Ludwig Shivani Gupta, Kiyohara Casey L, Carlucci Laura A, Kisiela Dagmara, Sokurenko Evgeni V, Thomas Wendy Evelyn
Department of Bioengineering, University of Washington, 3720 15th Ave NE. Foege N430P, Box 355061, Seattle, USA.
Department of Microbiology, University of Washington, HSB room J267a, Box 357735, Seattle, WA, USA.
J Biol Eng. 2021 Jan 12;15(1):3. doi: 10.1186/s13036-020-00253-2.
Recognition proteins are critical in many biotechnology applications and would be even more useful if their binding could be regulated. The current gold standard for recognition molecules, antibodies, lacks convenient regulation. Alternative scaffolds can be used to build recognition proteins with new functionalities, including regulated recognition molecules. Here we test the use of the bacterial adhesin FimH as a scaffold for regulated molecular recognition. FimH binds to its native small molecule target mannose in a conformation-dependent manner that can be regulated by two types of noncompetitive regulation: allosteric and parasteric.
We demonstrate that conformational regulation of FimH can be maintained even after reengineering the binding site to recognize the non-mannosylated targets nickel or Penta-His antibody, resulting in an up to 7-fold difference in K between the two conformations. Moreover, both the allosteric and parasteric regulatory mechanisms native to FimH can be used to regulate binding to its new target. In one mutant, addition of the native ligand mannose parasterically improves the mutant's affinity for Penta-His 4-fold, even as their epitopes overlap. In another mutant, the allosteric antibody mab21 reduces the mutant's affinity for Penta-His 7-fold. The advantage of noncompetitive regulation is further illustrated by the ability of this allosteric regulator to induce 98% detachment of Penta-His, even with modest differences in affinity.
This illustrates the potential of FimH, with its deeply studied conformation-dependent binding, as a scaffold for conformationally regulated binding via multiple mechanisms.
识别蛋白在许多生物技术应用中至关重要，如果其结合能够得到调控，将会更有用。目前识别分子的金标准——抗体，缺乏便捷的调控方式。可利用替代支架构建具有新功能的识别蛋白，包括可调控的识别分子。在此，我们测试将细菌粘附素FimH用作可调控分子识别的支架。FimH以构象依赖的方式与其天然小分子靶标甘露糖结合，这种结合可通过两种非竞争性调控方式进行调节：别构调控和顺式调控。
我们证明，即使在重新设计结合位点以识别非甘露糖基化靶标镍或五聚组氨酸抗体后，FimH的构象调控仍可维持，导致两种构象之间的解离常数（K）相差高达7倍。此外，FimH固有的别构和顺式调控机制均可用于调节其与新靶标的结合。在一个突变体中，添加天然配体甘露糖可顺式地将突变体对五聚组氨酸的亲和力提高4倍，即使它们的表位重叠。在另一个突变体中，别构抗体mab21可将突变体对五聚组氨酸的亲和力降低7倍。这种别构调节剂即使在亲和力差异不大的情况下也能诱导98%的五聚组氨酸解离，进一步说明了非竞争性调控的优势。
这说明了FimH凭借其深入研究的构象依赖结合，作为通过多种机制进行构象调控结合的支架的潜力。
