title: Energetic Coupling between Ligand Binding and Dimerization in Escherichia coli Phosphoglycerate Mutase.
abstract: Energetic coupling of two molecular events in a protein molecule is ubiquitous in biochemical reactions mediated by proteins, such as catalysis and signal transduction. Here, we investigate energetic coupling between ligand binding and folding of a dimer using a model system that shows three-state equilibrium unfolding of an exceptional quality. The homodimeric Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM) was found to be stabilized by ATP in a proteome-wide screen, although dPGM does not require or utilize ATP for enzymatic function. We investigated the effect of ATP on the thermodynamic stability of dPGM using equilibrium unfolding. We found that, in the absence of ATP, dPGM populates a partially unfolded, monomeric intermediate during equilibrium unfolding. However, addition of 1.0 mM ATP drastically reduces the population of the intermediate by selectively stabilizing the native dimer. Using a computational ligand docking method, we predicted ATP binds to the active site of the enzyme using the triphosphate group. By performing equilibrium unfolding and isothermal titration calorimetry with active-site variants of dPGM, we confirmed that active-site residues are involved in ATP binding. Our findings show that ATP promotes dimerization of the protein by binding to the active site, which is distal from the dimer interface. This cooperativity suggests an energetic coupling between the active site and the dimer interface. We also propose a structural link to explain how ligand binding to the active site is energetically coupled with dimerization.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, ‡Interdisciplinary Life Science Graduate Program, §Department of Biological Sciences, ∥Department of Computer Science, and ⊥Bindley Bioscience Center, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Nathan W
authors.initials: NW
authors.lastname: Gardner
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, ‡Interdisciplinary Life Science Graduate Program, §Department of Biological Sciences, ∥Department of Computer Science, and ⊥Bindley Bioscience Center, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Lyman K
authors.initials: LK
authors.lastname: Monroe
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, ‡Interdisciplinary Life Science Graduate Program, §Department of Biological Sciences, ∥Department of Computer Science, and ⊥Bindley Bioscience Center, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kihara
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, ‡Interdisciplinary Life Science Graduate Program, §Department of Biological Sciences, ∥Department of Computer Science, and ⊥Bindley Bioscience Center, Purdue University , West Lafayette, Indiana 47907, United States.
authors.forename: Chiwook
authors.initials: C
authors.lastname: Park
mesh_terms: D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D010736:Phosphoglycerate Mutase / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D055503:Protein Multimerization / Q000502:physiology*; D017433:Protein Structure, Secondary
chemical_list: D029968:Escherichia coli Proteins; D008024:Ligands; D010736:Phosphoglycerate Mutase