SixA is a phosphatase that dephosphorylates phosphohistidine in proteins involved in signaling in bacteria. Using NMR spectroscopy, the interaction of SixA was structurally characterized with one of its functional substrates, the phosphocarrier protein NPr, which shuttles a phosphate group between components of the nitrogen-related phosphotransferase system using a phosphorylated histidine residue. An ensemble structure of the complex was generated combining chemical shift perturbation data and intermolecular paramagnetic relaxation enhancement (PRE) via Gd-DOTA spin-labels attached to NPr, which shows two regions of interaction outside the SixA active site. Simulated molecular crowding did not enhance interaction with these regions. Phosphate, one of the products of the enzyme catalysis, was determined to bind at a site consistent with that seen for a tungstate ion in the SixA X-ray crystallography structure. SixA dephosphorylates NPr too rapidly for NMR characterization of the phosphorylated form; consequently, interaction with substrate analogs was studied, including a mutant of NPr, H16D, to mimic the negative charge of phosphate at the histidine, and non-cleavable analogs of phosphohistidine. The non-cleavable analogs bound in the active site, but at a location distinct from bound phosphate. A phosphohistidine-containing tripeptide was synthesized, and the activity of SixA was measured using liquid chromatography/mass spectrometry. In the presence of phosphate, the activity of SixA was enhanced.
