A dynamic homodimer structure of the response regulator FixJ from Bradyrhizobium japonicum in its phosphorylated state in aqueous solution.

The symbiotic nitrogen-fixing bacterium Bradyrhizobium japonicum possess a two component regulatory system comprising FixL, a histidine kinase with O2-sensor, and FixJ, a response regulator controlling the expression of nitrogen fixation-related genes. The phosphotransfer from FixL to the N-terminal receiver domain (REC) of FixJ promote the association of the C-terminal DNA-binding domain (DBD) to DNA. To understand the structural basis of the activation, solution NMR approaches were employed to FixJ in the acetyl phosphate-mediated phosphorylated and the BeF3--bound states. The backbone resonance assignments indicated the formation of symmetric homodimer in the activated states. Chemical shift changes caused by the activation were distributed on a half surface of REC as well as on a limited region in DBD, indicating that the phosphorylation propagates to DBD. Cross saturation experiments revealed a major dimerisation interface comprising helix α4 and strand β5, which is common to the Sinorhizobium meliloti FixJ, and additional dimerisation interfaces located on helices α3 and α5 of REC and on helices α7 and α10 on DBD. Considering that REC and DBD tumble separately both in the unphosphorylated and the phosphorylated states, the activation of FixJ can be delineated as the transition of one "inactive" ensemble structure to another "active" ensemble.