TNP-ATP and TNP-ADP as probes of the nucleotide binding site of CheA, the histidine protein kinase in the chemotaxis signal transduction pathway of Escherichia coli.
The interaction of CheA with ATP has important consequences in the chemotaxis signal transduction pathway of Escherichia coli. This interaction results in autophosphorylation of CheA, a histidine protein kinase. Autophosphorylation of CheA sets in motion a chain of biochemical events that enables the chemotaxis receptor proteins to communicate with the flagellar motors. As a result of this communication, CheA allows the receptors to control the cell swimming pattern in response to gradients of attractant and repellent chemicals. To probe CheA interactions with ATP, we investigated the interaction of CheA with the fluorescent nucleotide analogues TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate] and TNP-ADP. Spectroscopic studies indicated that CheA bound TNP-ATP and TNP-ADP with high affinity (micromolar Kd values) and caused a marked enhancement of the fluorescence of the TNP moiety of these modified nucleotides. Analysis of titration experiments indicated a binding stoichiometry of two molecules of TNP-ATP (TNP-ADP) per CheA dimer and suggested that the two binding sites on the CheA dimer operate independently. Binding of TNP-ATP to CheA was inhibited by ATP, and analysis of this inhibition indicated that the CheA dimer binds 2 molecules of ATP. Competition experiments also indicated that CheA binds TNP-ATP considerably more tightly than it binds unmodified ATP. Binding of TNP-ADP to CheA was inhibited by ADP in a similar manner. TNP-ATP was not a substrate for CheA and served as a potent inhibitor of CheA autophosphorylation (Ki < 1 microM). The glycine-rich regions (G1 and G2) of CheA and other histidine protein kinases have been presumed to play important roles in ATP binding and/or catalysis of CheA autophosphorylation, although few experimental tests of these functional assignments have been made. Here, we demonstrate that a CheA mutant protein with Gly-->Ala substitutions in G1 and G2 has a markedly reduced affinity for ATP and ADP, as measured by Hummel-Dreyer chromatography. This mutant protein also bound TNP-ATP and TNP-ADP very poorly and had no detectable autokinase activity. Surprisingly, a distinct single-site substitution in G2 (Gly470-->Lys) had no observable effect on the affinity of CheA for ATP and ADP, despite the fact that it rendered CheA completely inactive as an autokinase. This mutant protein also bound TNP-ATP and TNP-ADP with affinities and stoichiometries that were indistinguishable from those observed with wild-type CheA. These results provide some preliminary insight into the possible functional roles of G1 and G2, and they suggest that TNP-nucleotides are useful tools for exploring the effects of additional mutations on the active site of CheA.
Stewart
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Department of Cell Biology & Molecular Genetics, University of Maryland, College Park 20742, USA. rs224@umail.umd.edu
VanBruggen
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Ellefson
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Wolfe
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D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins; D001665:Binding Sites / Q000187:drug effects; D002633:Chemotaxis / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism / Q000502:physiology; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes / Q000378:metabolism* / Q000494:pharmacology; D006639:Histidine / Q000378:metabolism; D000071677:Histidine Kinase; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D016297:Mutagenesis, Site-Directed; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D047428:Protein Kinase Inhibitors; D006639:Histidine; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli