Molecular Biology and Biochemistry, University of California, Irvine, 1102 NS-2, Irvine, CA, 92697-2025, USA.
Departments of Chemistry, University of California, Irvine, 1102 NS-2, Irvine, CA, 92697-2025, USA.
Sci Rep. 2022 Jun 15;12(1):9956. doi: 10.1038/s41598-022-13617-z.
The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A's protease domain (LC/A) could expand its therapeutic applications; however, LC/A's extended substrate recognition (≈ 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A retains its ability to form full-length holotoxin, infiltrate neurons, and cleave SNAP23. The identification of substrate control loops outside BoNT/A's active site could guide the design of improved BoNT proteases and inhibitors.
A型肉毒神经毒素（BoNT/A）能切断 SNAP25 中的单一肽键，这一活性被用于治疗多种疾病。对 BoNT/A 蛋白酶结构域（LC/A）的底物特异性进行重新设计，可能会扩展其治疗应用；然而，LC/A 对底物的广泛识别（≈60 个残基）给传统方法带来了挑战。我们报告了一种定向进化方法，用于重新靶向 LC/A 并保留其精细的特异性。与之前报道的 LC/A 变体相比，所得的 8 个突变 LC/A（omLC/A）对 SNAP23 的切割特异性和催化效率分别提高了 1300 倍和 120 倍。重要的是，装备 omLC/A 的完整 BoNT/A 毒素保留了形成全长毒素、渗透神经元和切割 SNAP23 的能力。在 BoNT/A 活性位点之外发现的底物控制回路可以指导设计改进的 BoNT 蛋白酶和抑制剂。
