Client recruitment mechanism of the cytosolic Fe-S cluster assembly targeting complex.

Most cytosolic and nuclear eukaryotic Fe-S proteins acquire their critical Fe-S cofactor by interacting with the cytosolic Fe-S cluster assembly targeting complex (CTC). Despite the critical roles these Fe-S proteins play in fundamental biology, how they are specifically recognized by the CTC remains largely understudied. Here we identified a hidden consensus pentapeptide motif as a sequence signature dictating cluster acquisition in a majority of known human Fe-S proteins, particularly DNA/RNA processing enzymes for genome maintenance. The presence of this motif drives CTC-client engagement, while its defect impairs CTC recognition, iron incorporation, and enzymatic activities of these clients, ultimately compromising their cellular functions, such as in DNA repair. Furthermore, our studies revealed a conserved surface pocket of CTC dedicated to client recruitment in general. This single pocket recognizes two distinct sequence signatures in clients including the Pentapeptide motif and a previously reported C-tail motif. Subsequent structure-guided affinity-purification mass spectrometry (AP-MS) enabled us to investigate the pocket-dependent human CTC interactome, potentially unveiling unrecognized Fe-S proteins. Overall, our findings decipher the sequence signature-directed mechanism underlying CTC client recruitment and open an avenue for expanding the repertoire of Fe-S proteins.