6DG4
Structure of the Chaetomium thermophilum Ulp1-like SUMO protease catalytic domain
Summary for 6DG4
| Entry DOI | 10.2210/pdb6dg4/pdb |
| Descriptor | Ulp1-like SUMO protease, SULFATE ION, GLYCEROL, ... (4 entities in total) |
| Functional Keywords | sumo hydrolase, ubiquitin-like protease, smt3 hydrolase, desumoylating enzyme, cysteine protease, sumo processing enzyme, smt3 processing enzyme, hydrolase |
| Biological source | Chaetomium thermophilum |
| Total number of polymer chains | 1 |
| Total formula weight | 30010.11 |
| Authors | Lima, C.D.,Baytshtok, V. (deposition date: 2018-05-16, release date: 2018-07-11, Last modification date: 2023-10-11) |
| Primary citation | Lau, Y.K.,Baytshtok, V.,Howard, T.A.,Fiala, B.M.,Johnson, J.M.,Carter, L.P.,Baker, D.,Lima, C.D.,Bahl, C.D. Discovery and engineering of enhanced SUMO protease enzymes. J. Biol. Chem., 293:13224-13233, 2018 Cited by PubMed 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 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 to improve protein solubility. Second, we discovered an improved SUMO protease via genomic mining of the thermophilic fungus , as proteins from thermophilic organisms are commonly employed as reagent enzymes. Following expression in , 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 to 1.44 Å resolution. This structure revealed that this enzyme exhibits structural and functional conservation with the SUMO protease, despite exhibiting only 28% sequence identity. In summary, by re-engineering the Ulp1 protease and discovering a SUMO protease from , we have obtained proteases that are more soluble, more thermostable, and more efficient than the current commercially available Ulp1 enzyme. PubMed: 29976752DOI: 10.1074/jbc.RA118.004146 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.442 Å) |
Structure validation
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