Receptor Kinase Interactions In Bacterial Chemotaxis Revealed By Pulsed Dipolar Esr Spectroscopy

Abstract

Bacterial chemotaxis refers to the movement of bacteria under influence of attractants and repellents. This signal transduction pathway is characterized by incredible gain, sensitivity and co-operativity. The extra-cellular domains of receptors transmit the signal from outside the cell into the cytoplasm where it is further processed by the signaling complex of histidine kinase CheA, coupling protein CheW and cytoplasmic domains of receptors. Structural determination of individual protein components and their protein complexes is necessary to gain insight into the mechanism. In this work, we have used site-directed spin labeling and long-range distance restraints from Pulsed dipolar ESR spectroscopy to predict the structure of a ternary complex formed by CheA, CheW and the signaling domain of the receptor. We have developed a novel method to refine the structures of protein complexes from the distance restraints provided by pulsed dipolar ESR. Apart from determining the position of receptor in the ternary complex, our efforts have been directed towards understanding the change in orientations of CheA and CheW in the presence of receptor. On a macroscopic scale, CheA, CheW and receptors form dense clusters at the poles of cell. We observed that CheA also self-associates in solution to some extent. We speculate that this self-association property of CheA may play a crucial role in clustering of chemotaxis proteins in cell. We have successfully identified this binding interface with disulphide cross-linking and novel application of pulsed dipolar ESR signals that report on local spin concentrations.