Fig. 2

Overall structure of human USP14^CAT-IU1 and recognition of IU1 by USP14. a Overall structure of the USP14^CAT-IU1 complex. USP14^CAT comprises finger (green), palm (slate) and thumb (cyan) domains. The crucial surface loop BL2 is shown in orange. IU1 (yellow) binds the cleft between the palm and thumb domains. The catalytic center (Cys box) is shown in red. b Surface representation of the structure of the USP14^CAT-IU1 complex. c Structural alignment of USP14^CAT-IU1 and USP14^CAT-Ubal (PDB ID: 2AYO) shows that these complexes share a similar structure. The catalytic triad residues Cys114, His435, and Asp451 are magnified. Neither IU1 nor Ubal is shown in the figure. d Structural alignment of BL2 in apo USP14^CAT, USP14^CAT-Ubal and USP14^CAT-IU1. BL2 in USP14^CAT-IU1 exhibits a similar position as that in USP14^CAT-Ubal. e 2|Fo| − |Fc| electron density maps, contoured at 1.5 σ and covering all atoms of IU1. The catalytic center Cys114 is shown as red spheres. f Alignment of the structure of USP14^CAT-IU1 with that of USP14^CAT-Ubal shows that IU1 works blocks the access of the C terminus of ubiquitin to the catalytic center. Ubal is colored green. g The binding pocket for IU1 (yellow) in USP14 (slate). IU1 is 8.3 Å away from the catalytic center (red). h Recognition of IU1 by USP14. IU1 interacts with H426, Y436 and Y476 of USP14 via hydrophobic interactions and π-π stacking (π–π interactions). USP14 is colored in slate, and IU1 is colored in yellow. i H426 and Y436 are involved in IU1 recognition, as proved by the Ub-PA assay. The H426E or Y436A mutation rescued the Ub-PA covalent binding activity of USP14 upon the addition of 5 mM IU1. j Key residues involved in IU1 recognition were sequentially verified by an Ub-AMC hydrolysis assay: 15 μM IU1 significantly inhibited USP14 WT activity but exhibited no inhibition of the USP14 H426E, Y436A and Y476A mutants. All the values correspond to the averages of triplicate experiments, and the error bars represent SDs. Double asterisks denote p < 0.01. ns not significant