Active site phosphoryl groups in the biphosphorylated phosphotransferase complex reveal dynamics in a millisecond time scale

The N-terminal domain of Enzyme I (EIN) and phosphocarrier HPr can form a biphosphorylated complex when they are both phosphorylated by excess cellular phosphoenolpyruvate. Here we show that the electrostatic repulsion between the phosphoryl groups in the biphosphorylated complex results in characteristic dynamics at the active site in a millisecond time scale. The dynamics is localized to phospho-His15 and the stabilizing backbone amide groups of HPr, and does not impact on the phospho-His189 of EIN. The dynamics occurs with the kex of ∼500 s−1 which compares to the phosphoryl transfer rate of ∼850 s−1 between EIN and HPr. The conformational dynamics in HPr may be important for its phosphotransfer reactions with multiple partner proteins.Structured summary of protein interactionsEIN and HPrbind by nuclear magnetic resonance (View Interaction).

► Phosphoryl groups in the biphosphorylated complex exhibit characteristic dynamics.
► The dynamics occurs in a millisecond time scale at the active site of HPr.
► The time scale is comparable to the phosphoryl transfer rate between EIN and HPr.
► The dynamics of HPr may be important to interact with multiple partner proteins.