Bahar Ivet, Banerjee Anupam, Mathew Samuel, Naqvi Mohsin, Yilmaz Sema, Zachoropoulou Maria, Doruker Pemra, Kumita Janet, Yang Shang-Hua, Gur Mert, Itzhaki Laura, Gordon Reuven
Stony Brook University.
University of Victoria.
Res Sq. 2023 Nov 16:rs.3.rs-3599809. doi: 10.21203/rs.3.rs-3599809/v1.
PR65 is the HEAT-repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem-repeat protein, forming a spring-like architecture. PR65 conformational mechanics play a crucial role in PP2A function by opening/closing the substrate-binding/catalysis interface. Using saturation mutagenesis we identified "hinge" residues of PR65, whose substitutions are predicted to restrict its conformational adaptability and thereby disrupt PP2A function. Molecular simulations revealed that a subset of hinge mutations stabilized the extended/open conformation, whereas another had the opposite effect. By trapping in nanoaperture optical tweezer, we characterized PR65 motion and showed that the former mutants exhibited higher corner frequencies and lower translational scattering, indicating a shift towards extended conformations, whereas the latter showed the opposite behavior. Thus, experiments confirm the conformations predicted computationally. The study highlights the utility of nanoaperture-based tweezers for exploring structure and dynamics, and the power of integrating this single-molecule method with approaches.
PR65是异源三聚体蛋白磷酸酶2A（PP2A）的HEAT重复支架亚基，也是一种典型的串联重复蛋白，形成类似弹簧的结构。PR65的构象力学通过打开/关闭底物结合/催化界面在PP2A功能中起关键作用。利用饱和诱变，我们鉴定出PR65的“铰链”残基，预测其取代会限制其构象适应性，从而破坏PP2A功能。分子模拟显示，一部分铰链突变稳定了伸展/开放构象，而另一部分则有相反的效果。通过捕获在纳米孔径光镊中，我们表征了PR65的运动，结果表明，前一类突变体表现出更高的角频率和更低的平移散射，表明向伸展构象转变，而后者表现出相反的行为。因此，实验证实了计算预测的构象。该研究突出了基于纳米孔径的镊子在探索结构和动力学方面的实用性，以及将这种单分子方法与其他方法相结合的威力。
