Physical Chemistry, ETH Zürich, Zürich, Switzerland.
Department of Physics, Osnabrück University, Osnabrück, Germany.
Nat Commun. 2021 Sep 6;12(1):5293. doi: 10.1038/s41467-021-25599-z.
The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected P,H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds. F and Al MAS NMR spectra reveal a highly mobile, fast-rotating aluminum fluoride unit pointing to the capture of a late ATP hydrolysis transition state in which the phosphoryl unit is already detached from the arginine and lysine fingers.
马达蛋白的 ATP 水解过渡态是一种弱占据的蛋白质状态，可以通过用化学模拟物替代 ATP 来稳定和研究。我们介绍了一种由 ADP 铝氟化物结合幽门螺杆菌的细菌 DnaB 解旋酶形成的状态的原子水平结构和动态见解。我们使用电子顺磁共振确定了金属离子辅因子在活性位点内的定位，并使用质子探测 P,H 固态核磁共振光谱在 >100 kHz 的快速魔角旋转下，以及温度依赖的质子化学位移值来鉴定与 ADP 和 DNA 的磷酸基团配位的蛋白质质子，以证明它们参与氢键。F 和 Al MAS NMR 光谱揭示了一个高度移动、快速旋转的氟化铝单元，指向捕获晚期 ATP 水解过渡态，其中磷酸基已经从精氨酸和赖氨酸指上脱离。
