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 che motaxis signal transduction pathway of Escherichia coli. This interact ion results in autophosphorylation of CheA, a histidine protein kinase . Autophosphorylation of CheA sets in motion a chain of biochemical ev ents 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 grad ients of attractant and repellent chemicals. To probe CheA interaction s with ATP, we investigated the interaction of CheA with the fluoresce nt nucleotide analogues TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenos ine 5'-triphosphate] and TNP-ADP. Spectroscopic studies indicated that CheA bound TNP-ATP and TNP-ADP with high affinity (micromolar K-d val ues) and caused a marked enhancement of the fluorescence of the TNP mo iety 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 mann er. TNP-ATP was not a substrate for CheA and served as a potent inhibi tor of CheA autophosphorylation (K-i < 1 mu M). The glycine-rich regio ns (G1 and G2) of CheA and other histidine protein kinases have been p resumed to play important roles in ATP binding and/or catalysis of Che A autophosphorylation, although few experimental tests of these functi onal assignments have been made. Here, we demonstrate that a CheA muta nt protein with Gly-->Ala substitutions in G1 and G2 has a markedly re duced affinity for ATP and ADP, as measured by Hummel-Dreyer chromatog raphy. This mutant protein also bound TNP-ATP and TNP-ADP very poorly and had no detectable autokinase activity. Surprisingly, a distinct si ngle-site substitution in G2 (Gly470-->Lys) had no observable effect o n the affinity of CheA for ATP and ADP, despite the fact that it rende red CheA completely inactive as an autokinase. This mutant protein als o bound TNP-ATP and TNP-ADP with affinities and stoichiometries that w ere. 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 t ools for exploring the effects of additional mutations on the active s ite of CheA.