title: Crystal structure of a subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis: requirement of a C-terminal beta-jelly roll domain for hyperstability.
abstract: Tk-SP is a hyperthermostable subtilisin-like serine protease from Thermococcus kodakaraensis and is autoprocessed from its precursor (Pro-Tk-SP) with N- and C-propeptides. The crystal structure of the active-site mutant of Pro-Tk-SP lacking C-propeptide, ProN-Tk-S359A, was determined at 2.0 A resolution. ProN-Tk-S359A consists of the N-propeptide, subtilisin, and beta-jelly roll domains. Two Ca(2+) ions bind to the beta-jelly roll domain. The overall structure of ProN-Tk-S359A without the beta-jelly roll domain is similar to that of the bacterial propeptide:subtilisin complex, except that it does not contain Ca(2+) ions. To analyze the role of the beta-jelly roll domain of Tk-SP, we constructed a series of the active-site mutants of Tk-SP with (Tk-S359A/C) and without (Tk-S359A/CDeltaJ) beta-jelly roll domain. Both Tk-S359C and Tk-S359CDeltaJ exhibited protease activities in gel assay, indicating that the beta-jelly roll domain is not required for folding or activity. However, the T(m) value of Tk-S359ADeltaJ determined by far-UV CD spectroscopy in the presence of 10-mM CaCl(2) was lower than that of Tk-S359A by 29.4 degrees C. The T(m) value of Tk-S359A was decreased by 29.5 degrees C by the treatment with 10 mM ethylenediaminetetraacetic acid, indicating that the beta-jelly roll domain contributes to the stabilization of Tk-S359A only in a Ca(2+)-bound form. Tk-SP highly resembles subtilisin-like serine proteases from Pyrococcus furiosus, Thermococcus gammatolerans, and Thermococcus onnurineus in size and amino acid sequence. We propose that attachment of a beta-jelly roll domain to the C-terminus is one of the strategies of the proteins from hyperthermophiles to adapt to high-temperature environment.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tita
authors.initials: T
authors.lastname: Foophow
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Tanaka
authors.forename: Clement
authors.initials: C
authors.lastname: Angkawidjaja
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Koga
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Takano
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006358:Hot Temperature; D008958:Models, Molecular; D008970:Molecular Weight; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D011489:Protein Denaturation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000201:enzymology; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D057057:Serine Proteases / Q000737:chemistry* / Q000235:genetics; D019712:Thermococcus / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D002413:Cations, Divalent; D050505:Mutant Proteins; D057057:Serine Proteases; D002118:Calcium