Structure of full-length SUMO-1 bound to SENP1 C603A. (a) SENP1(C603A)–SUMO-1-FL complex. Cyan, SENP1; purple, SUMO-1-FL. SENP1 is effectively identical to earlier descriptions. (b) Superposition of SENP1(C603A)–RanGAP1–SUMO-1 complex with SENP1(C603A)–SUMO-1-FL complex. In the superposed RanGAP1–SUMO-1 complex, RanGAP1 is in red, SENP1 is in dark blue and SUMO-1 is in turquoise. Isopeptide bond is depicted as in Figure 1a. (c) Detail of the complex in a, with SENP1 in dark blue and carbons of SUMO-1-FL in pink. Residues mentioned in the text are indicated. Dotted line denotes hydrogen bond. (d) The same cis arrangement of nitrogens is seen in the SENP1(C603A)–SUMO-1-FL processing complex and in the SENP1(C603A)–RanGAP1–SUMO-1 deconjugating complex (colored as in b and c).

Thermodynamics of substrate and product binding by SENP1 C603A. ITC was used to study the thermodynamic changes effected by binding of SENP1 to SUMO-1-FL, SUMO-2-FL, RanGAP1–SUMO-1, SUMO-1-GG, SUMO-2-GG or RanGAP1 (as indicated). Experiments were repeated on three separate occasions with very similar results. Thermodynamic parameters are indicated in Table 1.

Proposed mechanism for cleavage of substrates by SENP1. It is likely that two reversible steps occur before catalysis. First, there is an association between substrate (SUMO-target) and SENP1 that stimulates the opening of the tryptophan tunnel. Second, closing of the tryptophan tunnel causes trans-cis isomerization of the amide nitrogens of the scissile bond of the substrate. Chemical catalysis can then proceed, with hydrolysis and dissociation of the target being essentially irreversible. Finally, the product, SUMO-GG, dissociates from the SUMO-binding site in SENP1 before another round of catalysis can occur.

Structure of the complex between SENP1 C603A and SUMO-1–modified RanGAP1. (a) Overall structure of the complex. RanGAP1 is shown in red ribbon, SENP1 C603A in blue and SUMO-1 in turquoise. Isopeptide bond between Lys524 of RanGAP1 and Gly97 of SUMO-1 is shown as sticks with carbon yellow, nitrogen blue and oxygen red. (b) A superposition of the SENP1(C603A)–RanGAP1–SUMO-1 complex with the Ubc9–RanBP2–RanGAP1–SUMO-1 complex²³. Only RanGAP1 (magenta) and SUMO-1 (teal) are shown from the Ubc9–RanBP2–RanGAP1–SUMO-1 complex; Ubc9 and RanBP2 are omitted. The isopeptide bond between Lys524 of RanGAP1 and Gly97 of SUMO-1 is shown as sticks with carbon green, nitrogen blue and oxygen red. The superposition is based on the 97 Cα atoms of SUMO-1. (c) Details of the complex in a. Residues mentioned in the text are indicated. (d) Details of the superposition in b. The more common trans orientation of the amide nitrogens in the isopeptide seen in the Ubc9–RanBP2–RanGAP1–SUMO-1 complex would result in severe steric clashes with Ser601 of SENP1. This forces the isopeptide to adopt a cis arrangement of the amide nitrogens.

SENP1 C603A induces a conformational change in substrates in solution. (a) Diagram of FRET substrates for SENP1 processing (C-terminal hydrolase) activity against full-length SUMO-1 or SUMO-2. Linear fusions YFP–SUMO-FL-ECFP (listed N terminus to C terminus) were purified from bacteria. The sequence on the C-terminal side of the scissile bond (arrowhead) is shown. (b) Diagram of FRET substrates for SENP1 deconjugation (isopeptidase) activity. Bacterially produced YFP-RanGAP1 was conjugated to either ECFP–SUMO-1-GG or ECFP–SUMO-2-GG in vitro and the conjugates purified. (c) Conformational change in RanGAP1–SUMO and SUMO-FL upon SENP1 C603A binding in solution. YFP–RanGAP1–SUMO-1–ECFP, YFP–RanGAP1–SUMO-2–ECFP, YFP–SUMO-1-FL–ECFP or YFP–SUMO-2-FL–ECFP (1,000 nM each) was mixed with indicated concentrations of the inactive protease mutant SENP1 C603A. FRET signals (Methods) were standardized against ‘buffer alone’ control. Binding of SENP1 C603A to the FRET SUMO constructs results in a change in the intensity of the FRET signal that represents a change in the distance between the two fluorophores. Measurements were in triplicate; error bars (±1 s.e.m.) are obscured by chart symbols.

Steady-state kinetic analysis of isopeptidase and C-terminal hydrolase activities of SENP1 for SUMO-1 and SUMO-2 substrates. (a,b) Relationship between initial rate of cleavage by SENP1 and concentration of substrate, for RanGAP1–SUMO-1, RanGAP–SUMO-2, SUMO-1-FL and SUMO-2-FL. FRET-based assays (Methods) used the substrates diagrammed in Figure 3a,b. Initial rates at different substrate concentrations were fit using nonlinear regression to the Michaelis-Menten equation. We used 0.625 nM SENP1 in all assays except those containing SUMO-2-FL, which contained 10 nM SENP1. (c,d) Effect of product inhibition on SENP1 isopeptidase and C-terminal hydrolase activities. FRET-based SENP1 protease assays had a fixed concentration of the SUMO substrate (500 nM) with indicated range of SUMO-1-GG and SUMO-2-GG concentrations. Charts contain error bars (±1 s.e.m.), which in most cases are obscured by chart symbols. Kinetic parameters are indicated in Table 2.