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J. Biol. Chem., Vol. 282, Issue 11, 8246-8255, March 16, 2007

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Crystal Structure of Unautoprocessed Precursor of Subtilisin from a Hyperthermophilic Archaeon

EVIDENCE FOR Ca²⁺-INDUCED FOLDING^*

Shun-ichi Tanaka, Kenji Saito, Hyongi Chon, Hiroyoshi Matsumura^¶, Yuichi Koga, Kazufumi Takano^¶, and Shigenori Kanaya¹

From the Departments of Material and Life Science and Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan, and ^¶CREST (Sosho Project), JST, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

The crystal structure of an active site mutant of pro-Tk-subtilisin (pro-S324A) from the hyperthermophilic archaeon Thermococcus kodakaraensis was determined at 2.3Å resolution. The overall structure of this protein is similar to those of bacterial subtilisin-propeptide complexes, except that the peptide bond linking the propeptide and mature domain contacts with the active site, and the mature domain contains six Ca²⁺ binding sites. The Ca-1 site is conserved in bacterial subtilisins but is formed prior to autoprocessing, unlike the corresponding sites of bacterial subtilisins. All other Ca²⁺-binding sites are unique in the pro-S324A structure and are located at the surface loops. Four of them apparently contribute to the stability of the central substructure of the mature domain. The CD spectra, 1-anilino-8-naphthalenesulfonic acid fluorescence spectra, and sensitivities to chymotryptic digestion of this protein indicate that the conformation of pro-S324A is changed from an unstable molten globule-like structure to a stable native one upon Ca²⁺ binding. Another active site mutant, pro-S324C, was shown to be autoprocessed to form a propeptide-mature domain complex in the presence of Ca²⁺. The CD spectra of this protein indicate that the structure of pro-S324C is changed upon Ca²⁺ binding like pro-S324A but is not seriously changed upon subsequent autoprocessing. These results suggest that the maturation process of Tk-subtilisin is different from that of bacterial subtilisins in terms of the requirement of Ca²⁺ for folding of the mature domain and completion of the folding process prior to autoprocessing.

Received for publication, October 30, 2006 , and in revised form, December 21, 2006.

The atomic coordinates and structure factors (code 2E1P) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by a Grant-in-aid for National Project on Protein Structural and Functional Analyses and by an Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel./Fax: 81-6-6879-7938; E-mail: kanaya{at}mls.eng.osaka-u.ac.jp.

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