Crystal Structure of a Subtilisin Homologue, Tk-SP, from Thermococcus kodakaraensis: Requirement of a C-terminal β-Jelly Roll Domain for Hyperstability

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 Å resolution. ProN-Tk-S359A consists of the N-propeptide, subtilisin, and β-jelly roll domains. Two Ca2+ ions bind to the β-jelly roll domain. The overall structure of ProN-Tk-S359A without the β-jelly roll domain is similar to that of the bacterial propeptide:subtilisin complex, except that it does not contain Ca2+ ions. To analyze the role of the β-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/CΔJ) β-jelly roll domain. Both Tk-S359C and Tk-S359CΔJ exhibited protease activities in gel assay, indicating that the β-jelly roll domain is not required for folding or activity. However, the Tm value of Tk-S359AΔJ determined by far-UV CD spectroscopy in the presence of 10-mM CaCl2 was lower than that of Tk-S359A by 29.4 °C. The Tm value of Tk-S359A was decreased by 29.5 °C by the treatment with 10 mM ethylenediaminetetraacetic acid, indicating that the β-jelly roll domain contributes to the stabilization of Tk-S359A only in a Ca2+-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 β-jelly roll domain to the C-terminus is one of the strategies of the proteins from hyperthermophiles to adapt to high-temperature environment.