Unusual zwitterionic catalytic site of SARS-CoV-2 main protease revealed by neutron crystallography.
Unusual zwitterionic catalytic site of SARS-CoV-2 main protease revealed by neutron crystallography.
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DOI：
10.1074/jbc.ac120.016154
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发表时间：
2020-12-11
期刊：
影响因子：
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通讯作者：
Kovalevsky A
中科院分区：
文献类型：
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作者：
Kneller DW;Phillips G;Weiss KL;Pant S;Zhang Q;O'Neill HM;Coates L;Kovalevsky A
SARS-CoV-2的主要蛋白酶（3CL Mpro）是COVID-19的病原体，是病毒复制的必需酶。3CL Mpro具有由Cys 145和His 41残基组成的不寻常的催化二联体。该领域的一个关键问题是底物结合活性位点空腔中可电离残基的质子化状态是什么;解决这一点将有助于了解酶的催化细节，并为针对这种有害病毒的合理药物开发提供信息。在这里，我们提出了室温中子结构的3CL Mpro，它允许直接确定氢原子的位置，因此，质子化状态的蛋白酶。我们观察到，催化位点天然采用两性离子反应形式，其中Cys 145是在带负电荷的硫醇盐状态和His 41是双质子化和带正电荷的，而不是通常设想的中性不反应状态。中子结构还确定了质子化状态，从而电荷，所有其他氨基酸残基，并揭示了复杂的氢键网络中的活性位点腔和二聚体界面。这种结构中存在的精细原子细节是由中子的独特散射特性实现的，中子是定位氢位置和在接近生理温度下通过实验确定质子化状态的理想探针。我们的观察结果为结构辅助和计算药物设计提供了关键信息，允许根据酶的静电环境精确定制抑制剂。
The main protease (3CL Mpro) from SARS–CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication. 3CL Mpro possesses an unusual catalytic dyad composed of Cys145 and His41 residues. A critical question in the field has been what the protonation states of the ionizable residues in the substrate-binding active-site cavity are; resolving this point would help understand the catalytic details of the enzyme and inform rational drug development against this pernicious virus. Here, we present the room-temperature neutron structure of 3CL Mpro, which allowed direct determination of hydrogen atom positions and, hence, protonation states in the protease. We observe that the catalytic site natively adopts a zwitterionic reactive form in which Cys145 is in the negatively charged thiolate state and His41 is doubly protonated and positively charged, instead of the neutral unreactive state usually envisaged. The neutron structure also identified the protonation states, and thus electrical charges, of all other amino acid residues and revealed intricate hydrogen-bonding networks in the active-site cavity and at the dimer interface. The fine atomic details present in this structure were made possible by the unique scattering properties of the neutron, which is an ideal probe for locating hydrogen positions and experimentally determining protonation states at near-physiological temperature. Our observations provide critical information for structure-assisted and computational drug design, allowing precise tailoring of inhibitors to the enzyme's electrostatic environment.