PDBsum entry 1fn0

Hydrolase inhibitor

PDB id: 1fn0

Contents

Protein chain

177 a.a. *

Ligands

SO4 ×4

Waters ×169

* Residue conservation analysis

PDB id:

1fn0

Name:

Hydrolase inhibitor

Title:

Structure of a mutant winged bean chymotrypsin inhibitor protein, n14d.

Structure:

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

Source:

Psophocarpus tetragonolobus. Winged bean. Organism_taxid: 3891. Organ: seed. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.00Å R-factor: 0.217 R-free: 0.287

Authors:

J.K.Dattagupta,C.Chakrabarti,S.Ravichandran,J.Dasgupta,S.Ghosh

Key ref:

S.Ravichandran et al. (2001). The role of Asn14 in the stability and conformation of the reactive-site loop of winged bean chymotrypsin inhibitor: crystal structures of two point mutants Asn14-->Lys and Asn14-->Asp. Protein Eng, 14, 349-357. PubMed id: 11438758

Date:

19-Aug-00 Release date: 19-Feb-01

Protein chain

P10822  (ICW3_PSOTE) -  Chymotrypsin inhibitor 3 from Psophocarpus tetragonolobus

Seq: 207 a.a.

Struc: 177 a.a.*

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

Protein Eng 14:349-357 (2001)

PubMed id: 11438758

The role of Asn14 in the stability and conformation of the reactive-site loop of winged bean chymotrypsin inhibitor: crystal structures of two point mutants Asn14-->Lys and Asn14-->Asp.

S.Ravichandran, J.Dasgupta, C.Chakrabarti, S.Ghosh, M.Singh, J.K.Dattagupta.

ABSTRACT

A double-headed chymotrypsin inhibitor, WCI, from winged bean seeds was cloned for structural and biochemical studies. The inhibitor was subjected to two point mutations at a conserved position, Asn14. This residue, known to have a pivotal role in stabilizing the first reactive-site loop (Gln63-Phe68) of the inhibitor, is highly conserved in the sequences of the other members of Kunitz (STI) family as well as in the sequences of Kazal family of serine protease inhibitors. The mutants, N14K and N14D, were subjected to biochemical assay and their characteristics were compared with those of the recombinant inhibitor (rWCI). Crystallographic studies of the recombinant and the mutant proteins are discussed. These studies were primarily aimed at understanding the importance of the protein scaffolding towards the conformational rigidity of the reactive-site loop. Our analysis reveals that, as the Lys14 side chain takes an unusual fold in N14K and the Asp14 side chain in N14D interacts with the loop residues by water-mediated hydrogen bonds, the canonical conformation of the loop has remained effectively intact in both the mutant structures. However, minor alterations such as a 2-fold increase in the inhibitory affinity towards the cognate enzyme were observed.