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Author Notes:

Email: arnold@cabm.rutgers.edu

Each author’s contributions to the paper are as follows: Conceptualization: E.A., P.R.G., F.X.R., R.Y., V.V.C., and S.K.D. Methodology: R.Y., V.V.C., S.K.D., J.J.E.K.H., J.T., J.B.H., F.X.R., and R.L.S. Investigation: R.Y., V.V.C., S.K.D., R.L.S., S.G.S., E.A., P.R.G., and F.X.R. Visualization: R.Y., V.V.C., S.K.D., and F.X.R. Supervision: E.A., P.R.G., and F.X.R. Writing—original draft: R.Y., V.V.C., F.X.R., E.A., and P.R.G. Writing—review and editing: All authors.

We thank M. Narwal and K. Murakami (Pennsylvania State University) for providing protein samples of the C145A Mpro mutant and the nsp7-10 polyprotein, numerous helpful discussions, and also critical reading of the manuscript. We thank B. Pascal and R. Vera Alvarez (Omics Informatics LLC) for help in running the nsp7-11 and nsp7-8 models through the HDXer software. We thank H. Zheng and P. Lobel (Biological Mass Spectrometry Facility of Robert Wood Johnson Medical School and Rutgers University) for performing the in-gel LC-MS/MS for the limited proteolysis assay of nsp7-11 polyprotein by Mpro. Molecular graphics and analyses were performed with (i) UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco; and (ii) PyMOL [DeLano WL (2002), The PyMOL Molecular Graphics System, San Carlos, CA; DeLano Scientific].

The authors declare that they have no competing interests.

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Research Funding:

We are grateful for support from National Institutes of Health (NIH) grants U54 AI150472 (to E.A. and P.R.G.), AI 027690 (to E.A.), F31 DK126394 (to V.V.C.), R01 AI167356 (to S.G.S.), and T32 AI157855 (to R.L.S.) and a research award from the Rutgers Center for COVID-19 Response and Pandemic Preparedness (to E.A.).

We are grateful to V. Nanda and P. Falkowski for providing financial support for J.T.’s efforts. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. This project was supported by grant P30 GM138395 from the National Institute of General Medical Sciences of the NIH.

Use of the Pilatus 3 1M detector was provided by grant 1S10OD018090-01 from NIGMS. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institute of General Medical Sciences or the NIH.

Keywords:

  • Science & Technology
  • Multidisciplinary Sciences
  • Science & Technology - Other Topics
  • MOUSE HEPATITIS-VIRUS
  • HYDROGEN/DEUTERIUM EXCHANGE
  • MASS-SPECTROMETRY
  • PROTEIN-STRUCTURE
  • PROTEOLYTIC PRODUCTS
  • HYDROGEN-EXCHANGE
  • REPLICATION
  • SCATTERING
  • IDENTIFICATION
  • RESOLUTION

Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro

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Journal Title:

SCIENCE ADVANCES

Volume:

Volume 8, Number 49

Publisher:

, Pages eadd2191-eadd2191

Type of Work:

Article | Final Publisher PDF

Abstract:

SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic. Its genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro). Polyprotein processing is essential yet incompletely understood.We studied Mpro-mediated processing of the nsp7- 11 polyprotein, whose mature products include cofactors of the viral replicase, and identified the order of cleavages. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and crosslinking) and x-ray scattering yielded a nsp7-11 structural ensemble, demonstrating shared secondary structural elements with individual nsps. The pattern of cross-links and HDX footprint of the C145A Mpro and nsp7-11 complex demonstrate preferential binding of the enzyme active site to the polyprotein junction sites and additional transient contacts to help orient the enzyme on its substrate for cleavage. Last, proteolysis assays were used to characterize the effect of inhibitors/binders on Mpro processing/inhibition using the nsp7-11 polyprotein as substrate.

Copyright information:

© 2022 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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