Enzyme I (EI) of the bacterial phosphoenolpyruvate:sugar phosphotransferase system can be auto-phosphorylated using PEP and transfers the phosphoryl group to the phosphocarrier protein HPr. Domain motions are central to biological functions of many proteins. EI which is the first protein to initiate a series of phosphotransfer reactions consists of two domains connected by the linker helix: the N-terminal domain (EIN) which is composed of a catalytic EINαβ subdomain, and an HPr binding EINα subdomain, and the C-terminal domain (EIC) which is a PEP binding domain. Here we employed different domain-deletion constructs to dissect and characterize the domain-domain motions coupled with ligand binding of unphosphorylated EI using isothermal titration calorimetry (ITC) .We demonstrated that the free energy of the hinge motion (△G = 1.5kcal/mol) is unfavorable energy, which can be overcome by the free energy of the swivel motion. The domain motions are entropy-driven, which is caused by the changes in the inter-domain interfaces upon ligand binding and domain motions. In addition, PEP binding and HPr binding did not crosstalk with each other. The fact that two substrates can bind independently suggests that EI:PEP:HPr ternary complex can be easily formed during phosphotransfer reactions. Our results will help understand the thermodynamics of large domain-domain motions associated with protein-ligand interactions.
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Thermodynamic characterization of domain motions coupled with ligand binding of Enzyme I in the bacterial phosphotransferase system
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