PDBsum entry 1j14

Hydrolase

PDB id: 1j14

Contents

Protein chain

223 a.a. *

Ligands

SO4

BEN

Metals

_CA

Waters ×64

* Residue conservation analysis

PDB id:

1j14

Name:

Hydrolase

Title:

Benzamidine in complex with rat trypsin mutant x99rt

Structure:

Trypsin ii, anionic. Chain: a. Synonym: trypsinogen, beta-trypsin, pretrypsinogen ii. Engineered: yes. Mutation: yes

Source:

Rattus norvegicus. Norway rat. Organism_taxid: 10116. Tissue: pancreas. Expressed in: saccharomyces cerevisiae. Expression_system_taxid: 4932.

Biol. unit:

Dimer (from PQS)

Resolution:

2.40Å R-factor: 0.193

Authors:

M.T.Stubbs

Key ref:

S.Reyda et al. (2003). Reconstructing the binding site of factor Xa in trypsin reveals ligand-induced structural plasticity. J Mol Biol, 325, 963-977. PubMed id: 12527302 DOI: 10.1016/S0022-2836(02)01337-2

Date:

30-Nov-02 Release date: 23-Dec-02

Protein chain

P00763  (TRY2_RAT) -  Anionic trypsin-2 from Rattus norvegicus

Seq: 246 a.a.

Struc: 223 a.a.*

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

Enzyme reactions

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

DOI no: 10.1016/S0022-2836(02)01337-2

J Mol Biol 325:963-977 (2003)

PubMed id: 12527302

Reconstructing the binding site of factor Xa in trypsin reveals ligand-induced structural plasticity.

S.Reyda, C.Sohn, G.Klebe, K.Rall, D.Ullmann, H.D.Jakubke, M.T.Stubbs.

ABSTRACT

In order to investigate issues of selectivity and specificity in protein-ligand interactions, we have undertaken the reconstruction of the binding pocket of human factor Xa in the structurally related rat trypsin by site-directed mutagenesis. Three sequential regions (the "99"-, the "175"- and the "190"- loops) were selected as representing the major structural differences between the ligand binding sites of the two enzymes. Wild-type rat trypsin and variants X99rT and X(99/175/190)rT were expressed in yeast, and analysed for their interaction with factor Xa and trypsin inhibitors. For most of the inhibitors studied, progressive loop replacement at the trypsin surface resulted in inhibitory profiles akin to factor Xa. Crystals of the variants were obtained in the presence of benzamidine (3), and could be soaked with the highly specific factor Xa inhibitor (1). Binding of the latter to X99rT results in a series of structural adaptations to the ligand, including the establishment of an "aromatic box" characteristic of factor Xa. In X(99/175/190)rT, introduction of the 175-loop results in a surprising re-orientation of the "intermediate helix", otherwise common to trypsin and factor Xa. The re-orientation is accompanied by an isomerisation of the Cys168-Cys182 disulphide bond, and burial of the critical Phe174 side-chain. In the presence of (1), a major re-organisation of the binding site takes place to yield a geometry identical to that of factor Xa. In all, binding of (1) to trypsin and its variants results in significant structural rearrangements, inducing a binding surface strongly reminiscent of factor Xa, against which the inhibitor was optimised. The structural data reveal a plasticity of the intermediate helix, which has been implicated in the functional cofactor dependency of many trypsin-like serine proteinases. This approach of grafting loops onto scaffolds of known related structures may serve to bridge the gap between structural genomics and drug design.

Selected figure(s)

Figure 3.
Figure 3. (a) Experimental 2Fo 2 Fc electron density, contoured at a level of 1s, for co-crystals of (3) with X99rT. One half of the desired aromatic box is formed by the side-chains of Y99 and W215. (b) Soaking of the crystals with (1) reveals the inhibitor to bind in an extended conformation, with its chloronaphthyl group buried deep in the primary specificity pocket and the piperidinyl and piperidinyl rings in the position of the hydrophobic box of factor Xa. The side-chain of Y217 rotates from its position seen in (3) --X99rT so that its phenolic moiety approaches the hydro- phobic/aromatic moieties of the inhibitor.

Figure 5.
Figure 5. (a) Ribbon representation showing the orientation of the intermediate helix (blue) in wild-type rat trypsin; colour coding and orientation as in Figure 1. ( p ) Indicates the cystine C168-C182, which is in a right-handed helical conformation. Only side-chains of selected residues are shown for clarity. (b) In X(99/175/190)rT --(3) (yellow), the helix is tilted by ca 208, with unwinding of the final turn. Cystine C168-C182 ( p ) isomerises to an extended fully trans form, while F174 becomes buried in the body of the enzyme. (c) Stereo overlay of X(99/175/190)rT -- (3) and wild- type rat trypsin, showing the cavity formed by the disulphide and the side-chains of I176, W215, P225 and V227. The aromatic side-chain of F174 in X(99/175/190)rT -- (3) superimposes with that of trypsin Y172.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 325, 963-977) copyright 2003.

Figures were selected by an automated process.