Bacterial Chemoreceptor Dynamics: Helical Stability in the Cytoplasmic Domain Varies with Functional Segment and Adaptational Modification

Abstract

Dynamics are thought to be important features of structure and signaling in the cytoplasmic domain of bacterial chemoreceptors. However, little is known about which structural features are dynamic. For this largely helical domain, comprising a four-helix bundle and an extended four-helix coiled coil, functionally important structural dynamics likely involves helical mobility and stability. To investigate, we used continuous wave EPR spectroscopy and site-specific spin labels that directly probed, in essentially physiological conditions, mobility of helical backbones in the cytoplasmic domain of intact chemoreceptor Tar homodimers inserted into lipid bilayers of Nanodiscs. We observed differences among functional regions, between companion helices in helical hairpins of the coiled coil and between receptor conformational states generated by adaptational modification. Increased adaptational modification decreased helical dynamics while preserving dynamics differences among functional regions and between companion helices. In contrast, receptor ligand occupancy did not have a discernable effect on dynamics to which our approach was sensitive, implying that the two sensory inputs alter different chemoreceptor features. Spectral fitting indicated that differences in helical dynamics we observed for ensemble spin-label mobility reflected differences in proportions of a minority receptor population in which the otherwise helical backbone was essentially disordered. We suggest that our measurements provided site-specific snapshots of equilibria between a majority state of well-ordered helix and a minority state of locally disordered polypeptide backbone. Thus, the proportion of polypeptide chain that is locally and presumably transiently disordered is a structural feature of cytoplasmic domain dynamics that varies with functional region and modification-induced signaling state.

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

“…This agrees with our previous HDX and NMR studies on similar samples, which found MH1 dynamics in both CF4E and CF4Q to be comparable to the C terminal tail dynamics (11,14). EPR studies of site-specific spin labels measured the mobility of helices of the cytoplasmic domain of intact Asp receptor inserted into nanodiscs, but lacking CheA and CheW (16). Consistent with our results, Bartelli and Hazelbauer observed that MH1 is more dynamic than MH2.…”

“…Peptides near the N and C termini exhibit very fast exchange, with 90-100% deuterium uptake in the first 3 minutes, suggesting these regions are highly flexible and solvent exposed. These findings are consistent with previous hydrogen exchange mass spectrometry (11) and NMR studies (14) on similar samples of functional complexes, and with electron paramagnetic resonance (EPR) on spin-labelled intact receptors in nanodiscs (16). The C-terminal tail is thought to be an unstructured, flexible tether for binding the methylation and demethylation enzymes (17).…”

“…Decreased HDX in both regions was also observed in our previous HDX study (11) on arrays of CF4E assembled with CheA and CheW at high density (kinase-on state) relative to those assembled at low density (kinaseoff state). An EPR study of intact Asp receptors in nanodiscs similarly observed decreased dynamics in the methylation region for the methylated state relative to the demethylated state (16). Finally, EPR/DEER studies of constructs containing the Asp receptor methylation and protein interaction domains linked to variants of the Aer HAMP domain observed decreased protein interaction region dynamics in the kinase-on constructs relative to the kinase-off constructs (32).…”

“…Consistent with our results, Bartelli and Hazelbauer observed that MH1 is more dynamic than MH2. Based on EPR lineshapes at sites throughout the receptor cytoplasmic domain, they suggested the protein conformation samples a disordered helical backbone, with the greatest fraction of disorder observed in MH1 of the 4E receptor (16). The high mobility of MH1 is likely to be characteristic of the native receptor, since it has been observed in both nanodisc-imbedded intact receptor dimers that lack CheA and CheW (EPR study) and in membranebound complexes of CF, CheA, and CheW that lack the periplasmic and transmembrane receptor domains but display native array structure and function (this study).…”

A zipped-helix cap potentiates HAMP domain control of chemoreceptor signaling

“…Several in vitro studies, including hydrogen-exchange MS (57), NMR dynamics approaches (58), and electron paramagnetic resonance (59)(60)(61), have demonstrated differential dynamic properties of the MH1 and MH2 helices that are consistent with the zipped-cap model. The in vivo signaling properties of truncated Tar receptors are also consistent with the zipped-cap model (62).…”