Differences between Signaling States of Chemotaxis Receptors Revealed by Hydrogen Exchange Mass Spectrometry

Abstract

Bacterial chemotaxis receptors provide the sensory input needed to direct swimming towards favorable environments. Ligand binding to the receptor periplasmic domain modulates activation of a kinase CheA bound at the cytoplasmic tip of the receptor. Receptors operate in complexes with CheA and a coupling protein CheW that together form extended arrays in the membrane. Understanding the differences in conformation and dynamics between the kinase-activating and kinase-deactivating signaling states of the receptor will provide insight into the mechanism of transmembrane signaling. We have used hydrogen exchange mass spectrometry to probe the differences between these signaling states of the receptor cytoplasmic domain assembled on membrane vesicles in functional complexes with CheA and CheW. Local exchange measurements reveal several differences between these states, including differences in the exchange pattern (EX1 vs EX2), the fraction undergoing fast exchange (exchanged in 3 minutes), and the fraction that are protected from exchange at long time (16 hours). Preliminary analysis shows that peptides corresponding to the adaptation region undergo EX1 exchange (long-lived unfolded state with complete exchange during each unfolding event) in the kinase-activating state and EX2 exchange (short-lived unfolded state with partial exchange) in the kinase-deactivating state. This suggests the adaptation region is destabilized in the kinase-activating state. Peptides corresponding to the signaling domain cytoplasmic tip of the receptor show greater protection from exchange in the kinase-activating state, which could be due to changes in interactions with CheA and CheW. This hydrogen exchange mass spectrometry approach is a promising means of detecting changes in structure and dynamics of a functional membrane-bound, multi-protein complex.This research supported by GM 47601, GM085288, and a Fellowship to Seena Koshy from the University of Massachusetts as part of the Chemistry-Biology Interface Training Program (NRSA T32 GM08515).