Abstract
In bacterial chemotaxis, chemoreceptors in signaling complexes modulate the activity of two-component histidine kinase CheA in response to chemical stimuli. CheA catalyzes phosphoryl transfer from ATP to a histidinyl residue of its P1 domain. That phosphoryl group is transferred to two response regulators. Receptor control is almost exclusively at autophosphorylation, but the aspect of enzyme action on which that control acts is unclear. We investigated this by a kinetic analysis of activated kinase in signaling complexes. We found that phosphoryl transfer from ATP to P1 is an ordered sequential reaction in which the binding of ATP to CheA is the necessary first step; the second substrate, the CheA P1 domain, binds only to an ATP-occupied enzyme; and phosphorylated P1 is released prior to the second product, namely, ADP. We confirmed the crucial features of this kinetically deduced ordered mechanism by assaying P1 binding to the enzyme. In the absence of a bound nucleotide, there was no physiologically significant binding, but the enzyme occupied with a nonhydrolyzable ATP analog bound P1. Previous structural and computational analyses indicated that ATP binding creates the P1-binding site by ordering the "ATP lid." This process identifies the structural basis for the ordered kinetic mechanism. Recent mathematical modeling of kinetic data identified ATP binding as a focus of receptor-mediated kinase control. The ordered kinetic mechanism provides the biochemical logic of that control. We conclude that chemoreceptors modulate kinase by controlling ATP binding. Structural similarities among two-component kinases, particularly the ATP lid, suggest that ordered mechanisms and control of ATP binding are general features of two-component signaling.IMPORTANCE Our work provides important new insights into the action of the chemotaxis signaling kinase CheA by identifying the kinetic mechanism of its autophosphorylation as an ordered sequential reaction, in which the required first step is binding of ATP. These insights provide a framework for integrating previous kinetic, mathematical modeling, structural, simulation, and docking observations to conclude that chemoreceptors control the activity of the chemotaxis kinase by regulating binding of the autophosphorylation substrate ATP. Previously observed conformational changes in the ATP lid of the enzyme active site provide a structural basis for the ordered mechanism. Such lids are characteristic of two-component histidine kinases in general, suggesting that ordered sequential mechanisms and regulation by controlling ATP binding are common features of these kinases.