Life Sciences Division, Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, Ontario, Canada.
Life Sciences Division, Human Health Therapeutics Research Centre, National Research Council Canada, Montréal, Quebec, Canada.
J Biol Chem. 2023 Nov;299(11):105278. doi: 10.1016/j.jbc.2023.105278. Epub 2023 Sep 22.
Most immunoglobulin (Ig) domains bear only a single highly conserved canonical intradomain, inter-β-sheet disulfide linkage formed between Cys23-Cys104, and incorporation of rare noncanonical disulfide linkages at other locations can enhance Ig domain stability. Here, we exhaustively surveyed the sequence tolerance of Ig variable (V) domain framework regions (FRs) to noncanonical disulfide linkages. Starting from a destabilized V domain lacking a Cys23-Cys104 disulfide linkage, we generated and screened phage-displayed libraries of engineered Vs, bearing all possible pairwise combinations of Cys residues in neighboring β-strands of the Ig fold FRs. This approach identified seven novel Cys pairs in V FRs (Cys4-Cys25, Cys4-Cys118, Cys5-Cys120, Cys6-Cys119, Cys22-Cys88, Cys24-Cys86, and Cys45-Cys100; the international ImMunoGeneTics information system numbering), whose presence rescued domain folding and stability. Introduction of a subset of these noncanonical disulfide linkages (three intra-β-sheet: Cys4-Cys25, Cys22-Cys88, and Cys24-Cys86, and one inter-β-sheet: Cys6-Cys119) into a diverse panel of V, V, and VH domains enhanced their thermostability and protease resistance without significantly impacting expression, solubility, or binding to cognate antigens. None of the noncanonical disulfide linkages identified were present in the natural human V repertoire. These data reveal an unexpected permissiveness of Ig V domains to noncanonical disulfide linkages at diverse locations in FRs, absent in the human repertoire, whose presence is compatible with antigen recognition and improves domain stability. Our work represents the most complete assessment to date of the role of engineered noncanonical disulfide bonding within FRs in Ig V domain structure and function.
大多数免疫球蛋白 (Ig) 结构域仅具有单个高度保守的典型结构域内，β-折叠片之间的二硫键连接，该连接由 Cys23-Cys104 形成，而在其他位置掺入罕见的非典型二硫键连接可以增强 Ig 结构域的稳定性。在这里，我们彻底调查了 Ig 可变 (V) 结构域框架区 (FR) 对非典型二硫键连接的序列耐受性。从缺乏 Cys23-Cys104 二硫键连接的不稳定 V 结构域开始，我们生成并筛选了工程化 V 的噬菌体展示文库，该文库带有 Ig 折叠 FR 中相邻 β-链上所有可能的 Cys 残基的成对组合。这种方法在 V FR 中鉴定了七个新的 Cys 对（Cys4-Cys25、Cys4-Cys118、Cys5-Cys120、Cys6-Cys119、Cys22-Cys88、Cys24-Cys86 和 Cys45-Cys100；国际免疫遗传学信息系统编号），它们的存在挽救了结构域折叠和稳定性。引入这些非典型二硫键中的一部分（三个在 β-片层内：Cys4-Cys25、Cys22-Cys88 和 Cys24-Cys86，一个在 β-片层之间：Cys6-Cys119）到各种 V、V 和 VH 结构域中，提高了它们的热稳定性和蛋白酶抗性，而对表达、溶解度或与同源抗原的结合没有显著影响。在天然人类 V 库中没有发现鉴定出的任何非典型二硫键。这些数据显示 Ig V 结构域对 FR 中不同位置的非典型二硫键连接具有出人意料的允许性，而在人类库中不存在，其存在与抗原识别兼容并提高了结构域稳定性。我们的工作代表了迄今为止对 FR 内工程化非典型二硫键键合在 Ig V 结构域结构和功能中的作用的最完整评估。
