxidized protein, being disulfide linked through Cys 62. As noted above, the interface in the oxidized crystals is through crystallographic symmetry, giving rise to a repeating unit that contains four thioredoxin chains [ Fig.…”

Section: Homodimer Interfacementioning

confidence: 79%

“…Both crystal structures were determined by molecular replacement using the program MOLREP in the CCP4 program suit, 33 and wild type thioredoxin as a starting model (PDB entry 1ERT). 18 Model adjustments were made with COOT 34 and refined with REFMAC5. 33 Anisotropic temperature factors and hydrogen atoms in their riding positions were used in the refinement of both structures.…”

Section: Crystal Structure Determinationsmentioning

confidence: 99%

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Crystal structure of human thioredoxin revealing an unraveled helix and exposed S‐nitrosation site

Weichsel¹,

Kem²,

Montfort³

2010

Protein Science

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Thioredoxins reduce disulfide bonds and other thiol modifications in all cells using a CXXC motif. Human thioredoxin 1 is unusual in that it codes for an additional three cysteines in its 105 amino acid sequence, each of which have been implicated in other reductive activities. Cys 62 and Cys 69 are buried in the protein interior and lie at either end of a short helix (helix 3), and yet can disulfide link under oxidizing conditions. Cys 62 is readily S-nitrosated, giving rise to a SNO modification, which is also buried. Here, we present two crystal structures of the C69S/C73S mutant protein under oxidizing (1.5 Å ) and reducing (1.1 Å ) conditions. In the oxidized structure, helix 3 is unraveled and displays a new conformation that is stabilized by a series of new hydrogen bonds and a disulfide link with Cys 62 in a neighboring molecule. The new conformation provides an explanation for how a completely buried residue can participate in SNO exchange reactions.

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Section: Homodimer Interfacementioning

confidence: 79%

Section: Crystal Structure Determinationsmentioning

confidence: 99%

Crystal structure of human thioredoxin revealing an unraveled helix and exposed S‐nitrosation site

Weichsel¹,

Kem²,

Montfort³

2010

Protein Science

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“…Comparing the structures of reduced wild-type human thioredoxin with that of the C32S/C3SS double mutant shows that positions of the oxygen atoms in the mutant are essentially the same as the sulfur atoms (to redoxin (Weichsel et al, 1996). This makes it likely that in the reaction with DTNB, the colinearity of the three sulfurs involved in thiol-disulfide interchange would be more easily maintained with the more exposed Cys3' than with Cys3'.…”

Section: Thiol Reactivities and Properties Of C135s-c3ss And Cl38s-c35smentioning

confidence: 99%

Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin: Evidence that cysteine‐138 functions to initiate dithiol‐disulfide interchange and to accept the reducing equivalent from reduced flavin

et al. 1998

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Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with CysI3' remaining or C138S with CysI3' remaining. The expression system for the genes encoding thioredoxin reductase, wild-type enzyme, C135S, and C138S has been re-engineered to allow for greater yields of protein. Wild-type enzyme and C135S were found to be as previously reported, whereas discrepancies were detected in the characteristics of C138S. It was shown that the original C138S was a heterogeneous mixture containing C138S and wild-type enzyme and that enzyme obtained from the new expression system is the correct species. C138S obtained from the new expression system having 0.1 % activity and 7% flavin fluorescence of wild-type enzyme was used in this study. Reductive titrations show that, as expected, only 1 mol of sodium dithionite/mol of FAD is required to reduce C138S. The remaining thiol in C135S and C138S has been reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) to form mixed disulfides. The half time of the reaction was <5 s for in C135S and approximately 300 s for C Y S '~~ in (2138s showing that CysI3' is much more reactive. The resulting mixed disulfides have been reacted with Cys32 in C35S mutant thioredoxin to form stable, covalent adducts C138S-C35S and C135S-C35S. The half times show that Cys"' is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cys 13' may be the thiol initiating dithiol-disulfide interchange between thioredoxin reductase and thioredoxin.Keywords: dithiol-disulfide interchange; flavoprotein; thioredoxin reductase Thioredoxin reductase from Escherichia coli is a pyridine nucleotidedisulfide oxidoreductase containing FAD and a disulfide in each active site. The redox active disulfide is composed of C Y S "~ and Cys'". Reducing equivalents move from NADPH to FAD, from Abbreviations: DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); TNB anion, 5-thio-2-nitrobenzoate anion: DTT, 1,4-dithiothreitol; DPDS, 4,4'-dithio-C138S and C135S, thioredoxin reductase in which C Y S '~~ or C Y S '~~ has dipyridine: PDS, 4-thiopyridone; IPTG, isopropyl P-D-thiogalactoside; been changed to Ser, respectively; C32S and C35S, thioredoxin in which Cys32 or has been changed to Ser, respectively: C138S-C35S, a covalent adduct where the remaining active site thiol of C138S is linked via a disulfide to the remaining active site thiol of C35S, analogous designations are used for the three other possible combinations of mutated thioredoxin reductases and thioredoxins. reduced flavin to the active site disulfide; dithiol-disulfide interchange effects reduction of the substrate thioredoxin. The redox active disulfide of thioredoxin is composed of Cys3' and Cys3'. Modification by site-directed mutagenesis of the active site in the enzyme has produced the single thiol mutants C138S (with CysI3' remaining from the active site disulfide) and C135S (with CysI3' remaining) (Prongay et al., 1989). The spectral characteristics...

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“…The human cytoplasmic thioredoxin crystal structure reveals a homodimer with Cys 73 forming an intermolecular disulphide bridge. 16 It is not known whether this dimer exits in vivo, or is an artefact of the crystallisation process, but it does obscure the active site groove and Cys 32. This dimer has also been shown to form in the crystal structure of the human thioredoxin mutant C73S, demonstrating that noncovalent interactions, other than the disulphide bridge, mediate the dimer.…”

Section: Introductionmentioning

confidence: 99%

Structure of human thioredoxin exhibits a large conformational change

Hall

Emsley

2010

Protein Science

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Thioredoxin is an oxidoreductase, which is ubiquitously present across phyla from humans to plants and bacteria. Thioredoxin reduces a variety of substrates through active site Cys 32, which is subsequently oxidized to form the intramolecular disulphide with Cys 35. The thioredoxin fold is known to be highly stable and conformational changes in the active site loops and residues Cys 32, Cys 35 have been characterized between ligand bound and free structures. We have determined a novel 2.0 Å resolution crystal structure for a human thioredoxin, which reveals a much larger conformational change than previously characterized. The principal change involves unraveling of a helix to form an extended loop that is linked to secondary changes in further loop regions and the wider area of the active site Cys 32. This gives rise to a more open conformation and an elongated hydrophobic pocket results in place of the helix. Buried residue Cys 62 from this helix becomes exposed in the open conformation. This provides a structural basis for observations that the Cys 62 sidechain can form mixed disulphides and be modified by thiol reactive small molecules.

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Crystal structures of reduced, oxidized, and mutated human thioredoxins: evidence for a regulatory homodimer

Cited by 362 publications

References 71 publications

Crystal structure of human thioredoxin revealing an unraveled helix and exposed S‐nitrosation site

Crystal structure of human thioredoxin revealing an unraveled helix and exposed S‐nitrosation site

Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin: Evidence that cysteine‐138 functions to initiate dithiol‐disulfide interchange and to accept the reducing equivalent from reduced flavin

Structure of human thioredoxin exhibits a large conformational change

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