The effect of Asp54 phosphorylation on the energetics and dynamics in the response regulator protein Spo0F studied by molecular dynamics.

The response regulator protein Spo0F acts as an intermediate phospho-messenger in the signal transduction pathway that controls initiation of the differentiation process of sporculation in the bacterium Bacillus subtilis. The regulatory domain of Spo0F contains a triad of three conserved aspartate residues, whereof one aspartate (Asp54) is phosphorylated. Using molecular dynamics simulations, we have studied the changes in flexibility induced by phosphorylation and estimated the free energy cost of introducing a phosphate group at this position using alchemical free energy calculations. The deduced conformational flexibility compares well with experimental NMR results. We find that the apo-conformation of the protein explores a rough energy landscape resulting in a broad population of conformational substates. Phosphorylation of Spo0F reduces protein flexibility, and in particular, the so-called anchor and recognition regions exhibit lower mobility relative to the apo-conformation. Phosphorylation of Asp54 (P-Asp54), in which the apo-structure coordinates to the magnesium ion, results in extension of the sidechain, and depending on which carboxylate oxygen is phosphorylated, distinct interactions between P-Asp54 and magnesium ion as well as residues in its proximity are established. However, phosphorylation does not affect the coordination number of the magnesium ion yielding, within the statistical uncertainties, the same free energy change.