A monomeric histidine kinase derived from EnvZ, an Escherichia coli osmosensor

Histidine kinases function as dimers. The kinase domain of the osmosensing histidine kinase EnvZ of Escherichia coli consists of two domains: domain A (67 residues) responsible for histidine phosphotransfer and dimerization, and domain B (161 residues) responsible for the catalytic and ATP-binding f unction. The individual structures of these two domains have been recently solved by NMR spectroscopy. Here, we demonstrate that an enzymatically func tional monomeric histidine kinase can be constructed by fusing in tandem tw o domains A and one domain B to produce a single polypeptide (A-A-B). We sh ow that this protein, EnvZc[AAB], is soluble and exists as a stable monomer . The autophosphorylation and OmpR kinase activities of the monomeric EnvZc [AAB] are similar to that of the wild-type EnvZ, while OmpR-binding and pho sphatase functions are reduced. V8 protease digestion and mutational analys es indicate that His-243 of only the amino proximal domain A is phosphoryla ted. Based on these results, molecular models are proposed for the structur es of EnvZc[AAB] and the kinase domain of EnvZ. The present results demonst rate for the first time the construction of a functional, monomeric histidi ne kinase, further structural studies of which may provide important insigh ts into the structure-function relationships of histidine kinases.