Ricin toxin's enzymatic subunit (RTA) has been subjected to intensive B cell epitope mapping studies using a combination of competition ELISAs, hydrogen exchange-mass spectrometry and X-ray crystallography. Those studies identified four spatially distinct clusters (I-IV) of toxin-neutralizing epitopes on the surface of RTA. Here we describe A9, a new single domain camelid antibody (VHH) that was proposed to recognize a novel epitope on RTA that straddles clusters I and III. The X-ray crystal structure of A9 bound to RTA (2.6 Å resolution) revealed extensive antibody contact with RTA's β-strand h (732 Å2 buried surface area; BSA), along with limited engagement with α-helix D (90 Å2) and α-helix C (138 Å2). Collectively, these contacts explain the overlap between epitope clusters I and III, as identified by competition ELISA. However, considerable binding affinity, and, consequently, toxin-neutralizing activity of A9 is mediated by an unusual CDR2 containing five consecutive Gly residues that interact with α-helix B (82 Å2), a known neutralizing hotspot on RTA. Removal of a single Gly residue from the penta-glycine stretch in CDR2 reduced A9's binding affinity by 10-fold and eliminated toxin-neutralizing activity. Computational modeling indicates that removal of a Gly from CDR2 does not perturb contact with RTA per se, but results in the loss of an intramolecular hydrogen bond network involved in stabilizing CDR2 in the unbound state. These results reveal a novel configuration of a CDR2 element involved in neutralizing ricin toxin.

中文翻译：

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单域抗体的不寻常 CDR2 元件对蓖麻毒素结合亲和力和中和活性的贡献。


结合竞争 ELISA、氢交换质谱法和 X 射线晶体学，对蓖麻毒素的酶亚基 (RTA) 进行了深入的 B 细胞表位作图研究。这些研究确定了 RTA 表面上四个空间上不同的毒素中和表位簇 (I-IV)。在这里，我们描述了 A9，一种新的单域骆驼抗体 (VHH)，它被提议用于识别跨簇 I 和 III 的 RTA 上的新表位。与 RTA 结合的 A9 的 X 射线晶体结构（2.6 Å 分辨率）揭示了抗体与 RTA 的 β 链 h（732 Å2 埋藏表面积；BSA）的广泛接触，以及与 α 螺旋 D (90 Å2) 和α-螺旋 C (138 Å2)。总的来说，这些接触解释了通过竞争 ELISA 鉴定的表位簇 I 和 III 之间的重叠。然而，A9 相当大的结合亲和力以及因此的毒素中和活性是由一个不寻常的 CDR2 介导的，该 CDR2 含有五个连续的甘氨酸残基，这些残基与 α 螺旋 B (82 Å2) 相互作用，α 螺旋 B (82 Å2) 是 RTA 上已知的中和热点。从 CDR2 中的五甘氨酸片段中去除单个甘氨酸残基可使 A9 的结合亲和力降低 10 倍，并消除毒素中和活性。计算模型表明，从 CDR2 中去除 Gly 不会干扰与 RTA 本身的接触，但会导致参与稳定 CDR2 处于未结合状态的分子内氢键网络的损失。这些结果揭示了参与中和蓖麻毒素的 CDR2 元件的新构型。