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

Abstract

Previously, we demonstrated that binding of a ligand to Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM), a homodimeric protein, is energetically coupled with dimerization. The equilibrium unfolding of dPGM occurs with a stable, monomeric intermediate. Binding of several nonsubstrate metabolites stabilizes the dimeric native form over the monomeric intermediate, reducing the population of the intermediate. Both the active site and the dimer interface appear to be unfolded in the intermediate. We hypothesized that a loop containing residues 118-152 was responsible for the energetic coupling between the dimer interface and the distal active site and was unfolded in the intermediate. Here, we investigated the structure of the dPGM intermediate by probing side-chain interactions and solvent accessibility of the peptide backbone. By comparing the effect of a mutation on the global stability and the stability of the intermediate, we determine an equilibrium φ value (φeq value), which provides information about whether side-chain interactions are retained or lost in the intermediate. Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was used to investigate differences in the solvent accessibility of the peptide backbone in the intermediate and native forms of dPGM. The results of φeq value analysis and HDX-MS reveal the least stable folding unit of dPGM, which is unfolded in the intermediate and links the active site to the dimer interface. The structure of the intermediate reveals how the cooperative network of residues in dPGM gives rise to the observed energetic coupling between dimerization and ligand binding.