Protein interactions and kinase regulation in template-assembled complexes of chemotaxis receptor fragments
The essential features of chemotactic signaling in E. coli include: high sensitivity over a wide dynamic range of chemoattractant concentrations, ability to integrate signals from different types of chemoreceptors, and a high gain. It is mediated by a central kinase, CheA, and an adaptor protein, CheW, which form noncovalent complexes with clusters of transmembrane receptors. In this study the molecular bases of these important features were elucidated utilizing functional complexes of template-assembled cytoplasmic fragments (CFs) of a chemoreceptor: Tar, Tsr or Trg (aspartate, serine or ribose/galactose receptors, respectively). Template-assembled complexes made with the wildtype CF (wt-CF) activated CheA, whereas complexes formed with any one of a number of inactivating point mutant CFs (in-CFs) did not. Measurements of kinase stimulation and complex formation revealed different mechanisms of kinase regulation involving competitive and cooperative interactions among receptor fragments. The apparent strength of the cooperative interactions and the composition of half-maximal inhibition (and binding) increased with adaptational covalent modification on the CF. Moreover, cooperative interactions between CFs from receptors of different ligand-binding specificities were also revealed. These interactions occur among CFs of high-abundance (HA) receptors (Tsr and Tar) and low-abundance (LA) receptor (Trg). There was also evidence of functional assistance to LA receptor Trg by HA receptors Tsr and Tar.
Receptor fragment clustering was probed by fluorescence resonance energy transfer (FRET). It was shown that receptor clustering is highly correlated with kinase stimulation and complex formation. On the other hand, CheW and CheA were not essential in forming tightly packed receptor clusters, but promoted the process at low densities of the CF-tethering lipid. Covalent modification affected the stability of forming tight clusters and modulated the disrupting effects of some kinase-inactivating CFs to receptor fragment clustering. The clustering states of the wt-CFs from the different receptor types of E. coli also provided an explanation for the conserved natural abundance of these receptors in the cell.
Overall, this study has shown the feasibility and utility of the template assembled system in probing functional interactions among proteins involved in bacterial chemotaxis signaling and has contributed to a better understanding of the essential features of this pathway.