The Cia1 and Cia2 subunits of the CTC mediate recognition of apo-FeS proteins with a C-terminal targeting complex recognition motif.

The cytosolic iron-sulfur cluster assembly (CIA) targeting complex is responsible for maturation of cytosolic and nuclear iron-sulfur enzymes, numbering >30 proteins critical for fundamental processes such as DNA replication and repair. Up to 25% of these client proteins terminate in a targeting complex recognition (TCR) motif. This carboxy-terminal tripeptide motif recruits the CIA targeting complex (CTC) to the client so that the metallocluster can be inserted. Herein, we use a combination of computational, biochemical and biophysical approaches to determine that the clients bearing a TCR motif docks at the interface of the Cia1 and Cia2 subunits of the CTC. Thus, mutations destabilizing the Cia1-Cia2 complex also disrupt TCR-based client identification by the CTC. Our study also reveals that the understudied human Cia2 paralog CIAO2A, which is proposed to be a specific targeting factor for iron regulatory protein 1, can recruit clients terminating in the TCR peptide. These data signal that CIAO2A plays a more general role in iron-sulfur protein maturation than previously appreciated. Taken together, our findings deepen our understanding of the molecular basis for client recognition by the CTC that is critical to understand the impact of CIA function in human health and disease.