PDBsum entry 1fn0

Hydrolase inhibitor

PDB id: 1fn0

Contents

Protein chain

177 a.a. *

Ligands

SO4 ×4

Waters ×169

* Residue conservation analysis

References listed in PDB file

Key reference

Title

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.

Authors

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

Ref.

Protein Eng, 2001, 14, 349-357.

PubMed id

11438758

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.

Secondary reference #1

Title

Cryocrystallography of a kunitz-Type serine protease inhibitor: the 90 k structure of winged bean chymotrypsin inhibitor (wci) at 2.13 a resolution.

Authors

S.Ravichandran, U.Sen, C.Chakrabarti, J.K.Dattagupta.

Ref.

Acta Crystallogr D Biol Crystallogr, 1999, 55, 1814-1821. [DOI no: 10.1107/S0907444999009877]

PubMed id

10531477

Figure 7.
Figure 7 A stereoview of the first reactive-site loop (Ser61-Leu67; labelled in green) of WCI. The scissile bond is between Leu65 and Ser66. The strong hydrogen-bonding network between the main-chain atoms of the first reactive-site loop and the side-chain atoms of Asn14 (labelled in pink) is important in maintaining the correct conformation of the loop.

Figure 10.
Figure 10 A stereoview of a sulfate ion (Sul195) identified on the protein surface where the side chains of basic residues are found.

The above figures are reproduced from the cited reference with permission from the IUCr

Secondary reference #2

Title

Refined crystal structure (2.3 a) of a double-Headed winged bean alpha-Chymotrypsin inhibitor and location of its second reactive site.

Authors

J.K.Dattagupta, A.Podder, C.Chakrabarti, U.Sen, D.Mukhopadhyay, S.K.Dutta, M.Singh.

Ref.

Proteins, 1999, 35, 321-331. [DOI no: 10.1002/(SICI)1097-0134(19990515)35:3<321::AID-PROT6>3.3.CO;2-P]

PubMed id

10328267

Figure 5.
Figure 5. The first reactive site of WCI (green) is docked at the enzyme (magenta) active site (van der Waals surface is shown). shows His57 at the center having a hydrophobic environment (partially shown for clarity in viewing).

Figure 8.
Figure 8. The canonical conformation of the second reactive site loop. The side-chain of Glu39 intrudes inside the loop and stabilizes it through hydrogen bonding.

The above figures are reproduced from the cited reference with permission from John Wiley & Sons, Inc.

Secondary reference #3

Title

Cdna cloning, Expression, And rapid purification of a kunitz-Type winged bean chymotrypsin inhibitor.

Authors

S.Ghosh, M.Singh.

Ref.

Protein Expr Purif, 1997, 10, 100-106. [DOI no: 10.1006/prep.1996.0707]

PubMed id

9179296