Structural and chemical requirements for histidine phosphorylation by the chemotaxis kinase CheA

Abstract

The CheA histidine kinase initiates the signal transduction pathway of bacterial chemotaxis by autophosphorylating a conserved histidine on its phosphotransferase domain (P1). Site-directed mutations of neighboring conserved P1 residues (Glu-67, Lys-48, and His-64) show that a hydrogen-bonding network controls the reactivity of the phospho-accepting His (His-45) in Thermotoga maritima CheA. In particular, the conservative mutation E67Q dramatically reduces phospho-transfer to P1 without significantly affecting the affinity of P1 for the CheA ATP-binding domain. High resolution crystallographic studies revealed that although all mutants disrupt the hydrogen-bonding network to varying degrees, none affect the conformation of His-45. N-15-NMR chemical shift studies instead showed that Glu-67 functions to stabilize the unfavored (NH)-H-delta 1 tautomer of His-45, thereby rendering the N-epsilon 2 imidazole unprotonated and well positioned for accepting the ATP phosphoryl group.

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