Chimeric sensory kinases containing O2 sensor domain of FixL and histidine kinase domain from thermophile.
To explore the functional mechanism of inter-domain interaction in a sensor histidine kinase, five chimeric sensory kinases were constructed. In each of these chimeric proteins (CskA254, CskA264, CskA274, CskA284, and CskA294), the sensor domain of heme-based O(2) sensor FixL, obtained from Sinorhizobium meliloti, was fused with the histidine kinase domain from a hyperthermophile, Thermotoga maritima, each at a systematically different position. The UV-visible (UV-vis), resonance Raman (RR), and circular dichroism (CD) spectral characteristics of the CskAs indicated that the secondary and heme environmental structures of all five CskAs examined are identical to those of FixL. In spite of these structural similarities, all CskAs did not exhibit O(2)-dependent regulation of autophosphorylation activity. Furthermore, their functional properties were much different from those of FixL: The O(2) binding affinity and the autophosphorylation activity for CskA254, CskA264, and CskA274 were similar to those of the truncated sensor and histidine kinase domain, whereas CskA284 and CskA294 display extremely low O(2) affinity and low autophosphorylation activity, as compared with each truncated domain. These observations indicated that the interdomain interaction was presented in those CskAs, and that interaction could be related to the O(2)-dependent regulatory interaction of FixL. In the present study, we demonstrated that the interaction in the physiological sensor histidine kinase would be strictly and finely controlled to mediate the signal ligation-dependent autophosphorylation activity in its histidine kinase domain.
Kumita
Hideyuki
H
RIKEN Harima Institute/SPring-8, 1-1-1 Koto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan. kumita@spring8.or.jp
Yamada
Seiji
S
Nakamura
Hiro
H
Shiro
Yoshitsugu
Y
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002942:Circular Dichroism; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics; D000071677:Histidine Kinase; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D015398:Signal Transduction; D016962:Sinorhizobium meliloti / Q000201:enzymology*; D013059:Spectrum Analysis, Raman; D020124:Thermotoga maritima / Q000201:enzymology*
D001426:Bacterial Proteins; D006420:Hemeproteins; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen