PDBsum entry 1v2v

Hydrolase

PDB id: 1v2v

Contents

Protein chain

223 a.a. *

Ligands

SO4

BEN

Metals

_CA

Waters ×53

* Residue conservation analysis

PDB id:

1v2v

Name:

Hydrolase

Title:

Benzamidine in complex with bovine trypsin variant x(ssai)bt.C1

Structure:

Trypsin. Chain: t. Synonym: beta-trypsin. Engineered: yes. Mutation: yes

Source:

Bos taurus. Cattle. Organism_taxid: 9913. Tissue: pancreas. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.80Å R-factor: 0.222 R-free: 0.249

Authors:

D.Rauh,G.Klebe,M.T.Stubbs

Key ref:

D.Rauh et al. (2004). Understanding protein-ligand interactions: the price of protein flexibility. J Mol Biol, 335, 1325-1341. PubMed id: 14729347 DOI: 10.1016/j.jmb.2003.11.041

Date:

17-Oct-03 Release date: 01-Jun-04

Protein chain

P00760  (TRY1_BOVIN) -  Serine protease 1 from Bos taurus

Seq: 246 a.a.

Struc: 223 a.a.*

* PDB and UniProt seqs differ at 4 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/j.jmb.2003.11.041

J Mol Biol 335:1325-1341 (2004)

PubMed id: 14729347

Understanding protein-ligand interactions: the price of protein flexibility.

D.Rauh, G.Klebe, M.T.Stubbs.

ABSTRACT

In order to design selective, high-affinity ligands to a target protein, it is advantageous to understand the structural determinants for protein-ligand complex formation at the atomic level. In a model system, we have successively mapped the factor Xa binding site onto trypsin, showing that certain mutations influence both protein structure and inhibitor specificity. Our previous studies have shown that introduction of the 172SSFI175 sequence of factor Xa into rat or bovine trypsin results in the destabilisation of the intermediate helix with burial of Phe174 (the down conformation). Surface exposure of the latter residue (the up conformation) is critical for the correct formation of the aromatic box found in factor Xa-ligand complexes. In the present study, we investigate the influence of aromatic residues in position 174. Replacement with the bulky tryptophan (SSWI) shows reduced affinity for benzamidine-based inhibitors (1) and (4), whereas removal of the side-chain (alanine, SSAI) or exchange with a hydrophilic residue (arginine, SSRI) leads to a significant loss in affinity for all inhibitors studied. The variants could be crystallised in the presence of different inhibitors in multiple crystal forms. Structural characterisation of the variants revealed three different conformations of the intermediate helix and 175 loop in SSAI (down, up and super-up), as well as a complete disorder of this region in one crystal form of SSRI, suggesting that the compromised affinity of these variants is related to conformational flexibility. The influence of Glu217, peripheral to the ligand-binding site in factor Xa, was investigated. Introduction of Glu217 into trypsin variants containing the SSFI sequence exhibited enhanced affinity for the factor Xa ligands (2) and (3). The crystal structures of these variants also exhibited the down and super-up conformations, the latter of which could be converted to up upon soaking and binding of inhibitor (2). The improved affinity of the Glu217-containing variants appears to be due to a shift towards the up conformation. Thus, the reduction in affinity caused by conformational variability of the protein target can be partially or wholly offset by compensatory binding to the up conformation. The insights provided by these studies will be helpful in improving our understanding of ligand binding for the drug design process.

Selected figure(s)

Figure 2.
Figure 2. Stereo view showing the alternative binding modes adopted by inhibitor (4) in the two structures (a) X(SSYI)bT.A4 and (b) X(SSYI)bT.B4. In a, the glycine spacer hydrogen bonds to Gly216; the tosyl group of the inhibitor occupies the S3/S4 site. In b, the glycine spacer hydrogen bonds to Gly219; the tosyl group points away from the enzyme, making contacts with a symmetry-related molecule in the crystal (not shown).

Figure 5.
Figure 5. a, Stereo view of the superposition of X(SSRI)bT.C1 (pink) and factor Xa (silver). For simplicity, the inhibitor (1) is not shown. Note the re-registration of residues Ser171-Ser178 and the formation of a hydrogen bond between Ser178 O and Asn233 Nd2 in the case of X(SSRI)bT.C1. b, Stereo diagram of the experimental electron density for X(SSRI)bT.B4; residues 169-175 (green) are disordered, and only partial density is present for Trp215 (violet). The ligand (4) is well defined, binding as seen in Figure 2b. Density for the cystine Cys168-Cys182 (orange) corresponds to the up conformation.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 335, 1325-1341) copyright 2004.

Figures were selected by an automated process.