Mechanism of GLI binding. (a, b, c) Interactions between MBPA216H_K220H-SUFU-ΔW61D_L62S_G63F_P453A_Δ454–456_K457A and GLI3p. (a) The peptide (blue) is clamped between the N-terminal (beige) and C-terminal (green) domains. (b) GLI3p (blue residue labels) binds in a channel with His336 and Lys337 protruding into deep pockets. (c) SUFU–GLI3p interactions, with side-chain hydrogen bonds highlighted in yellow, and comparison of GLI1p and GLI3p. Residues that are conserved in GLI1, GLI2 and GLI3 are shown in red. Blue boxes indicate residues with visible electron density. (d, e) Mutations around the GLI3p binding site have varying effects on the ability of SUFU-FL to repress GLI1-induced reporter gene activity in HEK 293 cells (d) and to suppress constitutive pathway activity in Sufu−/− cells (e).