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Contents 1 Structural Insights into SARS-CoV-2 Main Protease (Mpro) with a Covalent Inhibitor (PDB: 6XWD) 1.1 Overall Architecture 1.2 References 1.3 Catalytic Machinery 1.4 References 1.5 Inhibitor Recognition and Binding Mode 1.6 References 1.7 Your Heading Here (maybe something like 'Structure') 1.8 References 1.9 Why This Structure Was Important 1.10 Structural Highlights (Paper-Based Key Points) 1.11 Biological Relevance 1.12 References

Structural Insights into SARS-CoV-2 Main Protease (Mpro) with a Covalent Inhibitor (PDB: 6XWD)

The SARS-CoV-2 main protease (Mpro) is the central proteolytic enzyme responsible for processing the viral polyprotein into functional non-structural proteins. Because this step is essential for viral replication, Mpro became one of the earliest and most intensively targeted COVID-19 drug-design systems. The iScience 2020 study (DOI: 10.1016/j.isci.2020.101258) presented the high-resolution crystal structure of Mpro bound to a covalent peptide-like inhibitor (PDB: 6XWD), providing an immediate template for structure-based antiviral design.

Overall Architecture

Mpro exists as a functional homodimer. Each protomer contains three domains:

• **Domain I (residues 8–101)** – β-barrel catalytic scaffold
• **Domain II (residues 102–184)** – substrate-binding groove
• **Domain III (residues 201–303)** – α-helical region important for dimerization

Dimer formation is essential for activating the catalytic machinery, as seen in ==Your Heading Here (maybe something like 'Structure')==

spinqualitylabelsall models Caption for this structure Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image This is a default text for your page SceneLink. Click above on edit this page to modify. Be careful with the < and > signs. You may include any references to papers as in: the use of JSmol in Proteopedia [1] or to the article describing Jmol [2] to the rescue. Contents 1 Function 2 Disease 3 Relevance 4 Structural highlights Function Disease Relevance Structural highlights This is a sample scene created with SAT to by Group, and another to make of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

References

1. ↑ Hanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. and Sussman, J. L. (2013), JSmol and the Next-Generation Web-Based Representation of 3D Molecular Structure as Applied to Proteopedia. Isr. J. Chem., 53:207-216. doi:http://dx.doi.org/10.1002/ijch.201300024
2. ↑ Herraez A. Biomolecules in the computer: Jmol to the rescue. Biochem Mol Biol Educ. 2006 Jul;34(4):255-61. doi: 10.1002/bmb.2006.494034042644. PMID:21638687 doi:10.1002/bmb.2006.494034042644

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Catalytic Machinery

The active site lies between Domains I and II and contains the **His41–Cys145 catalytic dyad**, a hallmark of viral cysteine proteases. The paper highlights that unlike classical serine proteases, Mpro uses the thiol of Cys145 as the nucleophile. This dyad is displayed in ==Your Heading Here (maybe something like 'Structure')==

spinqualitylabelsall models Caption for this structure Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image This is a default text for your page SceneLink. Click above on edit this page to modify. Be careful with the < and > signs. You may include any references to papers as in: the use of JSmol in Proteopedia [1] or to the article describing Jmol [2] to the rescue. Contents 1 Function 2 Disease 3 Relevance 4 Structural highlights Function Disease Relevance Structural highlights This is a sample scene created with SAT to by Group, and another to make of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

References

1. ↑ Hanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. and Sussman, J. L. (2013), JSmol and the Next-Generation Web-Based Representation of 3D Molecular Structure as Applied to Proteopedia. Isr. J. Chem., 53:207-216. doi:http://dx.doi.org/10.1002/ijch.201300024
2. ↑ Herraez A. Biomolecules in the computer: Jmol to the rescue. Biochem Mol Biol Educ. 2006 Jul;34(4):255-61. doi: 10.1002/bmb.2006.494034042644. PMID:21638687 doi:10.1002/bmb.2006.494034042644

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Inhibitor Recognition and Binding Mode

The co-crystallized inhibitor in 6XWD fits tightly into the S1, S2, and S4 subsites. Key observations derived from the structure:

• **S1 pocket (His163, Glu166):** strict preference for glutamine at P1
• **S2 pocket (Met49, His41):** hydrophobic, favors Leu/Phe
• **S4 pocket:** accommodates bulky groups
• **Oxyanion hole (Gly143, Ser144, Cys145 backbone atoms):** stabilizes transition states

The inhibitor forms a **covalent thioether bond with Cys145**, blocking substrate entry. This interaction is visualized in ==Your Heading Here (maybe something like 'Structure')==

spinqualitylabelsall models Caption for this structure Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image This is a default text for your page SceneLink. Click above on edit this page to modify. Be careful with the < and > signs. You may include any references to papers as in: the use of JSmol in Proteopedia [1] or to the article describing Jmol [2] to the rescue. Contents 1 Function 2 Disease 3 Relevance 4 Structural highlights Function Disease Relevance Structural highlights This is a sample scene created with SAT to by Group, and another to make of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

References

1. ↑ Hanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. and Sussman, J. L. (2013), JSmol and the Next-Generation Web-Based Representation of 3D Molecular Structure as Applied to Proteopedia. Isr. J. Chem., 53:207-216. doi:http://dx.doi.org/10.1002/ijch.201300024
2. ↑ Herraez A. Biomolecules in the computer: Jmol to the rescue. Biochem Mol Biol Educ. 2006 Jul;34(4):255-61. doi: 10.1002/bmb.2006.494034042644. PMID:21638687 doi:10.1002/bmb.2006.494034042644

and

Your Heading Here (maybe something like 'Structure')

spinqualitylabelsall models Caption for this structure Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image This is a default text for your page SceneLink. Click above on edit this page to modify. Be careful with the < and > signs. You may include any references to papers as in: the use of JSmol in Proteopedia [1] or to the article describing Jmol [2] to the rescue. Contents 1 Function 2 Disease 3 Relevance 4 Structural highlights Function Disease Relevance Structural highlights This is a sample scene created with SAT to by Group, and another to make of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

References

1. ↑ Hanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. and Sussman, J. L. (2013), JSmol and the Next-Generation Web-Based Representation of 3D Molecular Structure as Applied to Proteopedia. Isr. J. Chem., 53:207-216. doi:http://dx.doi.org/10.1002/ijch.201300024
2. ↑ Herraez A. Biomolecules in the computer: Jmol to the rescue. Biochem Mol Biol Educ. 2006 Jul;34(4):255-61. doi: 10.1002/bmb.2006.494034042644. PMID:21638687 doi:10.1002/bmb.2006.494034042644

.

Why This Structure Was Important

The 6XWD structure was one of the earliest experimentally solved complexes of SARS-CoV-2 Mpro. Its impact includes:

• Revealing **exact inhibitor positioning** inside the substrate groove
• Showing how **covalent warheads** can effectively “lock” the enzyme
• Providing a **template for rapid in-silico screening**
• Guiding the design of later clinical candidates such as PF-07321332 (nirmatrelvir)

Structural Highlights (Paper-Based Key Points)

• Catalytic dyad geometry shows ideal alignment for nucleophilic attack
• Inhibitor occupies the canonical P1–P4 substrate positions
• Hydrogen-bond network anchors inhibitor in the active groove
• Covalent attachment makes the inhibition irreversible
• Conformational rigidity of domains I/II supports catalysis
• Domain III contributes to dimer stabilization rather than direct catalysis

Biological Relevance

Because Mpro lacks any human homologs with a similar cleavage specificity (Leu-Gln ↓), it provides an excellent therapeutic window. The 6XWD structure demonstrated that **covalent inhibitors are both specific and structurally compatible**, accelerating COVID-19 antiviral development.

References

1. Structural Basis of SARS-CoV-2 Main Protease Inhibition. iScience, 2020. DOI:10.1016/j.isci.2020.101258 2. Protein Data Bank: 6XWD