Rapid Phosphotransfer to CheY from a CheA Protein Lacking the CheY-Binding Domain

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Abstract
The histidine protein kinase CheA plays a central role in the bacterial chemotaxis signal transduction pathway. Autophosphorylated CheA passes its phosphoryl group to CheY very rapidly (kcat ∼ 750 s-1). Phospho-CheY in turn influences the direction of flagellar rotation. The autophosphorylation site of CheA (His48) resides in its N-terminal P1 domain. The adjacent P2 domain provides a high-affinity binding site for CheY, which might facilitate the phosphotransfer reaction by tethering CheY in close proximity to the phosphodonor located in P1. To explore the contribution of P2 to the CheA → CheY phosphotransfer reaction in the Escherichia coli chemotaxis system, we examined the transfer kinetics of a mutant CheA protein (CheAΔP2) in which the 98 amino acid P2 domain had been replaced with an 11 amino acid linker. We used rapid-quench and stopped-flow fluorescence experiments to monitor phosphotransfer to CheY from phosphorylated wild-type CheA and from phosphorylated CheAΔP2. The CheAΔP2 reaction rates were significantly slower and the Km value was markedly higher than the corresponding values for wild-type CheA. These results indicate that binding of CheY to the P2 domain of CheA indeed contributes to the rapid kinetics of phosphotransfer. Although phosphotransfer was slower with CheAΔP2 (kcat/Km ∼ 1.5 × 106 M-1 s-1) than with wild-type CheA (kcat/Km ∼ 108 M-1 s-1), it was still orders of magnitude faster than the kinetics of CheY phosphorylation by phosphoimidazole and other small molecule phosphodonors (kcat/Km ∼ 5−50 M-1 s-1). We conclude that the P1 domain of CheA also makes significant contributions to phosphotransfer rates in chemotactic signaling.