PDBsum entry 2z56

Hydrolase

PDB id: 2z56

Contents

Protein chains

318 a.a. *

65 a.a. *

Metals

_CA ×7

_ZN

Waters ×154

* Residue conservation analysis

PDB id:

2z56

Name:

Hydrolase

Title:

Crystal structure of g56s-propeptide:s324a-subtilisin complex

Structure:

Tk-subtilisin. Chain: a. Fragment: mature domain, residue 81-398. Engineered: yes. Mutation: yes. Tk-subtilisin. Chain: b. Fragment: propeptide domain, residue 5-69. Engineered: yes.

Source:

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

Resolution:

1.90Å R-factor: 0.190 R-free: 0.210

Authors:

M.A.Pulido,S.Tanaka,C.Sringiew,D.J.You,H.Matsumura,Y.Koga,K.Takano, S.Kanaya

Key ref:

M.A.Pulido et al. (2007). Requirement of left-handed glycine residue for high stability of the Tk-subtilisin propeptide as revealed by mutational and crystallographic analyses. J Mol Biol, 374, 1359-1373. PubMed id: 17988685 DOI: 10.1016/j.jmb.2007.10.030

Date:

29-Jun-07 Release date: 01-Jan-08

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.*

Protein chain

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

Seq: 422 a.a.

Struc: 65 a.a.*

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

Enzyme reactions

• Enzyme class: Chains A, B: E.C.3.4.21.- - ?????

DOI no: 10.1016/j.jmb.2007.10.030

J Mol Biol 374:1359-1373 (2007)

PubMed id: 17988685

Requirement of left-handed glycine residue for high stability of the Tk-subtilisin propeptide as revealed by mutational and crystallographic analyses.

M.A.Pulido, S.Tanaka, C.Sringiew, D.J.You, H.Matsumura, Y.Koga, K.Takano, S.Kanaya.

ABSTRACT

Tk-subtilisin [the mature domain of Pro-Tk-subtilisin in active form (Gly70-Gly398)] from the hyperthermophilic archaeon Thermococcus kodakaraensis is matured from Pro-Tk-subtilisin [a subtilisin homologue from T. kodakaraensis in pro form (Gly1-Gly398)] upon autoprocessing and degradation of propeptide. Pro-Tk-subtilisin is characterized by extremely slow maturation at mild temperatures, but this maturation rate is greatly increased by a single Gly56-->Ser mutation in the propeptide region. To analyze the role of Gly56, which assumes a left-handed conformation, Pro-Tk-subtilisin variants with complete amino acid substitutions at Gly56 were constructed. A comparison of their halo-forming activities suggests that all variants, except for Pro-G56W [Pro-G56X, Pro-Tk-subtilisin with Gly56-->X mutation (X = any amino acid)], mature faster than WT. Pro-G56W and Pro-G56E with the lowest and highest maturation rates, respectively, among 19 variants, as well as WT and Pro-G56S, were overproduced, purified, and characterized. SDS-PAGE analyses and Tk-subtilisin activity assay indicated that their maturation rates increased in the order WT < or = Pro-G56W < Pro-G56S < Pro-G56E. The propeptides of these variants were also overproduced, purified, and characterized. The stability and inhibitory potency of these propeptides decreased in the order Tk-propeptide [propeptide of Tk-subtilisin (Gly1-Leu69)] > or = G56W-propeptide > G56S-propeptide > G56E-propeptide, indicating that they are inversely correlated with the maturation rates of Pro7-Tk-subtilisin and its derivatives. The crystal structures of these propeptides determined in complex with S324A-subtilisin indicate that the conformation of the propeptide is altered by the mutation, such that nonglycine residues at position 56 assume a right-handed conformation and hydrophobic interactions at the core region decrease. These results indicate that Gly56 is required in stabilizing the propeptide fold. Stabilization of this fold leads to strong binding of Tk-propeptide to Tk-subtilisin, high resistance of Tk-propeptide to proteolytic degradation, and slow maturation of Pro-Tk-subtilisin.

Selected figure(s)

Figure 1.
Fig. 1. Halo-forming activities of the wild-type and mutant proteins of Pro-Tk-subtilisin. The halo-forming activities of Pro-Tk-subtilisin (WT), Pro-G56S, Pro-G56W, and Pro-G56E were analyzed at the indicated temperatures as described in Materials and Methods. The E. coli HB101 transformant with pBR322 serves as a negative control.

Figure 7.
Fig. 7. Stereo views of the three-dimensional structures of the propeptide:subtilisin complexes. (a) The entire structure of the G56S-propeptide:S324A-subtilisin complex is superimposed on that of the Tk-propeptide:S324A-subtilisin complex (Protein Data Bank code 2Z30). For the structure of the G56S-propeptide:S324A-subtilisin complex, G56S-propeptide is in red and S324A-subtilisin is in green. Two active-site residues (Asp115 and His153) and Ala324, which is substituted for the active-site serine residue, are indicated by yellow stick models, in which the oxygen and nitrogen atoms are in red and blue, respectively. Seven Ca^2 + are shown in cyan spheres. N and C represent the N- and C-termini, respectively. The entire structure of the Tk-propeptide:S324A-subtilisin complex, including seven Ca^2 +, is in gray. (b–d) G56S-propeptide (b), G56E-propeptide (c), and G56W-propeptide (d) in the structures of the G56S-propeptide:S324A-subtilisin, G56E-propeptide:S324A-subtilisin, and G56W-propeptide:S324A-subtilisin complexes are superimposed on Tk-propeptide in the structure of the Tk-propeptide:S324A-subtilisin complex. The view direction is changed from (a), such that the mature domain is located behind the propeptide. The mutant and wild-type propeptides are in red and gray, respectively. Nine of the 11 residues that form a hydrophobic core and Ser56 (b), Glu56 (c), or Trp56 (d) are indicated by blue and yellow stick models, respectively. Two other core-forming residues are not shown because they are hidden.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 374, 1359-1373) copyright 2007.

Figures were selected by an automated process.