(A) KIF4ALE,PE peptide (1192–1232; 1224CS1225 to LE, 1231AH1232 to PE; yellow sticks) binds B56 between heat repeats 3 and 4 (HR3 and 4; lavender and blue; B56 heat repeat schematic, with corresponding helices numbered, is shown below). (B) The KIF4ALE,PE:B56γ and AIM1:B56γ complexes are superimposed on the pS-RepoMan:B56γ complex (PDB: 5SW9) via B56γ. B56γ is colored according to electrostatic potential; KIF4A, AIM1 and RepoMan peptides are shown as yellow, purple and green sticks, respectively. The sequences of KIF4ALE,PE, AIM1, and RepoMan that include the basic patch (colored in blue) and the LxxIxE motif are shown below. The residues with electron density in the KIF4ALE,PE:B56γ, AIM1:B56γ, and pS-RepoMan:B56γ complexes are highlighted in yellow, purple and green, respectively. (C) Electrostatic and hydrogen bonding interactions between phosphomimetic KIF4ALE,PE S1225E (yellow) and B56γ H187/R188 (orange). (D) Electrostatic and hydrogen bonding interactions between RepoMan pS591 (green) and B56γ H187/R188 (blue, 5SW9). (E) The B56γ LxxIxE binding pocket (colored in light blue) and the electron density (2Fo–Fc, σ = 1.0) of the AIM1 peptide; 14 residues of the AIM1 peptide were modeled into the electron density (purple: 716KRKKARMPNSPAPHFAMPPIHEDHLE741). (F) The C-terminal residues of AIM1 peptide also interact with B56γ, with the formation of a bidentate salt bridge between AIM1 E741 and B56γ R201.