Dataset Information

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.

ABSTRACT: The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ?17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly.

SUBMITTER: Ranatunga W

PROVIDER: S-EPMC4858984 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Publications

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.

Ranatunga Wasantha W Gakh Oleksandr O Galeano Belinda K BK Smith Douglas Y DY Söderberg Christopher A G CA Al-Karadaghi Salam S Thompson James R JR Isaya Grazia G

The Journal of biological chemistry 20160303 19

The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this c ...[more]

PMID: 26941001

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Project description:Mitochondrial Fe-S cluster biosynthesis is accomplished within yeast utilizing the biophysical attributes of the "Isu1" scaffold assembly protein. As a member of a highly homologous protein family, Isu1 has sequence conservation between orthologs and a conserved ability to assemble [2Fe-2S] clusters. Regardless of species, scaffold orthologs have been shown to exist in both "disordered" and "structured" conformations, a structural architecture that is directly related to conformations utilized during Fe-S cluster assembly. During assembly, the scaffold helps direct the delivery and utilization of Fe(ii) and persulfide substrates to produce [2Fe-2S] clusters, however Zn(ii) binding alters the activity of the scaffold while at the same time stabilizes the protein in its structured state. Additional studies confirm Zn binds to the scaffold's Cys rich active site, and has an impact on the protein's ability to make Fe-S clusters. Understanding the interplay between Fe(ii) and Zn(ii) binding to Isu1 in vitro may help clarify metal loading events that occur during Fe-S cluster assembly in vivo. Here we determine the metal : protein stoichiometry for Isu1 Zn and Fe binding to be 1 : 1 and 2 : 1, respectively. As expected, while Zn binding shifts the Isu1 to its structured state, folding is not influenced by Fe(ii) binding. X-ray absorption spectroscopy (XAS) confirms Zn(ii) binds to the scaffold's cysteine rich active site but Fe(ii) binds at a location distinct from the active site. XAS results show Isu1 binding initially of either Fe(ii) or Zn(ii) does not significantly perturb the metal site structure of alternate metal. XAS confirmed that four scaffold orthologs bind iron as high-spin Fe(ii) at a site composed of ca. 6 oxygen and nitrogen nearest neighbor ligands. Finally, in our report Zn binding dramatically reduces the Fe-S cluster assembly activity of Isu1 even in the presence of frataxin. Given the Fe-binding activity we report for Isu1 and its orthologs here, a possible mechanism involving Fe(ii) transport to the scaffold's active site during cluster assembly has been considered.

Project description:Copper (Cu) ions serve as catalytic cofactors to drive key biochemical processes, and yet Cu levels that exceed cellular homeostatic control capacity are toxic. The underlying mechanisms for Cu toxicity are poorly understood. During pulmonary infection by the fungal pathogen Cryptococcus neoformans, host alveolar macrophages compartmentalize Cu to the phagosome, and the ability to detoxify Cu is critical for its survival and virulence. Here, we report that iron-sulfur (Fe-S) clusters are critical targets of Cu toxicity in both Saccharomyces cerevisiae and C. neoformans in a manner that depends on the accessibility of Cu to the Fe-S cofactor. To respond to this Cu-dependent Fe-S stress, C. neoformans induces the transcription of mitochondrial ABC transporter Atm1, which functions in cytosolic-nuclear Fe-S protein biogenesis in response to Cu and in a manner dependent on the Cu metalloregulatory transcription factor Cuf1. As Atm1 functions in exporting an Fe-S precursor from the mitochondrial matrix to the cytosol, C. neoformans cells depleted for Atm1 are sensitive to Cu even while the Cu-detoxifying metallothionein proteins are highly expressed. We provide evidence for a previously unrecognized microbial defense mechanism to deal with Cu toxicity, and we highlight the importance for C. neoformans of having several distinct mechanisms for coping with Cu toxicity which together could contribute to the success of this microbe as an opportunistic human fungal pathogen.IMPORTANCEC. neoformans is an opportunistic pathogen that causes lethal meningitis in over 650,000 people annually. The severity of C. neoformans infections is further compounded by the use of toxic or poorly effective systemic antifungal agents as well as by the difficulty of diagnosis. Cu is a natural potent antimicrobial agent that is compartmentalized within the macrophage phagosome and used by innate immune cells to neutralize microbial pathogens. While the Cu detoxification machinery of C. neoformans is essential for virulence, little is known about the mechanisms by which Cu kills fungi. Here we report that Fe-S cluster-containing proteins, including members of the Fe-S protein biogenesis machinery itself, are critical targets of Cu toxicity and therefore that this biosynthetic process provides an important layer of defense against high Cu levels. Given the role of Cu ionophores as antimicrobials, understanding how Cu is toxic to microorganisms could lead to the development of effective, broad-spectrum antimicrobials. Moreover, understanding Cu toxicity could provide additional insights into the pathophysiology of human diseases of Cu overload such as Wilson's disease.

Dataset Information

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.

Publications

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.

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