Decoupling the ATP-binding Domain in the CheA Kinase by Increasing Linker Flexibility Dramatically Alters Kinase Activity
In bacterial chemotaxis chemoreceptors regulate the cytosolic dimeric histidine kinase CheA. To test the role that interdomain linkers play in CheA regulation the linkers that connect the P4 kinase domain to the P3 dimerization domain (L3) and the P5 regulatory domain (L4) were extended and altered in variants of Thermotoga maritima (Tm) CheA. Flexible extensions of the L3 and L4 linkers in CheA-LV1 (linker variant 1) allow for a well-folded kinase domain that retains WT-like binding affinities
... for nucleotide and normal interactions with the receptor-coupling protein CheW. However, CheA-LV1 autophosphorylation activity registers ~50-fold lower compared to wild-type. Formation of the CheA-LV1 / CheA WT heterodimer fails to rescue CheA-LV1 autophosphorylation and instead reduces the activity of the WT subunit. Neither CheA WT nor CheA-LV1 can phosphorylate P1 in a CheA dimer that contains a single P4 domain. Rescue of autophosphorylation activity in variants with a poly-alanine L3 or an L3 that maintains a heptad repeat suggest that positioning and conformational transitions of P4 depend on L3 assuming helical structure. Pulse dipolar ESR measurements indicate that the CheA-LV1 P4 domains are in close proximity whereas broader distributions in other variants correlate with increased activity. CheA-LV1 has a substantially larger hydrodynamic radius than does CheA WT by SAXS, despite the P4 domains assuming a closed, inhibited conformation. These results explain negative cooperativity in CheA nucleotide binding, demonstrate coupling between P4 disposition and P1 / P2 dynamics and underscore the importance of P4-P4 interactions and an L3 a- helix in CheA activity and regulation.