Characterizing the Physiological Interfaces of the Histidine Kinase CheA: A TAM-IDS study

The core components of the bacterial chemosensory array, the transmembrane receptor, the histidine kinase CheA, and the adaptor protein CheW, organize into an extended hexagonal array typically located at one or both of the poles of bacterial cells. Transmembrane receptor signaling regulates kinase activity which in turn regulates the flagella which controls cell movement. The kinase CheA is made up of five separate domains. The regulatory domain of kinase and the adaptor protein bind at overlapping sites at the cytoplasmic tip of the receptor called the protein interaction region. The kinase regulatory domain and the adaptor protein share tandem SH3-like structural motifs, allowing the proteins to bind to receptor in similar ways. The present study compares the physiological binding interfaces between the kinase and other core components to the current in vitro structural array models by (i) determining the minimal binding domain of the kinase, (ii) characterizing a predicted interface between the kinase and the adaptor protein in vivo and (iii) characterizing the interface between the kinase and receptor in vivo predicted by two contradictory models. Current evidence is unable to determine which model more accurately describes the receptor-kinase interface in native chemosensory arrays. Model 1 is based on a crystal structure of a homologous Thermotoga complex between receptor fragment and adaptor protein, and Model 2 is based upon the newly solved Thermotoga complex between receptor fragment and the kinase regulatory domain. Physiological interfaces are probed using live cell fluorescent microscopy in tandem with a novel interface scanning method: Tryptophan and Alanine Mutatagenesis to Identify Docking sites (TAM-IDS). The results reveal the kinase regulatory domain is the minimal binding domain of the kinase, the current structural model of the kinase-adaptor protein interface is consistent with the physiological interface, and the physiological interface between kinase and receptor is more accurately described by Model 2