Fig 2: A CTC client recruitment pocket for exploring hidden Fe-S proteins.(A) Close-up view of a structural model showing a shared pocket in CTC for client recruitment generated by superposition of the structure of dmCIAO1-CIAO2B-hCDKAL1 pentapeptide and the binding model of CTC-C-tail motif recognition. The binding model is created by AlphaFold 3 using RSAD2 as a client. For clarity, only the last three residues at the C-terminus of RSAD2 are shown. CIAO1 and CIAO2B are shown in surface with CIAO1 in sage green and CIAO2B in wheat. CDKAL1 pentapeptide and RSAD2 C-tail are shown in ribbon with CDKAL1 pentapeptide in orange and RSAD2 C-tail in pink. The area of shared pocket is highlighted in light gray. See also Fig. EV5B for interaction details. (B) GST pull-down assay assessing the binding abilities of the core CTC in forms of WT and pocket-deficient variants toward the clients of CDKAL1 and RSAD2. (C) In vitro competition assays demonstrating the abilities of both CDKAL1 and RSAD2 to compete with each other for the CTC binding reciprocally. Dissociation of the pre-bound clients was monitored in the flowthrough. (D) Representative SDS-PAGE gel of affinity pull-down using the CTC in forms of WT and pocket-deficient variants (PD1: Pocket-deficient 1, containing CIAO1-R125A, PD2: Pocket-deficient 2, containing CIAO2B-R141A) as baits. The pulled down proteins against HEK293T cell lysate were detected by silver staining. (E) Dot plot generated in ProHits-viz program visualizing the average spectral counts of known human Fe-S proteins captured in the AP-MS by using the CTC WT and pocket-deficient variants PD1 and PD2 as baits. Fill color and size of dots represent AvgSpec and Relative abundance, respectively. The fill color represents the average spectral count of each protein, with darker shading indicating higher counts (maximum capped at 50; values above 50 are shown in black). The dot size reflects the relative abundance of each protein across the three datasets. CTC components, Pentapeptide-containing Fe-S proteins, C-tail-containing Fe-S proteins, Fe-S proteins with no sequence signature but pocket dependent, and pocket independent MMS19-driven Fe-S proteins are grouped, labeled and colored accordingly. Star (*) indicates the adapter or the associated protein of the Fe-S protein. PD1: Pocket-deficient 1, containing CIAO1-R125A, and PD2: Pocket-deficient 2, containing CIAO2B-R141A. (F) Network of PPIs showing the high-confidence CTC-interactors retrieved from Fig. EV5F visualized with Cytoscape. Edge thickness proportional to spectral counts. The CTC is shown as red diamond, and protein hits are shown either individually (circles) or grouped as protein complexes and biological processes (framed circles) according to their reported functions. The Pentapeptide-containing and C-tail-containing proteins are colored in orange and pink, respectively. TRMT1L harbors both motifs and is shown in bicolor. The protein complexes and biological processes are labeled and colored accordingly. (G) A schematic summary of mechanisms underlying the cluster acquisition of Fe-S proteins via the CTC. Pocket-dependent mechanisms comprising Pentapeptide-driven (shown in orange) and C-tail-driven (shown in pink) recognition represent the predominant CTC-client recognition strategies, while a few exceptions utilize either pocket-independent mechanism probably driven by MMS19 (shown in blue) or mechanism still to be investigated (shown in gray). In the schematic boxes illustrating pocket-dependent mechanisms, the left cartoon depicts direct recruitment of a client by the CTC, while the right cartoon represents recognition mediated by an adapter protein. Source data are available online for this figure.

Fig 3: The pentapeptide as a consensus motif for CTC-binding in Fe-S proteins.(A) The binding affinities of pentapeptide variants to the core CTC determined by ITC quantitatively. Residue substitutions in pentapeptide were indicated by orange circle and the preferred residues for each position were highlighted in orange. N.B.: No detectable binding activity. N.D.: Not determined due to the weak binding. See also Appendix Fig. S1 for ITC binding curves. (B) A consensus sequence of pentapeptide summarized from (A) is shown in orange. Indicated position of amino acid in pentapeptide is shown as subscript. A paired square bracket ‘[]’ represents a position where any residues in the bracket is accepted, while a paired curly bracket ‘{}’ represents a position of any amino acids except for those denoted inside. (C) Sequence alignment of the consensus pentapeptide motif found in known human CTC client Fe-S proteins. For comparison, the flanking sequences are also shown at both sides. Two consensus pentapeptide motifs in both EXO5 and POLE1 are identified and labeled accordingly. Residues are colored from red to black according to their conservations from low to high. A corresponding sequence logo is generated by WebLogo (Crooks et al, 2004) and presented beneath the alignment with all residues colored in black. The overall height of a stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino acid at that position. The most conserved four residues in the pentapeptide motif are highlighted. (D) HEK293T cells were transiently transfected with FLAG-tagged indicated Fe-S proteins in forms of either WT or consensus motif-deficient mutant. Whole cell extracts (WCE) were subjected to immunoprecipitations with anti-FLAG beads and immunoblotting against the antibodies of FLAG, MMS19, CIAO1 and CIAO2B. The transfected RTEL1 was RTEL11-767aa in mouse version, and the FANCJ was FANCJ1-1100aa. EV: empty vector. Source data are available online for this figure.

Fig 4: CDKAL1 pentapeptide mediating the CDKAL1-CTC interface.(A) ITC data showing the quantitative binding affinities of CDKAL1 fragment F3–F4 to the core CTC in both Drosophila and human versions. (B) Two orthogonal views of the complex comprising dmCIAO1-CIAO2B and hCDKAL1 pentapeptide shown in ribbon diagram with CIAO1 in sage green, CIAO2B in wheat, and CDKAL1 pentapeptide in orange, respectively. The side chains of pentapeptide are shown in sticks. The N and C termini and the top and bottom sides of CIAO1 are labeled accordingly in green. Select secondary structure/structural unit of different proteins are numbered and labeled in corresponding colors. (C) Structural model of CDKAL1 pentapeptide in complex with CTC complete complex composed of CIAO1, CIAO2B, and MMS19 generated by superposition of the structures of dmCIAO1-CIAO2B-hCDKAL1 pentapeptide and dmCIAO1-CIAO2B-mMMS19 (PDB: 6TC0). The model is shown in surface with the same color scheme as shown in (B), and MMS19 is shown in the color of light blue. (D) Close-up view of the interface between CDKAL1 pentapeptide and dmCIAO1-CIAO2B complex featuring supportive contacts targeting the backbone and side chain of CDKAL1 pentapeptide. CIAO1 and CIAO2B are shown in surface and ribbon with the same color scheme as shown in (B), and CDKAL1 pentapeptide is shown in orange sticks. Select interface residues are shown in sticks. Dashed lines in violet blue represent hydrogen bonds and polar interactions. (E, F) Sequence alignments of the regions enclosing the pentapeptide and the fragment F8–F9 in CDKAL1 orthologs from Homo sapiens, Mus musculus, Taeniopygia guttata, Xenopus tropicalis, Danio rerio, Branchiostoma belcheri, Oyster, Drosophila melanogaster, Trichoplax adhaerens, Arabidopsis thaliana and Archaeon. Residues are colored from red to black according to their conservations from low to high. The sequences of pentapeptide and fragment F8–F9 are highlighted by orange shade and with a frame in black, respectively.