Enzyme structure captures four cysteines aligned for disulfide relay

Gat, Yair; Vardi-Kilshtain, Alexandra; Grossman, Iris; Major, Dan Thomas; Fass, Deborah

doi:10.1002/pro.2496

Cited by 11 publications

(15 citation statements)

References 49 publications

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“…The location of C165 in a predicted disordered region of QSOX1 between the Trx1 and Trx2 domains, however, may allow for interactions with nearby basic resides [ 31 ]. A recently described mechanism proposes that the flexible domain architecture of QSOX1 is critical in allowing Trx1 to come into close contact with Erv to transfer electrons to the C-X-X-C in this domain [ 30 , 36 ]. Ebselen bound to C165 and C237 may interfere with the conformational change required for the interaction of the Trx1 and Erv domains.…”

Section: Discussionmentioning

confidence: 99%

Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines

et al. 2015

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Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a “proof-of-principle” that enzymatic inhibition of QSOX1 may have clinical relevancy.

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Section: Discussionmentioning

confidence: 99%

Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines

et al. 2015

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“…Participating in the QSOX electron relay are two redox-active di-cysteine (CXXC) motifs and a flavin adenine dinucleotide (FAD) cofactor 1 9 10 11 ( Fig. 1 ).…”

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confidence: 99%

“…Structural data showing the role of protein conformation in assembling the components of the QSOX electron relay 9 11 led us to inquire how conformational dynamics govern electron transfer. In particular, we sought to determine the contribution of the interdomain electron-transfer intermediate to the kinetics of turnover.…”

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confidence: 99%

Single-molecule spectroscopy exposes hidden states in an enzymatic electron relay

et al. 2015

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The ability to query enzyme molecules individually is transforming our view of catalytic mechanisms. Quiescin sulfhydryl oxidase (QSOX) is a multidomain catalyst of disulfide-bond formation that relays electrons from substrate cysteines through two redox-active sites to molecular oxygen. The chemical steps in electron transfer have been delineated, but the conformational changes accompanying these steps are poorly characterized. Here we use single-molecule Förster resonance energy transfer (smFRET) to probe QSOX conformation in resting and cycling enzyme populations. We report the discovery of unanticipated roles for conformational changes in QSOX beyond mediating electron transfer between redox-active sites. In particular, a state of the enzyme not previously postulated or experimentally detected is shown to gate, via a conformational transition, the entrance into a sub-cycle within an expanded QSOX kinetic scheme. By tightly constraining mechanistic models, smFRET data can reveal the coupling between conformational and chemical transitions in complex enzymatic cycles.

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“…A RMSD of 2.2 Å over 108 residues was observed compared to the fourth domain (the a′ domain) of Pdi1p (2B5E) [7] . The Eps1p Trx1 domain has familiar structural and functional features observed previously in PDI domains, including a proline five residues downstream of the second cysteine in the Cys-X-X-Cys motif, a buried glutamic acid probably involved in deprotonating the second Cys-X-X-Cys cysteine via an intermediary water molecule [8] , [9] , and the cis -proline spatially adjacent to the Cys-X-X-Cys disulfide bond [10] ( Figure 3 ). These features are major contributors to the dithiol/disulfide exchange mechanism, and their presence suggests that the Eps1p Trx1 domain is catalytically active.…”

Section: Resultsmentioning

confidence: 81%

The Eps1p Protein Disulfide Isomerase Conserves Classic Thioredoxin Superfamily Amino Acid Motifs but Not Their Functional Geometries

2014

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The widespread thioredoxin superfamily enzymes typically share the following features: a characteristic α-β fold, the presence of a Cys-X-X-Cys (or Cys-X-X-Ser) redox-active motif, and a proline in the cis configuration abutting the redox-active site in the tertiary structure. The Cys-X-X-Cys motif is at the solvent-exposed amino terminus of an α-helix, allowing the first cysteine to engage in nucleophilic attack on substrates, or substrates to attack the Cys-X-X-Cys disulfide, depending on whether the enzyme functions to reduce, isomerize, or oxidize its targets. We report here the X-ray crystal structure of an enzyme that breaks many of our assumptions regarding the sequence-structure relationship of thioredoxin superfamily proteins. The yeast Protein Disulfide Isomerase family member Eps1p has Cys-X-X-Cys motifs and proline residues at the appropriate primary structural positions in its first two predicted thioredoxin-fold domains. However, crystal structures show that the Cys-X-X-Cys of the second domain is buried and that the adjacent proline is in the trans, rather than the cis isomer. In these configurations, neither the “active-site” disulfide nor the backbone carbonyl preceding the proline is available to interact with substrate. The Eps1p structures thus expand the documented diversity of the PDI oxidoreductase family and demonstrate that conserved sequence motifs in common folds do not guarantee structural or functional conservation.

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Enzyme structure captures four cysteines aligned for disulfide relay

Cited by 11 publications

References 49 publications

Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines

Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines

Single-molecule spectroscopy exposes hidden states in an enzymatic electron relay

The Eps1p Protein Disulfide Isomerase Conserves Classic Thioredoxin Superfamily Amino Acid Motifs but Not Their Functional Geometries

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