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Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor
We recently demonstrated that inhibitor binding reorganizes the oxyanion loop of a monomeric catalytic domain of SARS CoV-2 main protease (MPro) from an unwound (E) to a wound (active, E*) conformation, independent of dimerization. Here we assess the effect of the flanking N-terminal residues, to im...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9628131/ https://www.ncbi.nlm.nih.gov/pubmed/36334779 http://dx.doi.org/10.1016/j.jmb.2022.167876 |
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author | Kovalevsky, Andrey Coates, Leighton Kneller, Daniel W. Ghirlando, Rodolfo Aniana, Annie Nashed, Nashaat T. Louis, John M. |
author_facet | Kovalevsky, Andrey Coates, Leighton Kneller, Daniel W. Ghirlando, Rodolfo Aniana, Annie Nashed, Nashaat T. Louis, John M. |
author_sort | Kovalevsky, Andrey |
collection | PubMed |
description | We recently demonstrated that inhibitor binding reorganizes the oxyanion loop of a monomeric catalytic domain of SARS CoV-2 main protease (MPro) from an unwound (E) to a wound (active, E*) conformation, independent of dimerization. Here we assess the effect of the flanking N-terminal residues, to imitate the MPro precursor prior to its autoprocessing, on conformational equilibria rendering stability and inhibitor binding. Thermal denaturation (T(m)) of C145A mutant, unlike H41A, increases by 6.8 °C, relative to wild-type mature dimer. An inactivating H41A mutation to maintain a miniprecursor containing TSAVL[Q or E] of the flanking nsp4 sequence in an intact form [((-6))MPro(H41A) and ((-6*))MPro(H41A), respectively], and its corresponding mature MPro(H41A) were systematically examined. While the H41A mutation exerts negligible effect on T(m) and dimer dissociation constant (K(dimer)) of MPro(H41A), relative to the wild type MPro, both miniprecursors show a 4–5 °C decrease in T(m) and > 85-fold increase in K(dimer) as compared to MPro(H41A). The K(d) for the binding of the covalent inhibitor GC373 to ((-6*))MPro(H41A) increases ∼12-fold, relative to MPro(H41A), concomitant with its dimerization. While the inhibitor-free dimer exhibits a state in transit from E to E* with a conformational asymmetry of the protomers’ oxyanion loops and helical domains, inhibitor binding restores the asymmetry to mature-like oxyanion loop conformations (E*) but not of the helical domains. Disorder of the terminal residues 1–2 and 302–306 observed in both structures suggest that N-terminal autoprocessing is tightly coupled to the E-E* equilibrium and stable dimer formation. |
format | Online Article Text |
id | pubmed-9628131 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-96281312022-11-03 Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor Kovalevsky, Andrey Coates, Leighton Kneller, Daniel W. Ghirlando, Rodolfo Aniana, Annie Nashed, Nashaat T. Louis, John M. J Mol Biol Research Article We recently demonstrated that inhibitor binding reorganizes the oxyanion loop of a monomeric catalytic domain of SARS CoV-2 main protease (MPro) from an unwound (E) to a wound (active, E*) conformation, independent of dimerization. Here we assess the effect of the flanking N-terminal residues, to imitate the MPro precursor prior to its autoprocessing, on conformational equilibria rendering stability and inhibitor binding. Thermal denaturation (T(m)) of C145A mutant, unlike H41A, increases by 6.8 °C, relative to wild-type mature dimer. An inactivating H41A mutation to maintain a miniprecursor containing TSAVL[Q or E] of the flanking nsp4 sequence in an intact form [((-6))MPro(H41A) and ((-6*))MPro(H41A), respectively], and its corresponding mature MPro(H41A) were systematically examined. While the H41A mutation exerts negligible effect on T(m) and dimer dissociation constant (K(dimer)) of MPro(H41A), relative to the wild type MPro, both miniprecursors show a 4–5 °C decrease in T(m) and > 85-fold increase in K(dimer) as compared to MPro(H41A). The K(d) for the binding of the covalent inhibitor GC373 to ((-6*))MPro(H41A) increases ∼12-fold, relative to MPro(H41A), concomitant with its dimerization. While the inhibitor-free dimer exhibits a state in transit from E to E* with a conformational asymmetry of the protomers’ oxyanion loops and helical domains, inhibitor binding restores the asymmetry to mature-like oxyanion loop conformations (E*) but not of the helical domains. Disorder of the terminal residues 1–2 and 302–306 observed in both structures suggest that N-terminal autoprocessing is tightly coupled to the E-E* equilibrium and stable dimer formation. Elsevier 2022-12-30 2022-11-02 /pmc/articles/PMC9628131/ /pubmed/36334779 http://dx.doi.org/10.1016/j.jmb.2022.167876 Text en Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. |
spellingShingle | Research Article Kovalevsky, Andrey Coates, Leighton Kneller, Daniel W. Ghirlando, Rodolfo Aniana, Annie Nashed, Nashaat T. Louis, John M. Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor |
title | Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor |
title_full | Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor |
title_fullStr | Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor |
title_full_unstemmed | Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor |
title_short | Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor |
title_sort | unmasking the conformational stability and inhibitor binding to sars-cov-2 main protease active site mutants and miniprecursor |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9628131/ https://www.ncbi.nlm.nih.gov/pubmed/36334779 http://dx.doi.org/10.1016/j.jmb.2022.167876 |
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