Hydrogen Exchange Mass Spectrometry of Functional Membrane-Bound Chemotaxis Receptor Complexes

Abstract

The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (~2 Å) piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen exchange mass spectrometry (HDX-MS) measurements of global exchange of CF demonstrate that CF exhibits significantly slower exchange in functional complexes than in solution. Since the exchange rates in functional complexes are comparable to that of other proteins of similar structure, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements, by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system.

Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346

“…To more precisely characterize the structural rearrangements upon light sensing, we used HDX-MS, which reports on changes in amide protection and, by extension, changes in protein structure (23). Deuterium uptake can mark transiently populated states for detection on the peptide level ("local HDX-MS"), making it a powerful tool that has been applied to studying dynamic signaling states of photoreceptors (24,25) and histidine kinases (26)(27)(28). Our protocol ( Fig.…”

Signaling and sensory adaptation in Escherichia coli chemoreceptors: 2015 update

“…How can those motions be documented and measured in working receptor molecules? Over the next five years of chemoreceptor research, methods capable of revealing protein dynamic behaviors, such as hydrogen/deuterium exchange rates [86, 87] and electron paramagnetic resonance [88], should make valuable contributions to our understanding of receptor signaling. In addition, novel combinations of experimental approaches, for example molecular dynamics simulations on well-characterized mutant receptors, will help to address the technical challenges posed by dynamics-based signaling mechanisms (Box 4).…”

Hydrogen exchange of chemoreceptors in functional complexes suggests protein stabilization mediates long-range allosteric coupling

“…Binding of the His-tagged CF to the surface of large unilamellar vesicles containing a mixture of DOPC and DGS-NTA(Ni), in the presence of CheA and CheW, restores function (CF becomes a good substrate for methylation and activates CheA) (8) and also yields native-like hexagonal arrays (9). We have previously used HDX-MS on such complexes to show that incorporation of CF into functional complexes reduces its rapid hydrogen exchange (10), and to measure differences in the exchange properties of functionally important receptor subdomains (11).…”