Structure, function and interfacial allosterism in phospholipase A2: insight from the anion-assisted dimer

Area of Science:

• Structural biology
• Biochemistry
• Enzymology

Background:

• Membrane-bound enzymes present challenges for structural and functional analysis in physiological conditions.
• Developing methods to mimic protein-surface interactions is key to understanding enzyme mechanisms.

Purpose of the Study:

• To characterize the allosteric nature of secreted phospholipase A2 (PLA2) at its substrate interface.
• To elucidate the structural basis for enzyme activation and catalytic mechanism.

Main Methods:

• Crystallization of pancreatic group-IB PLA2 in an anion-assisted dimer.
• Co-crystallization with a tetrahedral mimic inhibitor and substrate hydrolysis products.
• X-ray crystallography to determine enzyme structures.

Main Results:

• A novel anion-assisted dimer structure of PLA2 was obtained, sharing a mimic inhibitor and anions between subunits.
• The structure revealed inhibitor binding across the subunit interface, providing insight into the active site and anionic interactions.
• A critical active site water molecule was identified as essential for enzyme activation, with its presence or absence correlating to enzyme activity.
• Enzyme-product and zymogen (proPLA2) structures confirmed the proposed calcium-coordinated nucleophilic water mechanism.

Conclusions:

• The anion-assisted dimer crystallography approach effectively mimics physiological conditions for studying PLA2.
• A critical water molecule plays a key role in modulating PLA2 activity via its pKa.
• The findings support a calcium-coordinated nucleophilic water mechanism for PLA2, with implications for enzyme activation and substrate hydrolysis.