Journal of Biological Chemistry
Volume 295, Issue 50, 11 December 2020, Pages 17365-17373
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Molecular Bases of Disease
Unusual zwitterionic catalytic site of SARS–CoV-2 main protease revealed by neutron crystallography

https://doi.org/10.1074/jbc.AC120.016154Get rights and content
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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.

joint neutron
X-ray crystallography
room temperature
SARS–CoV-2
3CL main protease
3CL Mpro
protonation state
hydrogen bond
enzyme mechanism
drug design
neutron diffraction
viral protease
SARS–CoV-2 3CL main protease

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Author contributions—D. W. K., Q. Z., H. M. O., and L. C. formal analysis; D. W. K., K. L. W., S. P., Q. Z., and L. C. validation; D. W. K., G. P., K. L. W., S. P., and Q. Z. investigation; D. W. K. and L. C. visualization; D. W. K. and A. K. writing-original draft; G. P., K. L. W., S. P., and Q. Z. methodology; K. L. W., Q. Z., and H. M. O. resources; H. M. O. funding acquisition; H. M. O., L. C., and A. K. writing-review and editing; L. C. and A. K. conceptualization; L. C. data curation; A. K. supervision; A. K. project administration.

Funding and additional information—This work was supported by the Department of Energy Office of Science through the National Virtual Biotechnology Laboratory, a consortium of Department of Energy national laboratories focused on response to COVID-19, with funding provided by the Coronavirus CARES Act. This work used resources at the Spallation Neutron Source and the High Flux Isotope Reactor, which are Department of Energy Office of Science User Facilities operated by the Oak Ridge National Laboratory. The Office of Biological and Environmental Research supported research at the Oak Ridge National Laboratory Center for Structural Molecular Biology, a Department of Energy Office of Science user facility. This work also used resources at the Second Target Station, which is a Department of Energy Office of Science user facilities construction project at Oak Ridge National Laboratory.

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

    SARS

    severe acute respiratory syndrome

    CoV-2

    coronavirus 2

    PDB

    Protein Data Bank.