1V2N

Potent factor XA inhibitor in complex with bovine trypsin variant X(99/175/190)bT


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.196 
  • R-Value Work: 0.173 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Understanding protein-ligand interactions: the price of protein flexibility

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

(2004) J Mol Biol 335: 1325-1341

  • DOI: https://doi.org/10.1016/j.jmb.2003.11.041
  • Primary Citation of Related Structures:  
    1V2J, 1V2K, 1V2L, 1V2M, 1V2N, 1V2O, 1V2P, 1V2Q, 1V2R, 1V2S, 1V2T, 1V2U, 1V2V, 1V2W

  • PubMed 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.


  • Organizational Affiliation

    Institut für Pharmazeutische Chemie der Philipps, Universität Marburg, Marbacher Weg 6, D35032 Marburg, Germany.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
TrypsinA [auth T]223Bos taurusMutation(s): 7 
EC: 3.4.21.4
UniProt
Find proteins for P00760 (Bos taurus)
Explore P00760 
Go to UniProtKB:  P00760
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00760
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
BBA
Query on BBA

Download Ideal Coordinates CCD File 
C [auth T]2,7-BIS-(4-AMIDINOBENZYLIDENE)-CYCLOHEPTAN-1-ONE
C23 H28 N4 O
BXYGSMRTHHSAHZ-PMACEKPBSA-N
CA
Query on CA

Download Ideal Coordinates CCD File 
B [auth T]CALCIUM ION
Ca
BHPQYMZQTOCNFJ-UHFFFAOYSA-N
Binding Affinity Annotations 
IDSourceBinding Affinity
BBA PDBBind:  1V2N Ki: 1250 (nM) from 1 assay(s)
Binding MOAD:  1V2N Ki: 1250 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.196 
  • R-Value Work: 0.173 
  • Space Group: P 31 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 55.24α = 90
b = 55.24β = 90
c = 111.28γ = 120
Software Package:
Software NamePurpose
CrystalCleardata collection
CrystalCleardata reduction
CNSrefinement
CrystalCleardata scaling
CNSphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-06-01
    Type: Initial release
  • Version 1.1: 2008-04-27
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2021-11-10
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-12-27
    Changes: Data collection