Effects of histidine protonation and phosphorylation on histidine-containing phosphocarrier protein structure, dynamics, and physicochemical properties.

Previous structural studies of the histidine-containing phosphocarrier protein (HPr) have shown that active site residue His15 can adopt two distinct conformations which were termed OPEN and CLOSED. Using molecular dynamics simulations and protonation probability calculations, we were able to show that these two conformations correspond to different protonation forms of the histidine ring. The CLOSED-to-OPEN transition requires His15 to adopt a conformation with higher energy, which is compensated by the favorable energetic consequences of protonation. Calculations of the conformational energy of His15 show that HPr exists mainly in the CLOSED form at pH 7. The very low apparent pKa value (3.2-4.5) of the CLOSED conformation and the fact that the imidazole ring of residue 15 is primarily unprotonated at Ndelta1 at neutral pH ensure that His15 is ideally primed to be specifically phosphorylated at Ndelta1. In contrast to unphosphorylated HPr, the phosphorylated form exhibits no conformational transitions, and the CLOSED state is stable even for the protonated imidazole ring due to favorable interactions between the phosphate group and the backbone of Ala16 and Arg17. These observations from MD simulations are confirmed by a simple four-microstate model which can explain both the pH-dependent conformational change of unphosphorylated HPr and the conformational rigidity of phosphorylated HPr. Our study suggests that the predominant CLOSED conformation is relevant for HPr function in the phosphotransfer reaction, while the OPEN form of unphosphorylated HPr might be important for its additional regulatory function, in which an OPEN conformation of His15 is recognized by the transcriptional regulator CcpA.