1L7T

Crystal Structure Analysis of the anti-testosterone Fab fragment


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.275 
  • R-Value Work: 0.213 

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This is version 1.3 of the entry. See complete history


Literature

Crystal Structure of an in Vitro Affinity- and Specificity-matured Anti-testosterone Fab in Complex with Testosterone. IMPROVED AFFINITY RESULTS FROM SMALL STRUCTURAL CHANGES WITHIN THE VARIABLE DOMAINS

Valjakka, J.Hemminki, A.Niemi, S.Soderlund, H.Takkinen, K.Rouvinen, J.

(2002) J Biol Chem 277: 44021-44027

  • DOI: https://doi.org/10.1074/jbc.M208392200
  • Primary Citation of Related Structures:  
    1L7T, 1VPO

  • PubMed Abstract: 

    A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C(4)F(5)). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (K(d) = 3 x 10(-10) m) when compared with the wild-type (3-C(4)F(5)) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 A) and without testosterone (2.10 A) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.


  • Organizational Affiliation

    Department of Chemistry, University of Joensuu, P. O. Box 111, Finland. jarkko.valjakka@joensuu.fi


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
anti-testosterone (light chain)A [auth L]219Mus musculusMutation(s): 0 
UniProt
Find proteins for Q65ZC0 (Mus musculus)
Explore Q65ZC0 
Go to UniProtKB:  Q65ZC0
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ65ZC0
Sequence Annotations
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  • Reference Sequence
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
anti-testosterone (heavy chain)B [auth H]221Mus musculusMutation(s): 0 
UniProt
Find proteins for Q91Z05 (Mus musculus)
Explore Q91Z05 
Go to UniProtKB:  Q91Z05
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ91Z05
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.275 
  • R-Value Work: 0.213 
  • Space Group: C 2 2 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 71.695α = 90
b = 88.419β = 90
c = 143.868γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
CNSrefinement
CNSphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2002-10-02
    Type: Initial release
  • Version 1.1: 2008-04-28
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2018-04-04
    Changes: Data collection