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 replication and repair factors. The molecular mechanisms of client protein recognition and Fe–S cluster transfer remain unknown. Here, we report crystal structures of the CIA targeting complex (CTC), revealing that its CIAO2B subunit is centrally located and bridges CIAO1 and the client adaptor protein MMS19. Cryo-EM reconstructions of human CTC bound either to the DNA replication factor primase or to the DNA helicase DNA2, combined with biochemical, biophysical and yeast complementation assays, reveal an evolutionarily conserved, bipartite client recognition mode facilitated by CIAO1 and the structural flexibility of the MMS19 subunit. Unexpectedly, the primase Fe–S cluster is located ~70 Å away from the CTC reactive cysteine, implicating conformational dynamics of the CTC or additional maturation factors in the mechanism of Fe–S cluster transfer.

Fe-S簇插入胞质和核客户蛋白中需要胞质铁硫（Fe-S）组装（CIA）途径，包括许多DNA复制和修复因子。客户蛋白质识别和Fe–S团簇转移的分子机制仍然未知。在这里，我们报告了CIA靶向复合物（CTC）的晶体结构，揭示了其CIAO2B亚基位于中央，并桥接CIAO1和客户衔接蛋白MMS19。结合到DNA复制因子启动酶或DNA解旋酶DNA2上的人类CTC的Cryo-EM重建，结合生化，生物物理和酵母互补检测，揭示了CIAO1促进了进化上保守的二方客户识别模式及其结构灵活性MMS19亚基。不料，