Fe-S protein assembly involves bipartite client binding and conformational flexibility in the CIA targeting complex

The cytosolic iron sulfur (Fe-S) assembly (CIA) pathway is required for the insertion of Fe-S clusters into cytosolic and nuclear client proteins, including many DNA metabolism proteins. The molecular mechanisms of client protein recognition and Fe-S cluster transfer remain unknown. Here we report crystal structures of the CIA targeting complex and cryo-EM reconstructions of the complex bound either to the DNA replication factor primase or the DNA helicase DNA2. The structures, combined with biochemical, biophysical and yeast complementation assays, reveal an evolutionarily conserved, bipartite client binding mode facilitated by the structural flexibility of the MMS19 subunit. The primase Fe-S cluster is located [~]70 [A] away from the catalytic cysteine in the CIA targeting complex, pointing to a conformationally dynamic mechanism of Fe-S cluster transfer. Altogether, our studies suggest a model for Fe-S cluster insertion and thus provide a mechanistic framework to understand the biogenesis of critical DNA replication and repair factors.