Energetic Coupling between Ligand Binding and Dimerization in Escherichia coli Phosphoglycerate Mutase

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• Energetic coupling between molecular events is crucial for protein function in catalysis and signal transduction.
• Proteins often undergo conformational changes, including folding and unfolding, which can be influenced by external factors like ligand binding.

Purpose of the Study:

• To investigate the energetic coupling between ligand binding and protein dimerization using a model system.
• To elucidate the mechanism by which Adenosine triphosphate (ATP) affects the stability and quaternary structure of Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM).

Main Methods:

• Utilized equilibrium unfolding experiments to assess the thermodynamic stability of dPGM in the presence and absence of ATP.
• Employed computational ligand docking to predict ATP binding sites.
• Performed isothermal titration calorimetry and equilibrium unfolding with active-site variants to confirm ATP interaction.

Main Results:

• dPGM populates a partially unfolded monomeric intermediate during equilibrium unfolding without ATP.
• ATP (1.0 mM) significantly stabilizes the native dimer by reducing the population of the intermediate.
• ATP binds to the active site of dPGM, involving active-site residues in the binding process.
• ATP binding allosterically promotes dimerization, suggesting a link between the active site and the dimer interface.

Conclusions:

• ATP binding to the active site of dPGM energetically couples with and promotes protein dimerization.
• This cooperativity demonstrates a mechanism where ligand binding at a distal site influences quaternary structure.
• A structural link is proposed to explain the allosteric regulation of dimerization by active-site ligand binding.