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*
- Wasantha Ranatunga‡1,
- Oleksandr Gakh‡1,
- Belinda K. Galeano‡,
- Douglas Y. Smith IV‡,
- Christopher A. G. Söderberg§,
- Salam Al-Karadaghi§,
- James R. Thompson¶ and
- Grazia Isaya‡2
- From the ‡Departments of Pediatric and Adolescent Medicine and Biochemistry and Molecular Biology, and the Mayo Clinic Children's Research Center, and
- ¶Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905 and
- the §Center for Molecular Protein Science, Institute for Chemistry and Chemical Engineering, Lund University, P. O. Box 124, SE-221 00 Lund, Sweden
- ↵2 To whom correspondence should be addressed: Mayo Clinic, 200 First St. SW, Stabile 7-52, Rochester, MN 55905. Tel.: 507-266-0110; Fax: 507-266-9315; E-mail: isaya{at}mayo.edu.
-
↵1 Both authors contributed equally to this work.
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.
Footnotes
-
↵* This work was supported in part by National Institutes of Health Grant AG15709-19 from NIA (to G. I. and J. T.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
-
↵
This article contains supplemental Table S1.
- Received December 23, 2015.
- Revision received February 26, 2016.
- © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
