PDBsum entry 3e8l

Hydrolase inhibitor/hydrolase

PDB id: 3e8l

Contents

Protein chains

176 a.a. *

223 a.a. *

Ligands

GOL ×7

ACT ×4

SO4 ×2

EDO ×4

PEG

Metals

_NA ×3

_CA ×7

Waters ×160

* Residue conservation analysis

PDB id:

3e8l

Name:

Hydrolase inhibitor/hydrolase

Title:

The crystal structure of the double-headed arrowhead protease inhibitor a in complex with two trypsins

Structure:

Serine proteinase inhibitor a. Chain: c. Fragment: beta-trefoil fold. Engineered: yes. Cationic trypsin. Chain: a, b. Synonym: beta-trypsin. Ec: 3.4.21.4

Source:

Sagittaria sagittifolia. Arrowhead. Organism_taxid: 4451. Expressed in: escherichia coli. Expression_system_taxid: 562. Bos taurus. Bovine. Organism_taxid: 9913

Resolution:

2.48Å R-factor: 0.197 R-free: 0.245

Authors:

R.Bao,C.-H.Jiang,C.W.Chi,S.X.Lin,Y.X.Chen

Key ref:

R.Bao et al. (2009). The ternary structure of the double-headed arrowhead protease inhibitor API-A complexed with two trypsins reveals a novel reactive site conformation. J Biol Chem, 284, 26676-26684. PubMed id: 19640842

Date:

20-Aug-08 Release date: 28-Jul-09

Protein chain

Q7M1P4  (Q7M1P4_SAGSA) -  Serine proteinase inhibitor A from Sagittaria sagittifolia

Seq: 210 a.a.

Struc: 176 a.a.*

Protein chains

P00760  (TRY1_BOVIN) -  Serine protease 1 from Bos taurus

Seq: 246 a.a.

Struc: 223 a.a.

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

Enzyme reactions

• Enzyme class: Chains A, B: E.C.3.4.21.4 - trypsin.
• Reaction: Preferential cleavage: Arg-|-Xaa, Lys-|-Xaa.

J Biol Chem 284:26676-26684 (2009)

PubMed id: 19640842

The ternary structure of the double-headed arrowhead protease inhibitor API-A complexed with two trypsins reveals a novel reactive site conformation.

R.Bao, C.Z.Zhou, C.Jiang, S.X.Lin, C.W.Chi, Y.Chen.

ABSTRACT

The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a ternary complex at 2.48 A resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 A apart. The overall fold of API-A belongs to the beta-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The two P1 residues were unambiguously assigned as Leu(87) and Lys(145), and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met(83) to P5' Ala(92), adopts a novel conformation with the Leu(87) completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting of residues P5 Cys(141) to P5' Glu(150), binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering of the reactive sites.