Vajdos Felix F, Adams Camellia W, Breece Timothy N, Presta Leonard G, de Vos Abraham M, Sidhu Sachdev S
Department of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
J Mol Biol. 2002 Jul 5;320(2):415-28. doi: 10.1016/S0022-2836(02)00264-4.
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.
采用鸟枪法扫描组合诱变技术研究了Fab2C4的抗原结合位点，Fab2C4是一种人源化单克隆抗体片段，可与人癌基因产物ErbB2的细胞外结构域结合。利用噬菌体展示文库对Fab2C4互补决定区（CDR）中的几乎所有残基进行了丙氨酸扫描，该文库优先允许侧链以野生型或丙氨酸的形式变化。使用允许侧链仅以野生型或相似氨基酸残基形式变化的文库进行了单独的同源扫描。在进行结合筛选以分离功能克隆后，通过DNA测序确定每个可变位置的野生型/突变体比率，并利用这些比率评估每个侧链对抗原结合的贡献。丙氨酸扫描显示，对抗原结合有贡献的大多数侧链位于重链中，Fab2C4的三维结构表明这些残基可分为两组。第一组由溶剂暴露的残基组成，这些残基可能与抗原形成能量有利的接触，因此构成功能结合表位。第二组由埋藏的残基组成，其侧链与其他CDR残基堆积，显然起到支架作用，以维持功能表位处于有结合能力的构象。同源扫描涉及细微突变，因此，只有一部分对丙氨酸取代不耐受的侧链对同源取代也不耐受。特别是，丙氨酸扫描揭示的610 Ų功能表位表面在用同源取代作图时缩小到仅369 Ų，这表明这一较小的侧链子集可能参与与抗原更精确的接触。这些结果验证了鸟枪法扫描是一种快速准确的方法，可用于确定参与蛋白质-蛋白质相互作用的单个侧链的功能贡献。
