Fig. 3: Definition of interactions between Unkempt and CCR4-NOT.
From: Regulation by the RNA-binding protein Unkempt at its effector interface
A Schematic representation of CCR4-NOT. Yellow circles indicate points of contact with UNKIDR identified in this study. The numbering of contacts indicates SLiM 1 or SLiM 2 binding sites. Adapted from Raisch et al. 28. under permission provided by a Creative Commons Attribution 4.0 International License. B Pull-down assays with recombinant UNKFULL tagged with the StrepII (Strep) affinity tag upon incubation with different CCR4-NOT modules (n = 3). C As in (B) but with mutants of UNKIDR constructs fused to MBP and Strep after incubation with the NOT9 or the NOT module (n = 3). D Twenty-five AlphaFold predictions of interfaces of UNKIDR interacting with the NOT9 module. The predictions are aligned on the CNOT9/CNOT1 heterodimer51. The region of UNKIDR where the predictions converged is in dark blue. E The converged region of UNKIDR bound on the concave surface of CNOT9/CNOT1. F The same 25 predictions as in (D) but oriented to show the tryptophan (W)-binding pockets of CNOT9. G All 25 predictions of the converged region of UNKIDR close to the W-binding pockets of CNOT9. H Twenty-five AlphaFold predictions of UNKIDR interacting with the NOT module. The predictions are aligned on the CNOT1/CNOT2/CNOT3 heterotrimer58. The region of UNKIDR where predictions converged is in dark blue. I The converged region of UNKIDR bound on the surface of CNOT1. J Pull-down of WT or M1-M3 mutants of the NOT9 module by MBP-UNKIDR-Strep. Residues in CNOT9 mutated to alanines in M1 (Y203 and R244) line the W-pocket 1, and those mutated in M2 (R205 and H208) line the W-pocket 251. All residues (Y203, R205, H208, and R244) were mutated in M3 (n = 2). K Pull-down of the WT or M3 NOT9 module or the NOT module by WT MBP-UNKIDR-Strep or its mutant with key residues in SLiM 1 (V511, I515, L522) substituted with glutamic acid. In (E), (G), and (I), the C-alpha atoms of the key interacting residues are shown as yellow spheres. The sequences of the converged region are shown with the key interacting residues in yellow.
