Characterization of an unusual SARS-CoV-2 main protease natural variant exhibiting resistance to nirmatrelvir and ensitrelvir
Abstract We investigate the effects of two naturally selected substitution and deletion (Δ) mutations, constituting part of the substrate binding subsites S2 and S4, on the structure, function, and inhibition of SARS CoV-2 main protease. Comparable to wild-type, MProD48Y/ΔP168 undergoes N-terminal a...
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Nature Portfolio
2025-07-01
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| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-025-08487-w |
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| author | Dipendra Bhandari Oksana Gerlits Stephen Keable Leighton Coates Annie Aniana Rodolfo Ghirlando Nashaat T. Nashed Andrey Kovalevsky John M. Louis |
| author_facet | Dipendra Bhandari Oksana Gerlits Stephen Keable Leighton Coates Annie Aniana Rodolfo Ghirlando Nashaat T. Nashed Andrey Kovalevsky John M. Louis |
| author_sort | Dipendra Bhandari |
| collection | DOAJ |
| description | Abstract We investigate the effects of two naturally selected substitution and deletion (Δ) mutations, constituting part of the substrate binding subsites S2 and S4, on the structure, function, and inhibition of SARS CoV-2 main protease. Comparable to wild-type, MProD48Y/ΔP168 undergoes N-terminal autoprocessing essential for stable dimer formation and mature-like catalytic activity. The structures are similar, but for an open active site conformation in MProD48Y/ΔP168 and increased dynamics of the S2 helix, S5 loop, and the helical domain. Some dimer interface contacts exhibit shorter H bond distances corroborating the ~40-fold enhanced dimerization of the mutant although its thermal sensitivity to unfolding is 8 °C lower, relative to wild-type. ITC reveals a 3- and 5-fold decrease in binding affinity for nirmatrelvir and ensitrelvir, respectively, and similar GC373 affinity, to MProD48Y/ΔP168 relative to wild-type. Structural differences in four inhibitor complexes of MProD48Y/ΔP168 compared to wild-type are described. Consistent with enhanced dynamics, the S2 helix and S5 loop adopting a more open conformation appears to be a unique feature of MProD48Y/ΔP168 both in the inhibitor-free and bound states. Our results suggest that mutational effects are compensated by changes in the conformational dynamics and thereby modulate N-terminal autoprocessing, Kdimer, catalytic efficiency, and inhibitor binding. |
| format | Article |
| id | doaj-art-c3b92e4de95d48bc9ad1cff8483a8b19 |
| institution | DOAJ |
| issn | 2399-3642 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj-art-c3b92e4de95d48bc9ad1cff8483a8b192025-08-20T03:06:01ZengNature PortfolioCommunications Biology2399-36422025-07-018111410.1038/s42003-025-08487-wCharacterization of an unusual SARS-CoV-2 main protease natural variant exhibiting resistance to nirmatrelvir and ensitrelvirDipendra Bhandari0Oksana Gerlits1Stephen Keable2Leighton Coates3Annie Aniana4Rodolfo Ghirlando5Nashaat T. Nashed6Andrey Kovalevsky7John M. Louis8Neutron Scattering Division, Oak Ridge National LaboratoryDepartment of Natural Sciences, Tennessee Wesleyan UniversityNeutron Scattering Division, Oak Ridge National LaboratorySecond Target Station, Oak Ridge National LaboratoryLaboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHSLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHSLaboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHSNeutron Scattering Division, Oak Ridge National LaboratoryLaboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHSAbstract We investigate the effects of two naturally selected substitution and deletion (Δ) mutations, constituting part of the substrate binding subsites S2 and S4, on the structure, function, and inhibition of SARS CoV-2 main protease. Comparable to wild-type, MProD48Y/ΔP168 undergoes N-terminal autoprocessing essential for stable dimer formation and mature-like catalytic activity. The structures are similar, but for an open active site conformation in MProD48Y/ΔP168 and increased dynamics of the S2 helix, S5 loop, and the helical domain. Some dimer interface contacts exhibit shorter H bond distances corroborating the ~40-fold enhanced dimerization of the mutant although its thermal sensitivity to unfolding is 8 °C lower, relative to wild-type. ITC reveals a 3- and 5-fold decrease in binding affinity for nirmatrelvir and ensitrelvir, respectively, and similar GC373 affinity, to MProD48Y/ΔP168 relative to wild-type. Structural differences in four inhibitor complexes of MProD48Y/ΔP168 compared to wild-type are described. Consistent with enhanced dynamics, the S2 helix and S5 loop adopting a more open conformation appears to be a unique feature of MProD48Y/ΔP168 both in the inhibitor-free and bound states. Our results suggest that mutational effects are compensated by changes in the conformational dynamics and thereby modulate N-terminal autoprocessing, Kdimer, catalytic efficiency, and inhibitor binding.https://doi.org/10.1038/s42003-025-08487-w |
| spellingShingle | Dipendra Bhandari Oksana Gerlits Stephen Keable Leighton Coates Annie Aniana Rodolfo Ghirlando Nashaat T. Nashed Andrey Kovalevsky John M. Louis Characterization of an unusual SARS-CoV-2 main protease natural variant exhibiting resistance to nirmatrelvir and ensitrelvir Communications Biology |
| title | Characterization of an unusual SARS-CoV-2 main protease natural variant exhibiting resistance to nirmatrelvir and ensitrelvir |
| title_full | Characterization of an unusual SARS-CoV-2 main protease natural variant exhibiting resistance to nirmatrelvir and ensitrelvir |
| title_fullStr | Characterization of an unusual SARS-CoV-2 main protease natural variant exhibiting resistance to nirmatrelvir and ensitrelvir |
| title_full_unstemmed | Characterization of an unusual SARS-CoV-2 main protease natural variant exhibiting resistance to nirmatrelvir and ensitrelvir |
| title_short | Characterization of an unusual SARS-CoV-2 main protease natural variant exhibiting resistance to nirmatrelvir and ensitrelvir |
| title_sort | characterization of an unusual sars cov 2 main protease natural variant exhibiting resistance to nirmatrelvir and ensitrelvir |
| url | https://doi.org/10.1038/s42003-025-08487-w |
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