Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in Escherichia coli

We investigated the functional roles of putative active site residues in Es cherichia coli CheA by generating nine site-directed mutants, purifying the mutant proteins, and quantifying the effects of those mutations on autokin ase activity and binding affinity for ATP. We designed these mutations to a lter key positions in sequence motifs conserved in the protein histidine ki nase family, including the N box (H376 and N380), the G1 box (D420 and G422 ), the F box (F455 and F459), the G2 box (G470, G472, and G474), and the "G T block" (T499), a motif identified by comparison of CheA to members of the GHL family of ATPases. Four of the mutant CheA proteins exhibited no detec table autokinase activity (Kin(-)). Of these, three (N380D, D420N, and G422 A) exhibited moderate decreases in their affinities for ATP in the presence or absence of Mg2+. The other Kin(-) mutant (G470A/G472A/G474A) exhibited wild-type affinity for ATP in the absence of Mg2+, but reduced affinity (re lative to that of wild-type CheA) in the presence of Mg2+. The other five m utants (Kin) autophosphorylated at rates slower than that exhibited by wild -type CheA. Of these, three mutants (H376Q, D420E, and F455Y/F459Y) exhibit ed severely reduced k(cat) values, but preserved K-M(ATP) and K-d(ATP) valu es close to those of wild-type CheA. Two mutants (T499S and T499A) exhibite d only small effects on k(cat) and K-M(ATP). Overall, these results suggest that conserved residues in the N box, GI box, G2 box, and F box contribute to the ATP binding site and autokinase active site in CheA, while the GT b lock makes little, if any, contribution. We discuss the effects of specific mutations in relation to the three-dimensional structure of CheA and to bi nding I interactions that contribute to the stability of the complex betwee n CheA and Mg2+-bound ATP in both the around state and the transition state for the CheA autophosphorylation reaction.