Phosphotransfer site of the chemotaxis-specific protein kinase CheA as revealed by NMR

Bacterial chemotaxis involves autophosphorylation of a histidine kinase and transfer of the phosphoryl group to response regulators to control flagellar rotation and receptor adaptation. The phosphotransfer domain, CheA(1-134), of the chemotaxis-specific histidine autokinase CheA from Escherichia coli contains the site of phosphorylation, His48, and two other histidine residues, His26 and His67. Two-dimensional H-1-N-15 NMR techniques were applied to characterize the protonation states of these histidine residues and to evaluate the structural changes in the domain that occur upon phosphorylation of His48. The pK(a) of His48 was determined to be 7.8 (in 50 mM NaPO4 buffer at 30 degrees C). At high pH, its imidazole ring exists primarily as the normally unfavored (NH)-H-delta 1 tautomer, suggesting hydrogen bond formation to the ring nitrogen atom(s) to stabilize this state. The pK(a) values and predominant tautomeric states of the imidazole rings of His26 (pK(a) similar to 7.1, (NH)-H-epsilon 2 tautomer) and His67 (pK(a) similar to 6.5, (NH)-H-delta 1 tautomer) were also determined. His48 of CheA(1-134) and CheA(1-233) was phosphorylated by full-length CheA. The phosphorylation site was confirmed to be the N-epsilon 2 position in the imidazole ring. Phosphorylation of His48 only results in small changes in the amide H-1 and N-15 chemical shifts of a few residues from helices B and C, suggesting that only very small changes in structure are associated with phosphorylation of the phosphotransfer domain of CheA. These residues occupy a small surface area of the helix bundle and form the active site of the protein. At the active site, in addition to His48, residues Gly52, His67, and Glu70 are conserved in the CheA homologous phosphotransfer domains from 10 different organisms. Sequence comparison of these CheA homologs suggest that the phosphotransfer domains likely fold in a similar helix-bundle structure and the structural features at the active site are well-conserved.