Correy Galen J, Kneller Daniel W, Phillips Gwyndalyn, Pant Swati, Russi Silvia, Cohen Aina E, Meigs George, Holton James M, Gahbauer Stefan, Thompson Michael C, Ashworth Alan, Coates Leighton, Kovalevsky Andrey, Meilleur Flora, Fraser James S
Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA 94158, USA.
Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
bioRxiv. 2022 Feb 9:2022.02.07.479477. doi: 10.1101/2022.02.07.479477.
The NSP3 macrodomain of SARS CoV 2 (Mac1) removes ADP-ribosylation post-translational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the COVID-19 pandemic. Here, we determined neutron and X-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site, and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a re-evaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的NSP3宏结构域（Mac1）可去除翻译后修饰的ADP核糖基化，在导致新冠疫情的病毒免疫逃逸能力中起关键作用。在此，我们利用多种晶体形式、温度和pH值，在无配体和ADP核糖结合状态下，确定了SARS-CoV-2 NSP3宏结构域的中子和X射线晶体结构。我们对Mac1活性位点的广泛溶剂化进行了表征，并可视化了ADP核糖和非天然配体结合后水网络的重新组织，从而启发了置换水以提高Mac1抑制剂效力的策略。通过中子晶体学确定活性位点水分子的精确取向和关键催化位点残基的质子化状态，揭示了冠状病毒宏结构域不同于人类MacroD2所提出的底物辅助机制的催化机制。这些数据促使人们重新评估宏结构域的催化机制，并将指导Mac1抑制剂的优化。
