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X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action.
J Struct Biol. 2016 May; 194(2):180-90.JS

Abstract

The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica, the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution X-ray structures for E. histolytica thioredoxin (EhTrx) and thioredoxin reductase (EhTrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys(140)-Cys(143)) was invariably observed in crystals of EhTrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys(286) was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of EhTrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with Escherichia coli TrxR and Trx). We note that the -CXXC- catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group.

Authors+Show Affiliations

Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC, USA.Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA; Department of Pathology, University of California San Diego, La Jolla, CA, USA.Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.Department of Pathology, University of California San Diego, La Jolla, CA, USA; Department of Medicine, University of California San Diego, La Jolla, CA, USA.Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC, USA.Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA. Electronic address: lpodust@ucsd.edu.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

26876147

Citation

Parsonage, Derek, et al. "X-ray Structures of Thioredoxin and Thioredoxin Reductase From Entamoeba Histolytica and Prevailing Hypothesis of the Mechanism of Auranofin Action." Journal of Structural Biology, vol. 194, no. 2, 2016, pp. 180-90.
Parsonage D, Sheng F, Hirata K, et al. X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action. J Struct Biol. 2016;194(2):180-90.
Parsonage, D., Sheng, F., Hirata, K., Debnath, A., McKerrow, J. H., Reed, S. L., Abagyan, R., Poole, L. B., & Podust, L. M. (2016). X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action. Journal of Structural Biology, 194(2), 180-90. https://doi.org/10.1016/j.jsb.2016.02.015
Parsonage D, et al. X-ray Structures of Thioredoxin and Thioredoxin Reductase From Entamoeba Histolytica and Prevailing Hypothesis of the Mechanism of Auranofin Action. J Struct Biol. 2016;194(2):180-90. PubMed PMID: 26876147.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action. AU - Parsonage,Derek, AU - Sheng,Fang, AU - Hirata,Ken, AU - Debnath,Anjan, AU - McKerrow,James H, AU - Reed,Sharon L, AU - Abagyan,Ruben, AU - Poole,Leslie B, AU - Podust,Larissa M, Y1 - 2016/02/12/ PY - 2016/01/05/received PY - 2016/02/09/revised PY - 2016/02/11/accepted PY - 2016/2/16/entrez PY - 2016/2/16/pubmed PY - 2016/12/15/medline KW - Auranofin KW - Entamoeba histolytica KW - Human amebiasis KW - Thioredoxin KW - Thioredoxin reductase KW - X-ray structure SP - 180 EP - 90 JF - Journal of structural biology JO - J. Struct. Biol. VL - 194 IS - 2 N2 - The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica, the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution X-ray structures for E. histolytica thioredoxin (EhTrx) and thioredoxin reductase (EhTrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys(140)-Cys(143)) was invariably observed in crystals of EhTrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys(286) was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of EhTrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with Escherichia coli TrxR and Trx). We note that the -CXXC- catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group. SN - 1095-8657 UR - https://www.unboundmedicine.com/medline/citation/26876147/X_ray_structures_of_thioredoxin_and_thioredoxin_reductase_from_Entamoeba_histolytica_and_prevailing_hypothesis_of_the_mechanism_of_Auranofin_action_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1047-8477(16)30030-2 DB - PRIME DP - Unbound Medicine ER -