PDBsum entry 2z2x

Hydrolase

PDB id: 2z2x

Contents

Protein chain

318 a.a. *

Metals

_CA ×7

Waters ×299

* Residue conservation analysis

PDB id:

2z2x

Name:

Hydrolase

Title:

Crystal structure of mature form of tk-subtilisin

Structure:

Tk-subtilisin. Chain: a. Engineered: yes

Source:

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

Resolution:

1.70Å R-factor: 0.140 R-free: 0.160

Authors:

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

Key ref:

S.Tanaka et al. (2007). Four New Crystal Structures of Tk-subtilisin in Unautoprocessed, Autoprocessed and Mature Forms: Insight into Structural Changes during Maturation. J Mol Biol, 372, 1055-1069. PubMed id: 17706669 DOI: 10.1016/j.jmb.2007.07.027

Date:

29-May-07 Release date: 04-Dec-07

Protein chain

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

Seq: 422 a.a.

Struc: 318 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.4.21.- - ?????

DOI no: 10.1016/j.jmb.2007.07.027

J Mol Biol 372:1055-1069 (2007)

PubMed id: 17706669

Four New Crystal Structures of Tk-subtilisin in Unautoprocessed, Autoprocessed and Mature Forms: Insight into Structural Changes during Maturation.

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

ABSTRACT

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-subtilisin) is matured from Pro-Tk-subtilisin upon autoprocessing and degradation of the propeptide. The crystal structures of the autoprocessed and mature forms of Tk-subtilisin were determined at 1.89 A and 1.70 A resolution, respectively. Comparison of these structures with that of unautoprocessed Pro-Tk-subtilisin indicates that the structure of Tk-subtilisin is not seriously changed during maturation. However, one unique Ca(2+)-binding site (Ca-7) is identified in these structures. In addition, the N-terminal region of the mature domain (Gly70-Pro82), which binds tightly to the main body in the unautoprocessed form, is disordered and mostly truncated in the autoprocessed and mature forms, respectively. Interestingly, this site is formed also in the unautoprocessed form when its crystals are soaked with 10 mM CaCl(2), as revealed by the 1.87 A structure. Along with the formation of this site, the N-terminal region (Leu75-Thr80) is disordered, with the scissile peptide bond contacting with the active site. These results indicate that the calcium ion binds weakly to the Ca-7 site in the unautoprocessed form, but is trapped upon autoprocessing. We propose that the Ca-7 site is required to promote the autoprocessing reaction by stabilizing the autoprocessed form, in which the new N terminus of the mature domain is structurally disordered. Furthermore, the crystal structure of the Tk-propeptide:S324A-subtilisin complex, which was formed by the addition of separately expressed proteins, was determined at 1.65 A resolution. This structure is virtually identical with that of the autoprocessed form, indicating that the interaction between the two domains is highly intensive and specific.

Selected figure(s)

Figure 5.
Figure 5. The seventh Ca^2+-binding site. (a) A stereo view of electron density around the Ca-7 site of the mature form is shown. The 2F[o]–F[c] map contoured at the 1.5σ and 5.0σ levels are shown in blue and magenta, respectively. The calcium ion and the water molecules are represented by cyan and red spheres, respectively. The residues and water molecules that coordinate with the calcium ion are labeled. (b) The structures of the Ca-7 sites of the autoprocessed form (lime green), mature form (orange), and unautoprocessed form 2 (gray) are superimposed. The residues that coordinate with the calcium ion are shown as a stick model.

Figure 6.
Figure 6. Stereo view of the N-terminal region of the mature domain. The structures of the autoprocessed form (lime green), mature form (orange), and unautoprocessed form 2 (gray) shown as a stick model are superimposed. Two hydrogen bonds between Gln81 O^ε1 and Tyr317 O^γ, and between Gln81 N^ε2 and Asp315 O, are shown with broken lines.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 372, 1055-1069) copyright 2007.

Figures were selected by an automated process.