Gustavo Salinas1,2, Wei Gao3,4,5, Yang Wang3,4, Mariana Bonilla2,6, Long Yu3,4, Andrey Novikov3,4, Veridiana G Virginio7, Henrique B Ferreira7, Marisol Vieites8, Vadim N Gladyshev9, Dinorah Gambino8, Shaodong Dai3,4. 1. 1 Worm Biology Lab, Institut Pasteur de Montevideo , Montevideo, Uruguay . 2. 2 Cátedra de Inmunología, Facultad de Química, Instituto de Higiene, Universidad de la República , Montevideo, Uruguay . 3. 3 Department of Biomedical Research, National Jewish Health , Denver, Colorado. 4. 4 Department of Immunology and Microbiology, University of Colorado Denver , School of Medicine, Aurora, Colorado. 5. 5 School of Science, Beijing Forestry University , Beijing, China . 6. 6 Redox Biology of Trypanosomes, Institut Pasteur de Montevideo , Uruguay . 7. 7 Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul , Porto Alegre, RS, Brazil . 8. 8 Cátedra de Química Inorgánica, Facultad de Química, Universidad de la República , Montevideo, Uruguay . 9. 9 Brigham and Women's Hospital , Harvard Medical School, Boston, Massachusetts.
Abstract
AIMS: New drugs are needed to treat flatworm infections that cause severe human diseases such as schistosomiasis. The unique flatworm enzyme thioredoxin glutathione reductase (TGR), structurally different from the human enzyme, is a key drug target. Structural studies of the flatworm Echinococcus granulosus TGR, free and complexed with AuI-MPO, a novel gold inhibitor, together with inhibition assays were performed. RESULTS: AuI-MPO is a potent TGR inhibitor that achieves 75% inhibition at a 1:1 TGR:Au ratio and efficiently kills E. granulosus in vitro. The structures revealed salient insights: (i) unique monomer-monomer interactions, (ii) distinct binding sites for thioredoxin and the glutaredoxin (Grx) domain, (iii) a single glutathione disulfide reduction site in the Grx domain, (iv) rotation of the Grx domain toward the Sec-containing redox active site, and (v) a single gold atom bound to Cys519 and Cys573 in the AuI-TGR complex. Structural modeling suggests that these residues are involved in the stabilization of the Sec-containing C-terminus. Consistently, Cys→Ser mutations in these residues decreased TGR activities. Mass spectroscopy confirmed these cysteines are the primary binding site. INNOVATION: The identification of a primary site for gold binding and the structural model provide a basis for gold compound optimization through scaffold adjustments. CONCLUSIONS: The structural study revealed that TGR functions are achieved not only through a mobile Sec-containing redox center but also by rotation of the Grx domain and distinct binding sites for Grx domain and thioredoxin. The conserved Cys519 and Cys573 residues targeted by gold assist catalysis through stabilization of the Sec-containing redox center. Antioxid. Redox Signal. 27, 1491-1504.
AIMS: New drugs are needed to treat flatworm infections that cause severe human diseases such as schistosomiasis. The unique flatworm enzyme thioredoxin glutathione reductase (TGR), structurally different from the human enzyme, is a key drug target. Structural studies of the flatworm Echinococcus granulosusTGR, free and complexed with AuI-MPO, a novel gold inhibitor, together with inhibition assays were performed. RESULTS:AuI-MPO is a potent TGR inhibitor that achieves 75% inhibition at a 1:1 TGR:Au ratio and efficiently kills E. granulosus in vitro. The structures revealed salient insights: (i) unique monomer-monomer interactions, (ii) distinct binding sites for thioredoxin and the glutaredoxin (Grx) domain, (iii) a single glutathione disulfide reduction site in the Grx domain, (iv) rotation of the Grx domain toward the Sec-containing redox active site, and (v) a single gold atom bound to Cys519 and Cys573 in the AuI-TGR complex. Structural modeling suggests that these residues are involved in the stabilization of the Sec-containing C-terminus. Consistently, Cys→Ser mutations in these residues decreased TGR activities. Mass spectroscopy confirmed these cysteines are the primary binding site. INNOVATION: The identification of a primary site for gold binding and the structural model provide a basis for gold compound optimization through scaffold adjustments. CONCLUSIONS: The structural study revealed that TGR functions are achieved not only through a mobile Sec-containing redox center but also by rotation of the Grx domain and distinct binding sites for Grx domain and thioredoxin. The conserved Cys519 and Cys573 residues targeted by gold assist catalysis through stabilization of the Sec-containing redox center. Antioxid. Redox Signal. 27, 1491-1504.
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