1l7i
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(New page: 200px<br /> <applet load="1l7i" size="450" color="white" frame="true" align="right" spinBox="true" caption="1l7i, resolution 1.80Å" /> '''Crystal Structure o...)
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Revision as of 15:51, 12 November 2007
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Crystal Structure of the anti-ErbB2 Fab2C4
Overview
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.
About this Structure
1L7I is a Protein complex structure of sequences from Homo sapiens with SO4 as ligand. Full crystallographic information is available from OCA.
Reference
Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis., Vajdos FF, Adams CW, Breece TN, Presta LG, de Vos AM, Sidhu SS, J Mol Biol. 2002 Jul 5;320(2):415-28. PMID:12079396
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