A Cooperative Folding Unit as the Structural Link for Energetic Coupling within a Protein

Area of Science:

• Biochemistry
• Structural Biology
• Protein Folding

Background:

• Ligand binding to Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM) is energetically coupled with protein dimerization.
• dPGM equilibrium unfolding reveals a stable, monomeric intermediate where both the active site and dimer interface are unfolded.
• Nonsubstrate metabolite binding stabilizes the native dimeric form, reducing the intermediate population.

Purpose of the Study:

• To investigate the structure of the dPGM intermediate.
• To identify the specific region responsible for the energetic coupling between the dimer interface and the active site.
• To understand how cooperative residue networks influence ligand binding and dimerization.

Main Methods:

• Equilibrium unfolding experiments with site-directed mutagenesis to determine equilibrium φ values (φ_eq).
• Hydrogen/deuterium exchange mass spectrometry (HDX-MS) to probe peptide backbone solvent accessibility.
• Analysis of side-chain interactions and global/intermediate stability.

Main Results:

• φ_eq value analysis and HDX-MS identified the least stable folding unit in the dPGM intermediate.
• This unstable region, a loop containing residues 118-152, links the active site to the dimer interface and is unfolded in the intermediate.
• The study elucidates the structural basis for the energetic coupling between dimerization and ligand binding in dPGM.

Conclusions:

• The dPGM intermediate structure reveals that a specific loop (residues 118-152) is unfolded and mediates the coupling between the active site and dimer interface.
• This cooperative network of residues is crucial for the observed energetic linkage between ligand binding and protein dimerization.
• Understanding this mechanism provides insights into protein stability, allostery, and enzyme function.