Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland.
Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS/Université de Lyon, 69367, Lyon, France.
Nat Commun. 2019 Jan 3;10(1):31. doi: 10.1038/s41467-018-07968-3.
DnaB helicases are motor proteins that couple ATP-hydrolysis to the loading of the protein onto DNA at the replication fork and to translocation along DNA to separate double-stranded DNA into single strands during replication. Using a network of conformational states, arrested by nucleotide mimics, we herein characterize the reaction coordinates for ATP hydrolysis, DNA loading and DNA translocation using solid-state NMR spectroscopy. AMP-PCP is used as pre-hydrolytic, ADP:AlF as transition state, and ADP as post-hydrolytic ATP mimic. P and C NMR spectra reveal conformational and dynamic responses to ATP hydrolysis and the resulting DNA loading and translocation with single amino-acid resolution. This allows us to identify residues guiding the DNA translocation process and to explain the high binding affinities for DNA observed for ADP:AlF, which turns out to be optimally preconfigured to bind DNA.
DNA B 解旋酶是一种将 ATP 水解与蛋白质在复制叉处加载到 DNA 上以及沿着 DNA 易位以在复制过程中将双链 DNA 分离成单链结合起来的运动蛋白。使用由核苷酸模拟物捕获的构象状态网络，我们在此使用固态 NMR 光谱学来表征 ATP 水解、DNA 加载和 DNA 易位的反应坐标。AMP-PCP 用作预水解，ADP：AlF 用作过渡态，ADP 用作水解后的 ATP 模拟物。P 和 C NMR 谱揭示了对 ATP 水解以及由此产生的 DNA 加载和易位的构象和动态响应，具有单氨基酸分辨率。这使我们能够识别引导 DNA 易位过程的残基，并解释对于 ADP：AlF 观察到的高 DNA 结合亲和力，事实证明，ADP：AlF 被最佳地预配置以结合 DNA。
