Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS/Aix-Marseille Université, Campus Luminy, Marseille, France.
PLoS One. 2013 Oct 11;8(10):e77226. doi: 10.1371/journal.pone.0077226. eCollection 2013.
The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.
先前已经使用生化和突变方法定义了针对 Electrophorus electricus 乙酰胆碱酯酶（AChE）生成的单克隆抗体（mAbs） Elec403、Elec408 和 Elec410 的抑制特性和靶位。Elec403 和 Elec410 与彼此和肽毒素抑制剂 fasciculin 竞争性结合，针对位于 AChE 外周阴离子位点的独特但重叠的表位，该位点环绕活性位点峡谷的入口。Elec408 与其他两种 mAbs 或 fasciculin 均不具有竞争性，针对位于峡谷入口远处的后门区域的第二个表位。为了表征决定其结合位点特异性的分子决定因素，我们克隆和测序了 mAbs；生成保留亲本抑制特性的抗原结合片段（Fab）；并使用互补的 X 射线晶体学、同源建模和灵活对接方法探索了它们的结构-功能关系。Elec403 的一个互补决定区的超突变表明发生了抗原驱动的选择，以识别 AChE 外周位点。Fab408 的 1.9Å 分辨率结构和其 Fab403 和 Fab410 同源物的理论模型的比较分析表明，独特的表面形貌和各向异性的电荷分布，与它们各自的靶位和抑制特性一致。最后，经过验证的、基于数据的 Fab403-AChE 复合物对接模型表明了在 PAS 处的结合模式，该模式与功能数据完全相关。这项综合研究记录了 Fab403 和 Fab410 的分子特性，它们是针对外周位点的最大肽类抑制剂，以及 Fab408 的分子特性，它是针对 AChE 后门区域的第一个抑制剂，也是设计新型、特异性 AChE 催化调节剂的独特模板。
