Extended Data Fig. 4: Identifying the 'invisible' interface between Ctc1 OB-A and p50 peptide using NMR methods.

a, Schematic diagram of p50 and constructs of p50 peptide. The N-terminal 30 kDa and 25 kDa fragments of p50 are labelled as p50N30 and p50N25, respectively. Previous biochemical study showed that p50N30 could bind Ctc1, whereas p50N25 could not⁴³. The cryo-EM structure of p50 ends at residue 208 (Fig. 2b). On the basis of these facts, a series of p50 peptides in the range of residues 213-255 were designed to explore additional interface between p50 and Ctc1 OB-A that are 'invisible' in the cryo-EM structure. b, NMR binding study of p50 peptides with Ctc1 OB-A. Two regions of ¹H-¹⁵N HSQC spectra of ¹⁵N-labelled Ctc1 OB-A in the absence (apo) and presence of unlabelled p50 peptides were shown. Chemical shifts of T71 and A73 were chosen to illustrate the binding process in this and the following panels, c and d. p50_228-250 peptide is determined to be the optimal construct and was used for other NMR studies presented here. c, Titration series of p50 peptide into ¹⁵N-labelled Ctc1 OB-A. The binding is in the slow exchange regime and saturated at 1:1 stoichiometry. d, Truncations of two unstructured loops (residues 38-49 and 131-146) of Ctc1 OB-A individually have no effect on its binding with p50 peptide. e, Secondary-structure score of p50_228-250 in the presence of Ctc1 OB-A. f, CSP index of p50 peptide upon binding Ctc1 OB-A. ¹H-¹⁵N HSQC spectra shown in Fig. 2c were used for the CSP calculation. g, Model of the interactions between Ctc1 OB-A and p50. CS-Rosetta models of p50_228-250 are shown in the grey box with arrows pointing to the binding surface on Ctc1 OB-A. Unstructured linkers are shown as dashed lines.