Screening And Functional Study Of Hyperactive Mutations In An Interdomain Linker Connecting The Dimerization And Catalytic Domains Of Histidine Kinase CheA

In chemotaxis,motile bacteria sense surrounding chemical gradients and respond to them by swimming toward favorable environmental conditions and away from harmful ones.This phenomenon is realized by a signal transduction pathway.The extracellular parts of different types of chemoreceptors probe chemical signals in the environment.Chemoreceptors form a ternary signaling complex with the regulatory P5 domain of the unique and conserved histidine kinase CheA,and CheW,a small adaptor protein.In ternary signaling complexs,the autophosphorylation activity of CheA is strictly controlled by transmembrane receptors,and plays a central role in signal integration,conversion,and amplification.Despite recent advancements in the understanding of the architecture of the signaling complex,the molecular mechanism underlying CheA regulation remains elusive.The P3,P4,and P5 domains together form a kinase core,in which each domain seems to be responsible for a specific function,but actually works together to achieve the regulation of CheA.Previous studies in our laboratory have shown that the linkers connecting these three domains play an important role in the activity and regulation of the kinase.For example,single amino acid replacement in the P3-P4 linker could result in significant reduction in basal kinase activity as well as kinase activation.Based on this important finding,this study carried out a more in-depth study of the linker,aiming to explore whether the inter-domain linker really has the ability to influence the activity of kinase.Screening for hyperactive mutation in the region of linker showed that the activity of kinase could be increased significantly by changing the amino acid sequence of the linker,and the highest activity of the CheA variants we have obtained was 46 times higher than that of the wild-type CheA.In the absence of P5 alone or both P5 and the P4-P5 linker,P3-P4 linker mutants could not increase the activity of CheA,indicating that its effect on kinase activity requires the coordination of the P5 domain and even the P4-P5 linker.The combination of these P4-P5 linker mutants with the hyperactive P3-P4 linker mutants that we previously obtained did not achieve higher activity of kinase indicating that the two linkers had no synergetic effect on the activity,but acted together to modulate CheA activity.Despite high basal autophosphorylation activity,these P3-P4 linker mutants could not perform normal bacterial chemotaxis in Wvo.In vitro biochemical studies showed that mutations in the P3-P4 linker did not affect the natural receptor-mediated activation(kinase on)pathway,but undermined the attrattant-mediated inhibitory(kinase off)pathway.Therefore,CheA P3-P4 linker mutation may change the balance between "kinase-on" and "kinase-off' states in a free CheA by interfering with the inhibition of CheA,which results in the hyperactive basal kinase activity.