Discovery and engineering of enhanced SUMO protease enzymes

Abstract

Small ubiquitin-like modifier (SUMO) is commonly used as a protein fusion domain to facilitate expression and purification of recombinant proteins, and a SUMO-specific protease is then used to remove SUMO from these proteins. Although this protease is highly specific, its limited solubility and stability hamper its utility as an in vitro reagent. Here, we report improved SUMO protease enzymes obtained via two approaches. First, we developed a computational method and used it to re-engineer WT Ulp1 from Saccharomyces cerevisiae to improve protein solubility. Second, we discovered an improved SUMO protease via genomic mining of the thermophilic fungus Chaetomium thermophilum, as proteins from thermophilic organisms are commonly employed as reagent enzymes. Following expression in Escherichia coli, we found that these re-engineered enzymes can be more thermostable and up to 12 times more soluble, all while retaining WT-or-better levels of SUMO protease activity. The computational method we developed to design solubility-enhancing substitutions is based on the RosettaScripts application for the macromolecular modeling suite Rosetta, and it is broadly applicable for the improvement of solution properties of other proteins. Moreover, we determined the X-ray crystal structure of a SUMO protease from C. thermophilum to 1.44 Å resolution. This structure revealed that this enzyme exhibits structural and functional conservation with the S. cerevisiae SUMO protease, despite exhibiting only 28% sequence identity. In summary, by re-engineering the Ulp1 protease and discovering a SUMO protease from C. thermophilum, we have obtained proteases that are more soluble, more thermostable, and more efficient than the current commercially available Ulp1 enzyme.

Keywords: Rosetta; cysteine protease; enzyme kinetics; fusion protein; protein design; protein engineering; protein purification; protein solubility; small ubiquitin-like modifier (SUMO); thermostability.

Figure 1.

The SUMO protease Ulp1_WT (colored) is shown in complex with a substrate SUMO protein Smt3 (gray). Nonpolar amino acids of Ulp1_WT selected for computational design are colored orange, and residues that contact SUMO (yellow) or are part of the enzyme catalytic triad (green) were not permitted to change during design; all other residues are colored cyan. Models for Ulp1_WT and Smt3 are from PDB entry 1EUV. A, a molecular surface rendition. B, Smt3 is shown as a semitransparent molecular surface. The Ulp1_WT main chain is shown as a cartoon model, and amino acid side chains are shown as sticks. The side chains of the polar amino acid substitutions from Ulp1_R1–Ulp1_R4 are colored magenta; these and the corresponding side chains of Ulp1_WT are shown in boldface type for emphasis.

Figure 2.

A, structure-based sequence alignment of Ulp1/SENP family members. Residues belonging to the catalytic triad are marked with asterisks. Residues of Ulp1_WT that directly contact Smt3 (2) are marked above with blue dots. Vertical black lines in the SENP6 and SENP7 sequences denote gaps due to the absence of SENP6- and SENP7-specific loops in the other proteases. B, structure-based sequence alignment of Smt3/SUMO variants, including the potential SUMO ortholog from C. thermophilum (CTHT_0059470). Residues that contact Ulp1_WT in PDB entry 1EUV are marked above with blue dots. C, a cartoon model of Ulp1_WT (cyan) is shown aligned to the Robetta-predicted model of Cth SUMO protease (magenta). The substrate Smt3 from S. cerevisiae is shown as a transparent molecular surface (gray). Side chains for identically conserved active-site and substrate-binding residues are shown as sticks. Models for Ulp1_WT and Smt3 are from PDB entry 1EUV.

Figure 3.

Precipitation assays of SUMO proteases. Data points indicate the concentration of soluble protease remaining after equilibration at room temperature with increasing concentrations of the protein precipitant, PEG 8000. The y intercept of each log-linear trend line is S₀, and this value represents the theoretical maximum solubility of the protein in the absence of precipitant. Error bars, S.D. from three or more replicates.

Figure 4.

CD of SUMO proteases. A, steady-state wavelength spectra. B, thermal denaturation.

Figure 5.

Kinetic characterization of SUMO proteases. Rates of cleavage of EGFP-Smt3^GGGG-mCherry by Ulp1_WT and Rosetta-designed proteases (A) or the Cth SUMO protease (B). Rates were fit to the Michaelis–Menten equation to obtain V_max and K_m values (see Table 2). Error bars, S.D. from three or more replicates.

Figure 6.

Structure of Cth SUMO protease. A, two orthogonal views of the Cth SUMO protease (maroon; PDB code 6DG4) superimposed onto the Ulp1_WT structure (cyan; PDB code 1EUV, chain A) with the SUMO-binding interfaces colored in yellow and cyan, respectively, and depicted in stick representation. The arrow points to the catalytic cysteine in the view on the left. B, superimposed catalytic pockets of Ulp1_WT (cyan) and the apo-Cth SUMO protease (maroon/yellow) with several key residues shown in stick representation. Numbering is for the Cth protease. C, C-terminal region of Smt3 (purple) and Smt3-binding surface of Ulp1_WT (cyan) superimposed onto apo-Cth structure (maroon/yellow). Several side chains that differ between Cth and Ulp1_WT in the Ulp1_WT-Smt3 interface are shown in stick representation.