Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY 11794, USA.
Department of Electrical and Computer Engineering, University of Victoria, Victoria V8P 5C2, Canada.
Sci Adv. 2024 May 31;10(22):eadn2208. doi: 10.1126/sciadv.adn2208.
PR65 is the HEAT repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem repeat protein. Its conformational mechanics plays a crucial role in PP2A function by opening/closing substrate binding/catalysis interface. Using in silico saturation mutagenesis, we identified PR65 "hinge" residues whose substitutions could alter its conformational adaptability and thereby PP2A function, and selected six mutations that were verified to be expressed and soluble. Molecular simulations and nanoaperture optical tweezers revealed consistent results on the specific effects of the mutations on the structure and dynamics of PR65. Two mutants observed in simulations to stabilize extended/open conformations exhibited higher corner frequencies and lower translational scattering in experiments, indicating a shift toward extended conformations, whereas another displayed the opposite features, confirmed by both simulations and experiments. The study highlights the power of single-molecule nanoaperture-based tweezers integrated with in silico approaches for exploring the effect of mutations on protein structure and dynamics.
PR65 是三聚体蛋白磷酸酶 2A（PP2A）的 HEAT 重复支架亚基，也是典型的串联重复蛋白。其构象力学在 PP2A 功能中起着关键作用，通过打开/关闭底物结合/催化界面。我们使用计算机饱和诱变技术鉴定了 PR65 的“铰链”残基，其取代可以改变其构象适应性，从而改变 PP2A 的功能，并选择了六个经证实可表达和可溶解的突变。分子模拟和纳米孔径光镊实验揭示了这些突变对 PR65 结构和动力学的具体影响的一致结果。在模拟中观察到两种突变体稳定延伸/开放构象，实验中表现出更高的角频率和更低的平移散射，表明构象向延伸转变，而另一种则表现出相反的特征，模拟和实验均证实了这一点。该研究强调了基于单分子纳米孔径的镊子与计算机模拟方法相结合，用于探索突变对蛋白质结构和动力学影响的强大功能。
