Identification of a novel two component system in Thermotoga maritima. Complex stoichiometry and crystallization.
Two-component signal transduction systems, comprised of histidine kinase and its cognate response regulator, are the predominant mechanism by which microorganisms sense and respond to changes in many different environmental conditions. Different Thermotoga maritima histidine kinases have been used as prototypes; among them, the orphan TM0853 has been presented as a structural model of class I histidine kinases. We used phosphotransfer assays to identify TM0468 as the partner response regulator of TM0853. Since full-length TM0853 can be produced as a soluble protein in Escherichia coli, it was used to analyze the union stoichiometry in an intact two-component system for the first time. We demonstrate that TM0853, or its cytoplasmic catalytic portion, form a 1:1 complex with TM0468 with native PAGE. The complex band is unique, even in the presence of an excess of each individual protein, indicating that the union is cooperative. We corroborated these findings by using ultracentrifugation assays. Therefore, we propose that the general mode of interaction in an orthodox two-component system may be the stoichiometric and cooperative complex between a dimeric histidine kinase and two response regulators. Finally, we have been able to produce protein crystals of the complex between the cytoplasmic portion of TM0853 and TM0468 that diffract to 2.8 A Bragg spacing.
Casino
Patricia
P
Departamento de Genómica y Proteómica, Instituto de Biomedicina de Valencia (CSIC), Jaume Roig 11, 46010, Valencia, Spain.
Fernández-Alvarez
Ana
A
Alfonso
Carlos
C
Rivas
Germán
G
Marina
Alberto
A
D001483:Base Sequence; D003460:Crystallization; D017931:DNA Primers; D000071677:Histidine Kinase; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D020124:Thermotoga maritima / Q000737:chemistry* / Q000201:enzymology; D014461:Ultracentrifugation
D017931:DNA Primers; D011494:Protein Kinases; D000071677:Histidine Kinase