Unexpected Single-Ligand Occupancy and Negative Cooperativity in the SARS-CoV-2 Main Protease

Area of Science:

• Biochemistry
• Structural Biology
• Virology

Background:

• Homodimeric enzymes often regulate function through subunit cooperativity.
• Previous studies suggested positive cooperativity in SARS-CoV-2 Main protease (Mpro) based on crystallographic data.
• Understanding Mpro's cooperative mechanism is crucial for antiviral drug development.

Purpose of the Study:

• To investigate the cooperative mechanism of the SARS-CoV-2 Mpro homodimer.
• To determine whether Mpro exhibits positive or negative cooperativity.
• To elucidate the functional implications of Mpro's cooperativity for viral adaptability.

Main Methods:

• Macromolecular crystallography (MX) of Mpro in complex with covalent and noncovalent ligands.
• Ligand titration experiments to assess binding stoichiometry.
• Molecular dynamics (MD) simulations to analyze active site geometry and catalytic activity.

Main Results:

• Mpro active site occupation depends on ligand excess; 1:1 stoichiometry results in single occupation.
• The unoccupied binding site adopts a catalytically inactive conformation.
• Mpro exhibits negative cooperativity, with asymmetric catalytic site activity.

Conclusions:

• SARS-CoV-2 Mpro operates via negative cooperativity, challenging previous assumptions.
• This mechanism confers resistance to saturation and facilitates viral adaptability.
• The findings provide insights into Mpro's role in viral survival and potential therapeutic strategies.