The cytosolic iron sulfur cluster assembly (CIA) pathway is responsible for the maturation of >40 cytosolic and nuclear iron sulfur (FeS) proteins critical for fundamental processes such as DNA replication, transcription, and translation. The final stages of the pathway require the CIA targeting complex, which is composed of Cia1, Cia2, and Met18. This large multiprotein complex is proposed to recognize apo-enzyme substrates and insert their FeS clusters. However, it is unclear how these substrates are identified and how the CIA targeting complex mediates cofactor insertion. In this thesis, I mapped the protein-protein interaction sites critical for formation of the CIA targeting complex and discovered the first peptide motif that is both necessary and sufficient for recognition of a subset of FeS proteins by the CIA system.
Cia1’s seventh beta-propeller blade was found to bind to Cia2, while Cia2’s fifth conserved region mainly interacts with Cia1, via an in vitro affinity co-purification assay. A quantitative MicroScale Thermophoresis assay supported these findings, in addition this approach affirmed that Cia2’s N-terminal intrinsically disordered domain and hyperreactive cysteine are dispensable for CIA targeting complex assembly. In collaboration with the Drennan Lab at MIT, Met18 was discovered to form a hexamer via cryo-EM. Met18 is proposed to arrange into a hexamer before its CIA-related function. Hexamer formation and Cia2 binding depend on Met18’s C-terminus, whereas Leu1 recognition relies on Met18’s N-terminus.
A C-terminal W motif was demonstrated as both necessary and sufficient for identification of a subset of FeS proteins by the CIA targeting complex. A bioinformatics analysis revealed roughly 20% of CIA client proteins, including substrates, factors, and adaptors, terminate in a conserved [LTQ]-[DE]-[W]-COO- motif. CIA recognition depends on the C-terminal aromatic side chain and the carboxy terminus. This tripeptide motif is also sufficient for identification by the CIA system when attached to SUMO. Moreover, a series of competition experiments showed that the CIA targeting complex contains distinct, non-overlapping binding sites for client proteins where Cia1 serves as the docking site for the C-terminal W motif. Altogether, the first recognition motif is defined for one in five of CIA client proteins. / 2024-11-03T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/45300 |
| Date | 03 November 2022 |
| Creators | Marquez, Melissa Danae |
| Contributors | Perlstein, Deborah L. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution-NonCommercial-NoDerivatives 4.0 International, http://creativecommons.org/licenses/by-nc-nd/4.0/ |
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