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Impact of phosphorylation on structure and thermodynamics of the interaction between the N-terminal domain of enzyme I and the histidine phosphocarrier protein of the bacterial phosphotransferase system.

Suh JY, Cai M, Clore GM

Chemical Physics, NIDDK/NIH, Bethesda, MD 20892-0520.

The structural and thermodynamic impact of phosphorylation on the interaction of the N-terminal domain of enzyme I (EIN) and the histidine phosphocarrier protein HPr, the two common components of all branches of the bacterial phosphotransferase system (PTS), have been examined using NMR spectroscopy and isothermal titration calorimetry. His189 is located at the interface of the a and a/ss domains of EIN, resulting in rather widespread chemical shift perturbation upon phosphorylation, in contrast to the highly localized perturbations seen for HPr where His15 is fully exposed to solvent. Residual dipolar coupling (RDC) measurements, however, demonstrate unambiguously that no significant changes in backbone conformation of either protein occur upon phosphorylation: for EIN, the relative orientation of the a and a/ss domains remains unchanged; for HPr, the backbone f/ torsion angles of the active site residues are unperturbed within experimental error. HisGlu/Asp mutations of the active site histidines designed to mimic the phosphorylated states reveal binding equilibria that favor phosphoryl transfer from EIN to HPr. Although binding of phospho-EIN to phospho-HPr is reduced by a factor of ~21 relative to the unphosphorylated complex, RDC measurements reveal that the structures of the unphosphorylated and biphosphorylated complexes are the same. Hence, the phosphorylation states of EIN and HPr shift the binding equilibria predominantly by modulating intermolecular electrostatic interactions without altering either the backbone scaffold or binding interface. This facilitates highly efficient phosphoryl transfer between EIN and HPr, which is estimated to occur at a rate of ~850 s-1 from exchange spectroscopy.

Published 30 April 2008 in J Biol Chem.
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