Functional Role Of An Interdomain Linker Between The Catalytic And Regulatory Domains Of The Bacterial Chemotaxis Histidine Kinase CheA

Bacterial chemotaxis signal transduction pathway is typical two-component signal transduction system.In this pathway,the histidine kinase CheA performs autophosphorylation and transfer high-energy phosphoryl group to its homologous response regulator CheY.Phospho-CheY interacts with the flagellar rotary motor,resulting in clockwise(CW)rotation of flagella and a change in swimming direction of a bacterium.Beside CheA and CheY,the signal transduction requires the cooperation of the coupling protein CheW and the transmembrane chemoreceptors.The ultrastable chemotaxis signaling complexes are formed via the interactions among CheA's regulatory domain(P5),CheW and chemoreceptors.The kinase activity of CheA is regulated in chemotaxis signaling complexes.Transmembrane chemoreceptors sense the external environmental signal,and the signaling complexes exhibit kinase-on or kinase-off output states.The bacterial swimming behavior reflects the proportions of signaling complexes in the ON and OFF states.Thus,the histidine kinase CheA plays a central role in signal integration,conversion,and amplification in the bacterial chemotaxis signal transduction pathway.Despite recent advancements in the understanding of the architecture of the signaling complex,the molecular mechanism underlying this regulation remains elusive.Our previous results haven shown that an interdomain linker,P4-P5 linker,that connects the catalytic(P4)and regulatory domains of CheA,may mediate regulatory signals from the P5-CheW-receptor interactions to the catalytic domain.To investigate whether this interdomain linker is capable of affecting CheA's activity,that is to say whether it is able to increase or decrease CheA's activity under different conditions,we mutated the P4-P5 linker of the kinase CheA randomly and performed in vivo screens to search for P4-P5 linker mutations that result in different CheA autokinase activities(hyperactive or inactive).Several CheA variants were identified with kinase activities ranging from 30%to 670%of the activity of wild-type CheA.All of these CheA variants failed to support chemotaxis in vivo and defective in receptor-mediated kinase activation in vitro,while retaining their ability to form signaling complexes with CheW and chemoreceptor. The results indicates that the natural receptor-mediated signal transmission pathway was simultaneously affected by these mutations.In addition,the altered P4-P5 linkers in the context of the CheA-AP5 construct made changes in kinase activity similar to those in full-length CheA.This means the P4-P5 linkers are sufficient for making significant changes in kinase activity independent of the regulatory domain.Therefore,the interdomain linker is an active module that has the ability to impose regulatory effects on the catalytic activity of the P4 domain and CheA activity.These results suggest that chemoreceptors may manipulate the conformation of the P4-P5 linker to achieve CheA regulation in the platform of the signaling complex.