9EET | pdb_00009eet

Crystal structure of the SARS-CoV-2 nsp5 main protease (Mpro) E166V mutant in complex with inhibitor GC376

PDB DOI: https://doi.org/10.2210/pdb9EET/pdb

Classification: HYDROLASE/INHIBITOR
Organism(s): Severe acute respiratory syndrome coronavirus 2
Expression System: Escherichia coli BL21(DE3)
Mutation(s): Yes

Deposited: 2024-11-19 Released: 2025-06-18
Deposition Author(s): Neilsen, G., Kirby, K.A., Sarafianos, S.G.
Funding Organization(s): National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 2.39 ?
R-Value Free:
0.229 (Depositor), 0.228 (DCC)
R-Value Work:
0.193 (Depositor), 0.193 (DCC)
R-Value Observed:
0.195 (Depositor)

Starting Model: experimental
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wwPDB Validation 3D Report Full Report

Ligand Structure Quality Assessment

This is version 1.0 of the entry. See complete history.

Literature

Strategy to overcome a nirmatrelvir resistance mechanism in the SARS-CoV-2 nsp5 protease.

Neilsen, G., Lan, S., Slack, R.L., Lorson, Z.C., Emanuelli Castaner, A., Lee, R., Edwards, K.G., Zhang, H., Lee, J., Cantara, W.A., Cilento, M.E., Zhang, H., De, R., Amblard, F., Tedbury, P.R., Kirby, K.A., Schinazi, R.F., Sarafianos, S.G.

(2025) Sci Adv 11: eadv8875-eadv8875

PubMed: 40479048 Search on PubMedSearch on PubMed Central
DOI: https://doi.org/10.1126/sciadv.adv8875
Primary Citation of Related Structures:
9EEI, 9EET, 9EEV

PubMed Abstract:
E166V in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp5 protease confers strong resistance to the antiviral component of Paxlovid, nirmatrelvir (NIR), in passaging and clinical samples. In SARS-CoV-2 replicons, E166V drastically decreased Washington (WA1) but not Omicron (BA.1) fitness (20- versus 2-fold), suggesting a lower barrier to resistance in the BA.1 strain and consistent with observed differences in respective nsp5 dimerization affinities. Crystal structures reveal a steric clash between the rigid, bulky NIR tert -butyl group and the β-branched Val ¹⁶⁶, disrupting the covalent binding of NIR to the catalytic Cys ¹⁴⁵ and leading to high resistance in BA.1 and WA1 replicons. NIR-resistant replicons remained susceptible to GC376, which can still covalently bind Cys ¹⁴⁵ by avoiding a steric clash with Val ¹⁶⁶ through "wiggling and jiggling." Hence, strategic flexibility is a strategy that will help design second-generation antivirals against NIR-resistant viruses.

Organizational Affiliation

Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA.

Macromolecule Content

Total Structure Weight: 34.2 kDa
Atom Count: 2,503
Modeled Residue Count: 306
Deposited Residue Count: 306
Unique protein chains: 1

Macromolecules

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(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
3C-like proteinase nsp5	A	306	Severe acute respiratory syndrome coronavirus 2	Mutation(s): 1 Gene Names: rep, 1a-1b EC: 3.4.22.69
UniProt
Find proteins for P0DTD1 (Severe acute respiratory syndrome coronavirus 2) Explore P0DTD1 Go to UniProtKB: P0DTD1
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt Group	P0DTD1
Sequence Annotations Expand
Reference Sequence

Small Molecules

Ligands 1 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
UED (Subject of Investigation/LOI) Query on UED Download Ideal Coordinates CCD File SDF format, chain B [auth A] MOL2 format, chain B [auth A] mmCIF format, chain B [auth A]	B [auth A]	N~2~-[(benzyloxy)carbonyl]-N-{(2S)-1-hydroxy-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-L-leucinamide C₂₁ H₃₁ N₃ O₅ JUCVXDDMQHPCKT-BZSNNMDCSA-N		Interactions Focus chain B [auth A] Interactions & Density Focus chain B [auth A]