1L7I

Crystal Structure of the anti-ErbB2 Fab2C4


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.230 
  • R-Value Work: 0.197 

wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis.

Vajdos, F.F.Adams, C.W.Breece, T.N.Presta, L.G.de Vos, A.M.Sidhu, S.S.

(2002) J Mol Biol 320: 415-428

  • DOI: https://doi.org/10.1016/S0022-2836(02)00264-4
  • Primary Citation of Related Structures:  
    1L7I

  • PubMed Abstract: 

    Shotgun scanning combinatorial mutagenesis was used to study the antigen-binding site of Fab2C4, a humanized monoclonal antibody fragment that binds to the extracellular domain of the human oncogene product ErbB2. Essentially all the residues in the Fab2C4 complementarity determining regions (CDRs) were alanine-scanned using phage-displayed libraries that preferentially allowed side-chains to vary as the wild-type or alanine. A separate homolog-scan was performed using libraries that allowed side-chains to vary only as the wild-type or a similar amino acid residue. Following binding selections to isolate functional clones, DNA sequencing was used to determine the wild-type/mutant ratios at each varied position, and these ratios were used to assess the contributions of each side-chain to antigen binding. The alanine-scan revealed that most of the side-chains that contribute to antigen binding are located in the heavy chain, and the Fab2C4 three-dimensional structure revealed that these residues fall into two groups. The first group consists of solvent-exposed residues which likely make energetically favorable contacts with the antigen and thus comprise the functional-binding epitope. The second group consists of buried residues with side-chains that pack against other CDR residues and apparently act as scaffolding to maintain the functional epitope in a binding-competent conformation. The homolog-scan involved subtle mutations, and as a result, only a subset of the side-chains that were intolerant to alanine substitutions were also intolerant to homologous substitutions. In particular, the 610 A2 functional epitope surface revealed by alanine-scanning shrunk to only 369 A2 when mapped with homologous substitutions, suggesting that this smaller subset of side-chains may be involved in more precise contacts with the antigen. The results validate shotgun scanning as a rapid and accurate method for determining the functional contributions of individual side-chains involved in protein-protein interactions.


  • Organizational Affiliation

    Department of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
chimera of Fab2C4: "humanized" murine monoclonal antibodyA [auth L]214Homo sapiensMutation(s): 0 
UniProt
Find proteins for Q7Z3Y4 (Homo sapiens)
Explore Q7Z3Y4 
Go to UniProtKB:  Q7Z3Y4
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ7Z3Y4
Sequence Annotations
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  • Reference Sequence
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
chimera of Fab2C4: "humanized" murine monoclonal antibodyB [auth H]222Homo sapiensMutation(s): 0 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations
Expand
  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.230 
  • R-Value Work: 0.197 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 41.966α = 90
b = 64.249β = 105.44
c = 79.445γ = 90
Software Package:
Software NamePurpose
MOSFLMdata reduction
SCALAdata scaling
AMoREphasing
CNXrefinement
CCP4data scaling

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

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