Iron–Sulfur Cluster Binding by Mitochondrial Monothiol Glutaredoxin-1 ofTrypanosoma brucei: Molecular Basis of Iron–Sulfur Cluster Coordination and Relevance for Parasite Infectivity

Abstract

Aims: Monothiol glutaredoxins (1-C-Grxs) are small proteins linked to the cellular iron and redox metabolism. Trypanosoma brucei brucei, model organism for human African trypanosomiasis, expresses three 1-C-Grxs. 1-CGrx1 is a highly abundant mitochondrial protein capable to bind an iron-sulfur cluster (ISC) in vitro using glutathione (GSH) as cofactor. We here report on the functional and structural analysis of 1-C-Grx1 in relation to its ISC-binding properties. Results: An N-terminal extension unique to 1-C-Grx1 from trypanosomatids affects the oligomeric structure and the ISC-binding capacity of the protein. The active-site Cys104 is essential for ISC binding, and the parasite-specific glutathionylspermidine and trypanothione can replace GSH as the ligands of the ISC. Interestingly, trypanothione forms stable protein-free ISC species that in vitro are incorporated into the dithiol T. brucei 2-C-Grx1, but not 1-C-Grx1. Overexpression of the C104S mutant of 1-C-Grx1 impairs disease progression in a mouse model. The structure of the Grx-domain of 1-C-Grx1 was solved by nuclear magnetic resonance spectroscopy. Despite the fact that several residues-which in other 1-C-Grxs are involved in the noncovalent binding of GSH-are conserved, different physicochemical approaches did not reveal any specific interaction between 1-C-Grx1 and free thiol ligands. Innovation: Parasite Grxs are able to coordinate an ISC formed with trypanothione, suggesting a new mechanism of ISC binding and a novel function for the parasite-specific dithiol. The first 3D structure and in vivo relevance of a 1-C-Grx from a pathogenic protozoan are reported. Conclusion: T. brucei 1-C-Grx1 is indispensable for mammalian parasitism and utilizes a new mechanism for ISC binding. Antioxid. Redox Signal. 19,[665][666][667][668][669][670][671][672][673][674][675][676][677][678][679][680][681][682]

Selenoproteins of African trypanosomes are dispensable for parasite survival in a mammalian host

“…The role of the C-terminal selenocysteine-containing redox motif of Seltryp cannot be inferred; however, it is possible to speculate that it may substitute SQR by reacting with H 2 S forming a sulfur-selenide intermediate, streamlining an entire redox pathway in a single protein. The 180 amino acid N-terminal extension of Seltryp has neither predicted domains nor significant homology to eukaryotic or bacterial proteins, and therefore it may reflect a true kinetoplastid innovation probably playing a regulatory role as it is commonly observed in proteins of kinetoplastid origin [27,28].…”

“…In order to shed light into this question and taking into account a potential role for selenoproteins in redox or mitochondrial sulfur-related metabolism, we analyzed the content of representative proteins from thiol-dependent metabolic pathways in parasites isolated from animals infected with the SepSecS-KO and WT cell lines. Thus, we analyzed the expression of trypanothione reductase (TR), a NADPH-dependent flavoenzyme in charge of maintaining trypanothione reduced, which is the major low molecular mass redox co-substrate in trypanosomatids [7]; TXN-Px, a tryparedoxin-dependent peroxiredoxin that catalyzes the decomposition of hydroperoxides very efficiently [40]; TXN, a thioredoxin-like oxidoreductase that plays a key role in protein redox homeostasis [17]; Grx1, a class II glutaredoxin with an essential role in the mitochondrial iron-sulfur biosynthetic pathway [28]; and Grx3, a class II glutaredoxin fused to a thioredoxin domain with putative roles in cytosolic mobilization and/or assembly of iron-sulfur clusters [41,42]. The expression level of none of these proteins differed significantly between SepSecS-KO and WT parasites isolated from infected mice (Fig.…”

Kinetic studies reveal a key role of a redox-active glutaredoxin in the evolution of the thiol-redox metabolism of trypanosomatid parasites

“…Fractions containing the fusion protein (as assessed by SDS-PAGE) were pooled and treated with 5 mM DTT, 2 mM EDTA, and His-tagged 3C-type tobacco etch virus protease (prepared as described in Ref. 29) at a 70:1 protein: protease ratio (in mg) for 2 h at room temperature. The sample was then concentrated by ultrafiltration (Vivaspin, 5 kDa molecular weight cut-off), buffer exchanged with a HiPrep column coupled to an Ä KTA-FPLC system (GE Healthcare) equilibrated in buffer A and applied again onto a HisTrap pre-equilibrated in buffer A. Tag-free WT Grx1 or its Cys-to-Ser mutants was collected in the flow-through, treated with 5 mM DTT and 2 mM EDTA during 30 min at room temperature, and concentrated by ultrafiltration before loading it onto a preparative size exclusion column (HiLoad 26/60 Superdex 75 prep grade column, GE) equilibrated with 100 mM sodium phosphate, pH 7.4, with 150 mM NaCl (buffer B).…”

“…2CGrx1 (hereinafter Grx1) is a cytosolic and relatively abundant protein with a canonical CPYC active site motif and a third cysteine (Cys-78) that, in vitro, is target of glutathionylation (27). Similar to human Grx2 (12), the trypanosomal Grx1 has been shown to use its active site cysteine residues either to reduce GSH-containing disulfides or to bind an iron-sulfur cluster, employing indistinctly GSH and T(SH) 2 as redox cofactors or iron ligands, respectively (28,29). Grx1, and to a minor extent 2CGrx2, display a significant T(SH) 2 -GSSG oxidoreductase activity (28).…”

Crystal structure of yeast monothiol glutaredoxin Grx6 in complex with a glutathione-coordinated [2Fe–2S] cluster

“…Class II proteins are 1-C-Grxs that harbour a strict CGFS motif. Generally, 2-C-Grxs contribute greatly to reducing protein-GSH mixed disulfides and intermolecular or intramolecular disulfides, whereas most 1-C-Grxs show decreased activities in catalyzing thiol-disulfide exchange reactions but adopt more specialized functions in cells (Manta et al, 2013). For instance, class II 1-C-Grxs can coordinate [2Fe-2S] iron-sulfur clusters (ISCs), evolutionarily ancient prosthetic groups that are necessary to maintain essential life processes (Zhang, 2015).…”