A non-hydrolyzable ATP derivative generates a stable complex in a light-inducible two-component system.
Isothermal calorimetry (ITC) measurements yielded the binding constants during complex formation of light-inducible histidine kinases (HK) and their cognate CheY-type response regulators (RR). HK-RR interactions represent the core function of the bacterial two-component system, which is also present in many bacterial phytochromes. Here, we have studied the recombinant forms of phytochromes CphA and CphB from the cyanobacterium Tolypothrix PCC7601 and their cognate RRs RcpA and RcpB. The interaction between the two reaction partners (HK and RR) was studied in the presence and absence of ATP. A complex formation was observable in the presence of ATP, but specific interactions were only found when a non-hydrolyzable ATP derivative was added to the mixture. Also, the incubation of the HK domain alone (expressed as a recombinant protein) with the RR did not yield specific interactions, indicating that the HK domain is only active as a component of the full-length phytochrome. Considering also previous studies on the same proteins (Hübschmann, T., Jorissen, H. J. M. M., Börner, T., Gärtner, W., and de Marsac, N. (2001) Eur. J. Biochem. 268, 3383-3389) we now conclude that the HK domains of these phytochromes are active only when the chromophore domain is in its Pr form. The formerly documented phosphate transfer between the HK domain and the RR takes place via a transiently formed protein-protein complex, which becomes detectable by ITC in the presence of a non-hydrolyzable ATP derivative. This finding is of interest also in relation to the function of some (blue light-sensitive) photoreceptors that carry the HK domain and the RR fused together in one single protein.
Sharda
Shivani
S
Max Planck Institute for Bioinorganic Chemistry, 45413 Mülheim, Germany.
Koay
Melissa S T
MS
Kim
Young-Jun
YJ
Engelhard
Martin
M
Gärtner
Wolfgang
W
D000255:Adenosine Triphosphate / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D002151:Calorimetry / Q000379:methods; D000458:Cyanobacteria / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D008027:Light; D008565:Membrane Proteins / Q000737:chemistry; D000072236:Methyl-Accepting Chemotaxis Proteins; D008954:Models, Biological; D010777:Photochemistry / Q000379:methods; D010834:Phytochrome / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature
D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011994:Recombinant Proteins; D010834:Phytochrome; D000255:Adenosine Triphosphate