Thermal domain motions of CheA kinase in solution: Disulfide trapping reveals the motional constraints leading to trans-autophosphorylation.

The histidine kinase CheA is a central component of the bacterial chemotaxis signaling cluster, in which transmembrane receptors regulate CheA autokinase activity. CheA is a homodimer, and each of the two identical subunits possesses five different domains with distinct structures and functions. The free enzyme, like the receptor-bound enzyme, catalyzes a trans-autokinase reaction in which the catalytic domain (P4) of one subunit phosphorylates the substrate domain (P1) of the other subunit. Molecular analysis of CheA domain motions has important implications for the mechanism of CheA trans-autophosphorylation, for CheA assembly into the signaling cluster and for receptor regulation of CheA activity. In this initial study of the free CheA dimer, we employ disulfide trapping to analyze collisions between pairs of domains, thereby mapping out the ranges and kinetics of domain motions. A library of 33 functional single-cysteine CheA mutants, all retaining normal autokinase activity, is used to analyze intradimer collisions between symmetric domain pairs. The homodimeric structure of CheA ensures that each mutant contains a pair of symmetric, surface-exposed cysteine residues. Cysteine-cysteine collisions trapped by disulfide bond formation indicate that P1 is the most mobile CheA domain, but large amplitude P2, P4, and P5 domain motions are also detected. The mobility of P1 is further analyzed using a library of 17 functional dicysteine CheA mutants, wherein each mutant subunit possesses one cysteine at a fixed probe position on the P1 domain and a second cysteine on a different domain. The resulting CheA homodimers contain four cysteine residues; thus disulfide trapping yields multiple products that are identified by assignment methods. The findings reveal that the P1 substrate domain collides rapidly with residues on the P4' catalytic domain in the sister subunit, but no intrasubunit collisions are detected. This observation provides a direct, motional explanation for CheA trans-autophosphorylation, explains why the long linkers of the P1-P2 region do not become tangled in the dimer, and has important implications for other aspects of CheA function. Finally, a working model is proposed for the motional constraints that limit the P1 domain to the region of space near the P4' catalytic domain of the sister subunit.