Further insights into the mechanism of function of the response regulator CheY from crystallographic studies of the CheY–CheA_124–257 complex

New crystallographic structures of the response regulator CheY in association with CheA_124–257, its binding domain in the kinase CheA, have been determined. In all crystal forms, the molecular interactions at the heterodimer interface are identical. Soaking experiments have been performed on the crystals using acetyl phosphate as phosphodonor to CheY. No phosphoryl group attached to Asp57 of CheY is visible from the electron density, but the response regulator in the CheY–CheA_124–257 complex may have undergone a phosphorylation–dephosphorylation process. The distribution of water molecules and the geometry of the active site have changed and are now similar to those of isolated CheY. In a second soaking experiment, imido-diphosphate, an inhibitor of the phosphorylation reaction, was used. This compound binds in the vicinity of the active site, close to the N-terminal part of the first α-­helix. Together, these results suggest that the binding of CheY to CheA_124–257 generates a geometry of the active site that favours phosphorylation and that imido-diphosphate interferes with phosphorylation by precluding structural changes in this region.