title: Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis.
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.
authors.affiliation: Department of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Felix F
authors.initials: FF
authors.lastname: Vajdos
authors.forename: Camellia W
authors.initials: CW
authors.lastname: Adams
authors.forename: Timothy N
authors.initials: TN
authors.lastname: Breece
authors.forename: Leonard G
authors.initials: LG
authors.lastname: Presta
authors.forename: Abraham M
authors.initials: AM
authors.lastname: de Vos
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D015723:Gene Library; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library; D017354:Point Mutation; D011485:Protein Binding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry* / Q000276:immunology*
chemical_list: D000409:Alanine; D000906:Antibodies; D000939:Epitopes; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases