Identifying the Binding Interface between Rad3 and the Cytosolic Iron Sulfur Cluster Assembly Targeting Complex

Abstract

The Cytosolic Iron Sulfur Cluster Assembly (CIA) pathway is a highly conserved pathway that assembles and inserts iron sulfur (FeS) cluster cofactors into a variety of target proteins. These targets are involved in many cellular processes including DNA repair, iron homeostasis, and nucleotide metabolism. For CIA targets to receive the FeS cluster, they must be recruited for FeS cluster insertion by the CIA targeting complex. Although it is known that the targeting complex recognizes and binds to targets, the mechanisms of target recognition are not fully understood. In this work, mutagenesis and affinity co‐purification were utilized to elucidate the binding interface of Met18, a protein in the CIA targeting complex, and Rad3, a DNA helicase that receives an FeS cluster from the CIA pathway. Although this interaction has been investigated by other groups, there has been contradiction as to which portion of Rad3 binds to the targeting complex. Vashist et al. (J. Biol. Chem., 2012, 287, 43351) reported that the N‐terminus of Rad3 is responsible for binding to the targeting complex, while Ito et al. (Mol. Cell., 2010, 39, 632) reported that the C‐terminus of Rad3 is responsible for binding. To help resolve this discrepancy and gain insight into the targeting mechanism of the CIA targeting complex, truncated versions of Rad3 were expressed and isolated, and it was found that the N‐terminal region of the protein is responsible for binding to the targeting complex. Our current work involves investigating a 10 amino acid N‐terminal region of Rad3 identified by Vashist et al. Their group identified this region as participating in binding to Met18. In this work, we split the region into multiple alanine scans to further narrow down the Rad3‐Met18 binding interface. The identification of a region involved in Rad3‐Met18 binding could help define a target recognition motif for the CIA targeting complex.Support or Funding InformationBU Undergraduate Research Opportunities Program; NIH R01 GM121673This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.