Protein histidine phosphorylation exists widely in vertebrates, and it plays important roles in signal transduction and other cellular functions. However, knowledge about eukaryotic PHPT (protein histidine phosphatase) is still very limited. To date, only one vertebrate PHPT has been discovered, and two crystal structures of hPHPT1 (human PHPT1) have been solved. However, these two structures gave different ligand-binding sites and co-ordination patterns. In the present paper, we have solved the solution structures of hPHPT1 in both Pi-free and Pi-bound states. Through comparison of the structures, along with a mutagenesis study, we have determined the active site of hPHPT1. In contrast with previous results, our results indicate that the active site is located between helix α1 and loop L5. His53 was identified to be the catalytic residue, and the NH groups of residues His53, Ala54 and Ala96 and the OH group of Ser94 should act as anchors of Pi or substrate by forming H-bonds with Pi. On the basis of our results, a catalytic mechanism is proposed for hPHPT1: the imidazole ring of His53 serves as a general base to activate a water molecule, and the activated water would attack the substrate as a nucleophile in the catalysis; the positively charged side chain of Lys21 can help stabilize the transition state. No similar catalytic mechanism can be found in the EzCatDB database.
The Solution Structure of the Human ETS1-DNA Complex Reveals a Novel Mode of Binding and True Side Chain Intercalation