PDBsum entry 3vv2

Hydrolase

PDB id: 3vv2

Contents

Protein chains

320 a.a.

65 a.a.

Metals

_CL

_ZN

_CA ×7

Waters ×109

PDB id:

3vv2

Name:

Hydrolase

Title:

Crystal structure of complex form between s324a-subtilisin and mutant tkpro

Structure:

Tk-subtilisin. Chain: a. Fragment: unp residues 94-422. Engineered: yes. Mutation: yes. Propeptide from tk-subtilisin. Chain: b. Fragment: unp residues 25-93. Engineered: yes.

Source:

Thermococcus kodakarensis. Organism_taxid: 69014. Strain: kod1. Gene: tk1675. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.83Å R-factor: 0.221 R-free: 0.268

Authors:

R.Uehara,Y.Ueda,D.J.You,K.Takano,Y.Koga,S.Kanaya

Key ref:

R.Uehara et al. (2013). Accelerated maturation of Tk-subtilisin by a Leu→Pro mutation at the C-terminus of the propeptide, which reduces the binding of the propeptide to Tk-subtilisin. Febs J, 280, 994. PubMed id: 23237738 DOI: 10.1111/febs.12091

Date:

12-Jul-12 Release date: 06-Mar-13

Protein chain

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

Seq: 422 a.a.

Struc: 320 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: 10.1111/febs.12091

Febs J 280:994 (2013)

PubMed id: 23237738

Accelerated maturation of Tk-subtilisin by a Leu→Pro mutation at the C-terminus of the propeptide, which reduces the binding of the propeptide to Tk-subtilisin.

R.Uehara, Y.Ueda, D.J.You, Y.Koga, S.Kanaya.

ABSTRACT

Tk-subtilisin, a subtilisin homologue (Gly70-Gly398) from Thermococcus kodakarensis, is matured from its precursor, Pro-Tk-subtilisin [Tk-subtilisin in a pro form (Gly1-Gly398)], by autoprocessing and degradation of propeptide [Tk-propeptide, a propeptide of Tk-subtilisin (Gly1-Leu69)]. The scissile peptide bond between Leu69 and Gly70 of Pro-Tk-subtilisin is first self-cleaved to produce an inactive Tk-propeptide:Tk-subtilisin complex, in which the C-terminal region of Tk-propeptide binds to the active-site cleft of Tk-subtilisin. Tk-propeptide is then dissociated from Tk-subtilisin and degraded by Tk-subtilisin to release active Tk-subtilisin. To examine whether the mutation of Leu69 to Pro, which is the most unfavourable residue in the P1 position for subtilisins, affects the maturation of Pro-Tk-subtilisin, the Pro-Tk-subtilisin and Tk-propeptide derivatives with this mutation (Pro-L69P and L69P-propeptide) were constructed and characterized. Pro-L69P was autoprocessed more slowly than Pro-Tk-subtilisin. Nevertheless, it matured to Tk-subtilisin more rapidly than Pro-Tk-subtilisin because L69P-propeptide was degraded by Tk-subtilisin more rapidly than Tk-propeptide. The chaperone function and stability of L69P-propeptide were comparable to those of Tk-propeptide, whereas the inhibitory potency and binding ability of L69P-propeptide were considerably reduced compared to those of Tk-propeptide. The crystal structure of the complex between L69P-propeptide and S324A-subtilisin (i.e. a protease activity-defective mutant) revealed that the C-terminal region of L69P-propeptide does not well fit into the substrate binding pockets of Tk-subtilisin (S1-S4 subsites) as a result of a conformational change caused by the mutation. These results suggest that the Leu→Pro mutation accelerates the maturation of Pro-Tk-subtilisin by reducing the binding ability of Tk-propeptide to Tk-subtilisin. DATABASE: The coordinates and structure factors have been deposited in the RCSB Protein Data Bank under ID code: 3VV2. STRUCTURED DIGITAL ABSTRACT: Pro-Tk-subtilisin and Pro-Tk-subtilisin cleave by enzymatic study (View interaction) L69P-propeptide and S324A-subtilisin bind by x-ray crystallography (View interaction) Tk-propeptide binds to S324A-subtilisin by surface plasmon resonance (View interaction).