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Journal of Bacteriology, February 2001, p. 1359-1368, Vol. 183, No. 4
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.4.1359-1368.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Protein-Protein Interactions in the Complex between the Enhancer Binding Protein NIFA and the Sensor NIFL from Azotobacter vinelandii

Tracy Money, Jason Barrett, Ray Dixon, and Sara Austin*

Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, Norfolk, United Kingdom

Received 2 October 2000/Accepted 23 November 2000

The enhancer binding protein NIFA and the sensor protein NIFL from Azotobacter vinelandii comprise an atypical two-component regulatory system in which signal transduction occurs via complex formation between the two proteins rather than by the phosphotransfer mechanism, which is characteristic of orthodox systems. The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant homology to the histidine protein kinase transmitter domains. Adenosine nucleotides, particularly MgADP, also stimulate complex formation between NIFL and NIFA in vitro, allowing isolation of the complex by cochromatography. Using limited proteolysis of the purified proteins, we show here that changes in protease sensitivity of the Q linker regions of both NIFA and NIFL occurred 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. Experiments with truncated versions of NIFA demonstrate that the central domain of NIFA is sufficient to cause protection of the Q linker of NIFL, although in this case, stable protein complexes are not detectable by cochromatography.

* Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, Norfolk, United Kingdom. Phone: 44 (0)1603 450748. Fax: 44 (0)1603 450778. E-mail: sara.austin{at}bbsrc.ac.uk

Journal of Bacteriology, February 2001, p. 1359-1368, Vol. 183, No. 4
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.4.1359-1368.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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