PDBsum entry 3a3o

Hydrolase

PDB id: 3a3o

Contents

Protein chains

318 a.a. *

60 a.a. *

Metals

_CA ×7

_ZN

Waters ×270

* Residue conservation analysis

PDB id:

3a3o

Name:

Hydrolase

Title:

Crystal structure of complex between sa-subtilisin and tk-propeptide with deletion of the fivE C-terminal residues

Structure:

Tk-subtilisin. Chain: a. Fragment: residue in unp 94-422. Engineered: yes. Mutation: yes. Tk-subtilisin. Chain: b. Fragment: tk-propeptide, residue in unp 25-88. Engineered: yes

Source:

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

Resolution:

1.90Å R-factor: 0.166 R-free: 0.208

Authors:

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

Key ref:

S.Tanaka et al. (2009). Identification of the interactions critical for propeptide-catalyzed folding of Tk-subtilisin. J Mol Biol, 394, 306-319. PubMed id: 19766655 DOI: 10.1016/j.jmb.2009.09.028

Date:

15-Jun-09 Release date: 18-Aug-09

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: 60 a.a.

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

Enzyme reactions

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

DOI no: 10.1016/j.jmb.2009.09.028

J Mol Biol 394:306-319 (2009)

PubMed id: 19766655

Identification of the interactions critical for propeptide-catalyzed folding of Tk-subtilisin.

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

ABSTRACT

Tk-subtilisin requires Ca(2+) for folding. This folding is accelerated by the chaperone function of its propeptide (Tkpro). Several Tkpro and Tk-subtilisin derivatives were constructed to examine whether the interactions between the C-terminal extended region of Tkpro and Tk-subtilisin and Glu61/Asp63- and Glu201-mediated hydrogen bonds at the domain interface are important for the chaperone function of Tkpro. The Tkpro derivatives with a series of C-terminal truncations and double mutations at Glu61 and Asp63 exhibited weaker chaperone functions than Tkpro for SA-subtilisin (active-site mutant of Tk-subtilisin). Good correlation was observed between their chaperone functions and binding abilities to the folded SA-subtilisin protein. These results suggest that the C-terminal extended region, Glu61, and Asp63 of Tkpro are not critical for folding of Tk-subtilisin but accelerate it by binding to a folding intermediate of Tk-subtilisin with a native-like structure at their binding sites. In contrast, Tkpro exhibited little chaperone function for E201A/SA-subtilisin. It could bind to the folded E201A/SA-subtilisin protein with a lower association constant than that for SA-subtilisin. These results suggest a loop of Tkpro, which interacts with Glu201 of Tk-subtilisin through hydrogen bonds and is required for folding of Tk-subtilisin by binding to a folding intermediate of Tk-subtilisin with a nonnative structure. Because this loop is fairly hydrophobic and tightly packs to the surface parallel helices of the central alphabetaalpha substructure of Tk-subtilisin, binding of this loop to Glu201 may induce association of these two helices and thereby formation of the alphabetaalpha substructure. We propose that Glu201-mediated interactions are critical for initiation of Tkpro-catalyzed folding of Tk-subtilisin.

Selected figure(s)

Figure 1.
Fig. 1. Schematic representation of the primary structure of Pro-Tk-subtilisin. The shaded box represents a propeptide, and the open box represents a mature domain. The locations of the active-site residues and the regions of the recombinant proteins analyzed in this study are shown. The location of the mutated residue and the regions of the mutant proteins are shown in parentheses. The N- and C-terminal residues of the propeptide and mature domain regions are also shown. The GenBank accession number of this sequence is AB056701.

Figure 2.
Fig. 2. Stereo view of the three-dimensional structure of the Tkpro:SA-subtilisin complex. The entire structure of the Tkpro:SA-subtilisin complex (PDB code: 2Z30) (a) and the structure around Glu201 (b) are shown. Tkpro and SA-subtilisin are colored pink and lime green, respectively. The Ca^2+ ions are shown in cyan spheres. For the entire structure, two acidic residues (Glu61 and Asp63), six C-terminal residues of Tkpro (His64-Leu69), and Glu201 are indicated by stick models. 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 these stick models, the oxygen and nitrogen atoms are colored red and blue, respectively. Glu61/Asp63-mediated hydrogen bonds are shown by orange broken lines. N and C represent the N- and C-termini, respectively. For the structure around Glu201, Glu201, Tyr31, and Phe33-Pro37 are indicated by stick models, in which the oxygen and nitrogen atoms are colored red and blue, respectively. Glu201-mediated hydrogen bonds are shown by orange broken lines.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 394, 306-319) copyright 2009.

Figures were selected by an automated process.