PDBsum entry 2esu

Hydrolase inhibitor

PDB id: 2esu

Contents

Protein chain

173 a.a. *

Ligands

SO4

Metals

_NI

Waters ×304

* Residue conservation analysis

PDB id:

2esu

Name:

Hydrolase inhibitor

Title:

Crystal structure of asn to gln mutant of winged bean chymotrypsin inhibitor protein

Structure:

Chymotrypsin inhibitor 3. Chain: a. Synonym: wci-3. Engineered: yes. Mutation: yes

Source:

Psophocarpus tetragonolobus. Winged bean. Organism_taxid: 3891. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.94Å R-factor: 0.219 R-free: 0.238

Authors:

J.K.Dattagupta,U.Sen,J.Dasgupta,S.Khamrui

Key ref:

J.Dasgupta et al. (2006). Spacer Asn determines the fate of Kunitz (STI) inhibitors, as revealed by structural and biochemical studies on WCI mutants. Biochemistry, 45, 6783-6792. PubMed id: 16734415 DOI: 10.1021/bi060374q

Date:

27-Oct-05 Release date: 13-Jun-06

Protein chain

P10822  (ICW3_PSOTE) -  Chymotrypsin inhibitor 3 from Psophocarpus tetragonolobus

Seq: 207 a.a.

Struc: 173 a.a.*

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

DOI no: 10.1021/bi060374q

Biochemistry 45:6783-6792 (2006)

PubMed id: 16734415

Spacer Asn determines the fate of Kunitz (STI) inhibitors, as revealed by structural and biochemical studies on WCI mutants.

J.Dasgupta, S.Khamrui, J.K.Dattagupta, U.Sen.

ABSTRACT

The scaffold of serine protease inhibitors plays a significant role in the process of religation which resists proteolysis of the inhibitor in comparison to a substrate. Although the role of the conserved scaffolding Asn residue was previously implicated in the maintenance of the binding loop conformation of Kunitz (STI) inhibitors, its possible involvement in the prevention of proteolysis is still unexplored. In this paper, we have investigated the specific role of the spacer Asn in the prevention of proteolysis through structural and biochemical studies on the mutants where Asn14 of winged bean chymotrypsin inhibitor (WCI) has been replaced by Gly, Ala, Thr, Leu, and Gln. A residue having no side chain or beta-branching at the 14th position creates deformation and insufficient protrusion of the binding loop, and as a result N14G and N14T lose the ability to recognize proteases. Although the reactive site loop conformation of N14A and N14Q are almost identical to WCI, biochemical results present N14A as a substrate indicating that the methyl group of Ala14 is not suitable to capture the cleaved parts together for religation. The poor inhibitory power of N14L points toward the chemical incompatibility of Leu at the 14th position, although its size is the same as Asn; on the other hand, slight loss of inhibitory potency of N14Q is attributed to the inappropriate placement of the Gln14 polar head, caused by the strained accommodation of its bigger side chain. These observations collectively allow us to conclude that the side chain of spacer Asn fits snugly into the concave space of the reactive site loop cavity and its ND2 atom forms hydrogen bonds with the P2 and P1' carbonyl O at either side of the scissile bond holding the cleaved products together for religation. Through database analysis, we have identified such spacer asparagines in five other families of serine protease inhibitors with a similar disposition of their ND2 atoms, which supports our proposition.