Protein-protein interactions in the complex between the enhancer binding protein NIFA and the sensor NIFL from Azotobacter vinelandii

The enhancer binding protein NIFA and the sensor protein NIFL from Azotobac ter vinelandii comprise an atypical two-component regulatory system in whic h signal transduction occurs via complex formation between the two proteins rather than by the phosphotransfer mechanism, which is characteristic of o rthodox systems. The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant h omology to the histidine protein kinase transmitter domains. Adenosine nucl eotides, particularly MgADP, also stimulate complex formation between NIFL and NIFA in vitro, allowing isolation of the complex by cochromatography. U sing limited proteolysis of the purified proteins, we show here that change s in protease sensitivity of the Q linker regions of both NIFA and NIFL occ urred when the complex was formed in the presence of MgADP. The N-terminal domain of NIFA adjacent to the Q linker was also protected by NIFL. Experim ents with truncated versions of NIFA demonstrate that the central domain of NIFA is sufficient to cause protection of the Q linker of NIFL, although i n this case, stable protein complexes are not detectable by cochromatograph y.