Purification crystallization and preliminary X-ray analysis of bifunctional isocitrate dehydrogenase kinase/phosphatase in complex with its substrate isocitrate dehydrogenase from Escherichia coli

摘要

Escherichia coli isocitrate dehydrogenase (ICDH) can be phosphorylated and dephosphorylated by a single bifunctional protein, isocitrate dehydrogenase kinase/phosphatase (AceK), which is encoded by the aceK gene. In order to investigate the regulatory mechanism of (de)phosphorylation of ICDH by AceK, AceK was successfully cocrystallized in complex with its intact protein substrate, ICDH, in the presence of ATP. The complex crystal was obtained by the hanging-drop vapour-diffusion technique using PEG 300 as a precipitant and magnesium sulfate as an additive. SDS–PAGE analysis of dissolved crystals showed that the crystals contained both AceK and ICDH proteins. The complex crystals diffracted to a resolution of 2.9 Å in space group P63, with unit-cell parameters a = b = 196.80, c = 156.46 Å.