(A) Cartoon depicting Ki-67 and RepoMan domains. The only region of homology between the two proteins is indicated in blue. The sequences corresponding to the homologous regions are shown below, with conserved residues highlighted in grey. Ki-67 residues that interact directly with PP1 are underlined. The sequences corresponding to the RVxF and ΦΦ SLiMs (blue highlight) and the newly discovered KiR-SLiM (orange) are shown. (B) left, binding isotherm of Ki-67496-536 with PP1γ7–323 (KD, 193 ± 16 nM; the KD of the corresponding domain of RepoMan383-423 with PP1γ7–323 is 133 ± 16 nM); right, binding isotherm of the ΔKiR-SLIM, RepoMan383-404, with PP1γ7–323 (KD, 661 ± 160 nM). (C) Crystal structure of the Ki-67:PP1γ holoenzyme. PP1γ is in grey and Ki-67496-536 is in pink with the 2Fo–Fc electron density map contoured at 1σ (2.0 Å); no electron density was observed for Ki-67 residues 496–503 (pink dotted line) and 536. PP1 residues in green correspond to PP1 secondary structure elements helix A’, loop 1 and helix B. (D) Close-up of the Ki-67 (pink) and RepoMan (blue) interaction with PP1. Ki-67 residues 503–516 and RepoMan residues 390–403 bind the PP1 RVxF and ΦΦ binding pockets (cyan surface; the Ki-67 and RepoMan RVxF and ΦΦ SLiM residues are labeled and in italics). Ki-67 residues 517–535 and RepoMan residues 404–422 bind the newly defined KiR-SLiM binding pocket (beige surface). The black dotted line highlights the area shown in E. (E) The hydrophobic and polar interactions between Ki-67 (pink sticks) and PP1γ (grey sticks; surface). Hydrogen bonds and salt bridge interactions are indicated by dotted lines with the interacting residues labeled. (F) HMMER-derived sequence logo of the Ki-67/RepoMan PP1 binding domain, with the KiR-SLIM highlighted in beige (hydrophobic residues, black; acidic residues, red; basic residues blue; glycine/serine/threonine, green; asparagine/glutamine, pink).