PDBsum entry 2zwp

Hydrolase

PDB id: 2zwp

Contents

Protein chains

389 a.a. *

Metals

_CA ×11

Waters ×418

* Residue conservation analysis

PDB id:

2zwp

Name:

Hydrolase

Title:

Crystal structure of ca3 site mutant of pro-s324a

Structure:

Tk-subtilisin. Chain: a, b. Engineered: yes. Mutation: yes

Source:

Thermococcus kodakaraensis. Organism_taxid: 311400. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.40Å R-factor: 0.159 R-free: 0.202

Authors:

Y.Takeuchi,S.Tanaka,H.Matsumura,Y.Koga,K.Takano,S.Kanaya

Key ref:

Y.Takeuchi et al. (2009). Requirement of a unique Ca(2+)-binding loop for folding of Tk-subtilisin from a hyperthermophilic archaeon. Biochemistry, 48, 10637-10643. PubMed id: 19813760

Date:

17-Dec-08 Release date: 23-Jun-09

Protein chains

P58502  (TKSU_THEKO) -  Tk-subtilisin from Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)

Seq: 422 a.a.

Struc: 389 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.21.- - ?????

Biochemistry 48:10637-10643 (2009)

PubMed id: 19813760

Requirement of a unique Ca(2+)-binding loop for folding of Tk-subtilisin from a hyperthermophilic archaeon.

Y.Takeuchi, S.Tanaka, H.Matsumura, Y.Koga, K.Takano, S.Kanaya.

ABSTRACT

Tk-subtilisin from the hyperthermophiolic archaeon Thermococcus kodakaraensis matures from Pro-Tk-subtilisin upon autoprocessing and degradation of Tk-propeptide [Tanaka, S., Saito, K., Chon, H., Matsumura, H., Koga, Y., Takano, K., and Kanaya, S. (2007) J. Biol. Chem. 282, 8246-8255]. It requires Ca(2+) for folding and assumes a molten globule-like structure in the absence of Ca(2+) even in the presence of Tk-propeptide. Tk-subtilisin contains seven Ca(2+)-binding sites. Four of them (Ca2-Ca5) are located within a long loop, which mostly consists of a unique insertion sequence of this protein. To analyze the role of this Ca(2+)-binding loop, three mutant proteins, Deltaloop-Tk-subtilisin, DeltaCa2-Pro-S324A, and DeltaCa3-Pro-S324A, were constructed. These proteins were designed to remove the Ca(2+)-binding loop, Ca2 site, or Ca3 site of Pro-Tk-subtilisin or its active site mutant Pro-S324A. Far-UV CD spectra of these proteins refolded in the absence and presence of Ca(2+) indicated that Deltaloop-Tk-subtilisin completely lost the ability to fold into a native structure. In contrast, two other proteins retained this ability, although their refolding rates were greatly decreased compared to that of Pro-S324A. Determination of the crystal structures of these proteins purified in a Ca(2+)-bound form indicates that the structures of DeltaCa2-Pro-S324A and DeltaCa3-Pro-S324A are virtually identical to that of Pro-S324A, except that they lack the Ca2 and Ca3 sites, respectively, and the structure of the Ca(2+)-binding loop is destabilized. Nevertheless, these proteins were slightly more stable than Pro-S324A. These results suggest that the Ca(2+)-binding loop is required for folding of Tk-subtilisin but does not seriously contribute to the stabilization of Tk-subtilisin in a native structure.