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HYDROGEN EXCHANGE IDENTIFIES PROTEIN INTERFACES AND SIGNALING-RELATED CHANGES IN FUNCTIONAL CHEMORECEPTOR ARRAYS

Abstract
Chemotaxis is an ideal system for studying membrane protein signal transduction. Chemoreceptors are transmembrane proteins that sense chemicals in the environment and use this information to control a phosphorylation cascade that enables the cell to swim towards favorable environments. The receptors form a ternary complex with a histidine kinase, CheA, and an adaptor protein, CheW. These complexes assemble into membrane-bound hexagonal arrays that transmit the signal that controls CheA. It is widely accepted that ligand binding to the receptor causes a 2Å piston motion of a helix that extends through the periplasmic and transmembrane domains. But it is unclear how the signal then propagates through the cytoplasmic domain to inhibit CheA that is bound to the membrane- distal tip of the receptor, ~200Å away. Previous studies have suggested that signal propagation through the cytoplasmic domain involves inverse changes in the dynamics of the receptor. In this study, we employ hydrogen deuterium exchange mass spectrometry (HDX- MS) to measure differences in structure and dynamics between defined states of the receptor. Functional complexes of a His-tagged cytoplasmic fragment (CF) are assembled on vesicles with CheA and CheW in three states for HDX-MS. Widespread correlated exchange is observed, which indicates that the CF in functional complexes populates a long-lived unfolded state. Exchange is rapid throughout the CF except in the protein interaction region where CF binds CheA and CheW. These observations lead us to propose that signaling involves modulation of a folding equilibrium: binding of CheA (and possibly CheW) stabilizes the receptor, and CheA is bound in a kinase-on conformation. Thus, destabilization of the receptor will release this contact with CheA, which then adopts a kinase-off conformation. Both the kinase-off and demethylated samples of CF complexes exhibit faster HDX and less protection from exchange at long times at the binding interfaces with CheA. Thus we proposed that both the ligand-induced piston and demethylation destabilize the receptor, which releases its contact with CheA to turn off the kinase. Preliminary HDX results for CheA also set a stage for future analysis of the domain interactions of CheA in the functional complexes, and the differences that correlate with kinase activity. Ultimately, HDX-MS results will provide important information for deducing the signaling mechanism.
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