Fig. 3: Puf3 interacts with multiple binding sites on Ccr4–Not.
From: Phosphorylation-dependent tuning of mRNA deadenylation rates
a, Sulfo-SDA-based CLMS analysis of Ccr4–Not bound to Puf3. Crosslinks highlighted in blue show interprotein crosslinks between Puf3 and Ccr4–Not subunits. Other intersubunit crosslinks are shown in gray. Subunits are colored and arranged according to the subcomplex architecture of Ccr4–Not. The N-terminal and C-terminal residue numbers for each subunit are shown. b, AlphaFold2 screen of 50-residue fragments of Puf3, tiled in three-residue increments, with the Not9 module (Not9 plus Not1 residues 1099–1318). The pLDDT score per residue is plotted. c, AlphaFold2 structure prediction showing overlay of the ten highest-scoring Puf3 fragments based on pLDDT scores (purple and green) on Not9–Not1 (white surface). The Puf3 fragments are predicted to bind in the peptide-binding groove and tryptophan (aromatic, Ω) pockets of Not9. Hydrophobic residues that insert into the Ω pockets in the predicted structures are shown as dark-red sticks. d, Deadenylation activities (plotted as t1/2) of wild-type (WT) Ccr4–Not and complexes containing mutations impacting IDR-binding pockets with WT Puf3. Bars denote the mean of three independent assays (filled circles). The mutants (listed in Extended Data Fig. 6a) do not substantially affect the activity of Ccr4–Not without Puf3 (Extended Data Fig. 6c).
