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

## TL;DR

The study identifies a pentapeptide motif that helps the CTC complex recognize and deliver iron-sulfur clusters to proteins involved in DNA repair and genome maintenance.

## Contribution

Discovery of a conserved pentapeptide motif and a dedicated CTC surface pocket for client recruitment, enabling identification of new Fe-S proteins.

## Key findings

- A pentapeptide motif in Fe-S proteins is crucial for CTC recognition and cellular functions like DNA repair.
- A conserved surface pocket in CTC recognizes two distinct sequence signatures, including the pentapeptide and C-tail motifs.
- Structure-guided AP-MS analysis identified previously unannotated Fe-S proteins.

## Abstract

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.

The cytosolic Fe-S cluster assembly targeting complex (CTC) delivers Fe-S cofactors to diverse clients via poorly understood mechanisms. This study identified a consensus pentapeptide motif in known Fe-S proteins that targets a distinctive binding pocket within the CTC, elucidating its client recruitment mechanism and enabling the discovery of previously unrecognized Fe-S proteins.

A conserved pentapeptide motif in CDKAL1, a known CTC client, defines CTC recognition at both biochemical and structural levels.This pentapeptide acts as a consensus signature dictating CTC recognition in Fe-S proteins, being crucial for their cellular functions, including roles in DNA repair.A newly revealed surface pocket of the CTC is dedicated to general client recruitment, accommodating two distinct sequence signatures: the pentapeptide motif and a previously reported C-tail motif.Pocket-directed affinity-purification/mass-spec analysis allows prospective identification of previously unannotated Fe-S proteins.

A conserved pentapeptide motif in CDKAL1, a known CTC client, defines CTC recognition at both biochemical and structural levels.

This pentapeptide acts as a consensus signature dictating CTC recognition in Fe-S proteins, being crucial for their cellular functions, including roles in DNA repair.

A newly revealed surface pocket of the CTC is dedicated to general client recruitment, accommodating two distinct sequence signatures: the pentapeptide motif and a previously reported C-tail motif.

Pocket-directed affinity-purification/mass-spec analysis allows prospective identification of previously unannotated Fe-S proteins.

A consensus pentapeptide motif in known iron-sulfur-cluster proteins that targets a distinctive binding pocket within the CTC allows identification of previously unrecognized Fe-S proteins.