Structural and chemical requirements for histidine phosphorylation by the chemotaxis kinase CheA.
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 phosphotransfer 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. 15N-NMR chemical shift studies instead showed that Glu-67 functions to stabilize the unfavored N(delta1)H tautomer of His-45, thereby rendering the N(epsilon2) imidazole unprotonated and well positioned for accepting the ATP phosphoryl group.
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Cindy M
CM
Quezada
Damon J
DJ
Hamel
Cristian
C
Gradinaru
Alexandrine M
AM
Bilwes
Frederick W
FW
Dahlquist
Brian R
BR
Crane
Melvin I
MI
Simon
D000255:Adenosine Triphosphate / Q000737:chemistry; D002633:Chemotaxis; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D006639:Histidine / Q000737:chemistry*; D000071677:Histidine Kinase; D006859:Hydrogen; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry / Q000502:physiology*; D017434:Protein Structure, Tertiary; D020124:Thermotoga maritima / Q000201:enzymology* / Q000502:physiology
D006639:Histidine; D006859:Hydrogen; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000071677:Histidine Kinase