title: X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action.
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.affiliation: Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC, USA.
authors.forename: Derek
authors.initials: D
authors.lastname: Parsonage
authors.affiliation: Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.
authors.forename: Fang
authors.initials: F
authors.lastname: Sheng
authors.affiliation: 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.
authors.forename: Ken
authors.initials: K
authors.lastname: Hirata
authors.affiliation: Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.
authors.forename: Anjan
authors.initials: A
authors.lastname: Debnath
authors.affiliation: Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.
authors.forename: James H
authors.initials: JH
authors.lastname: McKerrow
authors.affiliation: Department of Pathology, University of California San Diego, La Jolla, CA, USA; Department of Medicine, University of California San Diego, La Jolla, CA, USA.
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Reed
authors.affiliation: Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA.
authors.forename: Ruben
authors.initials: R
authors.lastname: Abagyan
authors.affiliation: Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC, USA.
authors.forename: Leslie B
authors.initials: LB
authors.lastname: Poole
authors.affiliation: 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.
authors.forename: Larissa M
authors.initials: LM
authors.lastname: Podust
mesh_terms: D000595:Amino Acid Sequence; D000981:Antiprotozoal Agents / Q000737:chemistry*; D018501:Antirheumatic Agents / Q000737:chemistry; D001310:Auranofin / Q000737:chemistry*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry; D004748:Entamoeba histolytica / Q000737:chemistry* / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000981:Antiprotozoal Agents; D018501:Antirheumatic Agents; D004220:Disulfides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D001310:Auranofin; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase
keywords: Auranofin; Entamoeba histolytica; Human amebiasis; Thioredoxin; Thioredoxin reductase; X-ray structure