Figure 2.

Cys loop mobility regulates DUB activity

(A) Representation of the Cys loop in a superposition of MINDY1^apo (cyan) and MINDY1^C137A:K48-Ub₂ complex (pink). The isopeptide bond between K48 of Ub^prox (orange) and G76 of Ub^dist (yellow) is shown in sticks.

(B) Close-up view of (A) showing amino acid side chain rearrangements (side view).

(C) Surface representation of the hydrophobic pocket in MINDY1^apo that accommodates the Cys loop residue P138.

(D) Coomassie-stained gel comparing activity of MINDY1 WT and P138A to Ub^Prg in a time course.

(E) Steady-state kinetics of K48-linked pentaUb cleavage by MINDY1 WT and P138A mutant derived from reactions with varying concentrations of fluorescently labeled Ub₅ (n = 2; means ± SDs).

(F) Silver-stained gel comparing cleavage of K48-Ub₃ by MINDY1 WT and indicated mutants.

(G) DUB assay comparing cleavage of K48-Ub₂ by MINDY1 WT and P138A mutants.

(H) DUB assay monitoring cleavage of diUb of 7 different linkage types by MINDY1 P138A.

See also Figure S3.