PDBsum entry 2e1p

Hydrolase

PDB id: 2e1p

Contents

Protein chain

395 a.a. *

Metals

_CA ×6

Waters ×270

* Residue conservation analysis

PDB id:

2e1p

Name:

Hydrolase

Title:

Crystal structure of pro-tk-subtilisin

Structure:

Tk-subtilisin. Chain: a. Synonym: pro-tk-subtilisin. Engineered: yes. Mutation: yes

Source:

Thermococcus kodakarensis. Organism_taxid: 69014. Strain: kod1. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.30Å R-factor: 0.192 R-free: 0.243

Authors:

S.Tanaka,K.Saito,H.Chon,H.Matsumura,Y.Koga,K.Takano,S.Kanaya

Key ref:

S.Tanaka et al. (2007). Crystal structure of unautoprocessed precursor of subtilisin from a hyperthermophilic archaeon: evidence for Ca2+-induced folding. J Biol Chem, 282, 8246-8255. PubMed id: 17237225 DOI: 10.1074/jbc.M610137200

Date:

27-Oct-06 Release date: 16-Jan-07

Protein chain

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

Seq: 422 a.a.

Struc: 395 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.4.21.- - ?????

DOI no: 10.1074/jbc.M610137200

J Biol Chem 282:8246-8255 (2007)

PubMed id: 17237225

Crystal structure of unautoprocessed precursor of subtilisin from a hyperthermophilic archaeon: evidence for Ca2+-induced folding.

S.Tanaka, K.Saito, H.Chon, H.Matsumura, Y.Koga, K.Takano, S.Kanaya.

ABSTRACT

The crystal structure of an active site mutant of pro-Tk-subtilisin (pro-S324A) from the hyperthermophilic archaeon Thermococcus kodakaraensis was determined at 2.3 A 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 alphabetaalpha 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.

Selected figure(s)

Figure 2.
FIGURE 2. A stereo view of electron density around the active site (A) and Ca-1 site (B) of pro-S324A. In A, the 2F[o] - F[c] map contoured at the 1.5 level is shown. In B, the 2F[o] - F[c] maps contoured at the 2.0 and 5.0 levels are shown in blue and magenta, respectively. The active site residues, the nitrogen and oxygen atoms, and the Ca^2+ ions are indicated as in Fig. 1.

Figure 3.
FIGURE 3. Ca^2+ binding sites. The structures of the Ca-1 (A), Ca-2 to Ca-5 (B), and Ca-6 (D) sites are shown. The Ca^2+ ions and water molecules are represented by spheres. The residues that bind to the Ca^2+ ions are labeled. In C, a stereo view of the central substructure consisting of the 6m-helix, 5m-strand, and 7m-helix and the 1m-strand are shown in a ribbon drawing. One of the active site residues, Asp^115, is indicated by a stick model.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 8246-8255) copyright 2007.

Figures were selected by an automated process.