School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Toxins (Basel). 2024 Mar 4;16(3):133. doi: 10.3390/toxins16030133.
Mastering selective molecule trafficking across human cell membranes poses a formidable challenge in healthcare biotechnology while offering the prospect of breakthroughs in drug delivery, gene therapy, and diagnostic imaging. The cholera toxin B-subunit (CTB) has the potential to be a useful cargo transporter for these applications. CTB is a robust protein that is amenable to reengineering for diverse applications; however, protein redesign has mostly focused on modifications of the N- and C-termini of the protein. Exploiting the full power of rational redesign requires a detailed understanding of the contributions of the surface residues to protein stability and binding activity. Here, we employed Rosetta-based computational saturation scans on 58 surface residues of CTB, including the GM1 binding site, to analyze both ligand-bound and ligand-free structures to decipher mutational effects on protein stability and GM1 affinity. Complimentary experimental results from differential scanning fluorimetry and isothermal titration calorimetry provided melting temperatures and GM1 binding affinities for 40 alanine mutants among these positions. The results showed that CTB can accommodate diverse mutations while maintaining its stability and ligand binding affinity. These mutations could potentially allow modification of the oligosaccharide binding specificity to change its cellular targeting, alter the B-subunit intracellular routing, or impact its shelf-life and in vivo half-life through changes to protein stability. We anticipate that the mutational space maps presented here will serve as a cornerstone for future CTB redesigns, paving the way for the development of innovative biotechnological tools.
掌握人类细胞膜中选择性分子的转运是医疗保健生物技术中的一个巨大挑战，但也为药物输送、基因治疗和诊断成像方面的突破提供了前景。霍乱毒素 B 亚基 (CTB) 有可能成为这些应用的有用货物转运蛋白。CTB 是一种强大的蛋白质，可通过重新设计用于各种应用；然而，蛋白质的重新设计主要集中在蛋白质的 N 和 C 末端的修饰上。要充分利用合理的重新设计，需要详细了解表面残基对蛋白质稳定性和结合活性的贡献。在这里，我们对 CTB 的 58 个表面残基（包括 GM1 结合位点）进行了基于 Rosetta 的计算饱和扫描，以分析配体结合和无配体结构，以破译突变对蛋白质稳定性和 GM1 亲和力的影响。来自差示扫描荧光法和等温滴定量热法的互补实验结果提供了这些位置中 40 个丙氨酸突变体的熔点和 GM1 结合亲和力。结果表明，CTB 可以容纳多种突变，同时保持其稳定性和配体结合亲和力。这些突变可能允许修饰寡糖结合特异性以改变其细胞靶向性，改变 B 亚基细胞内途径，或通过改变蛋白质稳定性来影响其保质期和体内半衰期。我们预计，这里呈现的突变空间图谱将成为未来 CTB 重新设计的基石，为开发创新的生物技术工具铺平道路。
