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Biochemistry. 2016 Mar 29;55(12):1711-23. doi: 10.1021/acs.biochem.5b00980. Epub 2016 Mar 10.

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

Author information

1
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.

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.

PMID:
26919584
PMCID:
PMC5015447
DOI:
10.1021/acs.biochem.5b00980
[Indexed for MEDLINE]
Free PMC Article

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