Concerted Differential Changes of Helical Dynamics and Packing upon Ligand Occupancy in a Bacterial Chemoreceptor

Abstract

Transmembrane receptors are central components of the chemosensory systems by which motile bacteria detect and respond to chemical gradients. An attractant bound to the receptor periplasmic domain generates conformational signals that regulate a histidine kinase interacting with its cytoplasmic domain. Ligand-induced signaling through the periplasmic and transmembrane domains of the receptor involves a piston-like helical displacement, but the nature of this signaling through the >200 Å four-helix coiled coil of the cytoplasmic domain had not yet been identified. We performed single-molecule Förster resonance energy transfer measurements on Escherichia coli aspartate receptor homodimers inserted into native phospholipid bilayers enclosed in nanodiscs. The receptors were labeled with fluorophores at diagnostic positions near the middle of the cytoplasmic coiled coil. At these positions, we found that the two N-helices of the homodimer were more distant, that is, less tightly packed and more dynamic than the companion C-helix pair, consistent with previous deductions that the C-helices form a stable scaffold and the N-helices are dynamic. Upon ligand binding, the scaffold pair compacted further, while separation and dynamics of the dynamic pair increased. Thus, ligand binding had asymmetric effects on the two helical pairs, shifting mean distances in opposite directions and increasing the dynamics of one pair. We suggest that this reflects a conformational change in which differential alterations to the packing and dynamics of the two helical pairs are coupled. These coupled changes could represent a previously unappreciated mode of conformational signaling that may well occur in other coiled-coil signaling proteins.

WITHDRAWN: Signal Transduction and Kinase Control in Nanodisc-ReconstitutedE. coliAerotaxis Receptor

“…The KCD motional changes detected using PDS-ESR measurements in this study are not inconsistent with rotational and piston motions, however the role reversal of the N- and C-terminal KCD helices in Aer (discussed above) might suggest that helical rotations and piston movements differ from other chemoreceptors. Furthermore, the dramatic change observed at position 488 combined with the longer distances observed in the C-terminal helix of the KCD may indicate a 4- to 2-helix transition in the HAMP-adjacent region of the KCD, similar to other helical bundle changes proposed for other E. coli MCPs 45,46,48 . Piston motions and rotations relevant to kinase control in the distal regions of the KCD may be elicited by this 4- to 2-helix transition.…”

“…Previous in vitro FRET studies of the E. coli aspartate receptor, Tar, have demonstrated opposite behaviors for the N- and C-terminal KCD helices over the course of signal switching 45 . In the case of Tar, the C-terminal helices function as a scaffold and the N-terminal helices become more dynamic in the kinase-off state, increasing their separation distance and becoming less tightly packed.…”

Structural signatures of Escherichia coli chemoreceptor signaling states revealed by cellular crosslinking

“…Much of the experimental in vitro work on MH cap structure, primarily in the closely related Tar (aspartate) receptor, indicates that the MH1 helices have dynamic behaviors that reflect fluctuations in helicity and helix-packing strength. These bundle breathing motions are generally less pronounced in the MH2 helices ( 15 – 18 , 20 – 22 ). Moreover, the OFF state cap may enhance MH2 structural interactions ( 17 ), whereas MH1 structural interactions become less favorable ( 17 , 18 ).…”

“…Dynamics-based mechanisms of this sort could allow small input energies from ligand binding to propagate over the ∼200-Å distance to the CheA-controlling hairpin tip ( 5 , 6 , 11 – 13 ). Experiments that have been used to investigate structural and dynamic changes in the chemoreceptor signaling domain include hydrogen-deuterium exchange mass spectrometry ( 14 – 16 ), solid-state NMR ( 17 , 18 ), and tagging receptor molecules with electron paramagnetic resonance (EPR) reporters ( 19 – 21 ) or reporters for measuring Förster resonance energy transfer (FRET) interactions ( 22 ). Those studies were exclusively done in vitro using full-length Tar or soluble Tar chimeras or fragments.…”

Resolving the binding-kinase discrepancy in bacterial chemotaxis: A nonequilibrium allosteric model and the role of energy dissipation

“…More structural insight into the conformation states is needed to more concretely disentangle the differing effects of ligand binding and methylation. For example, repeating recent FRET measurements of nanodisc-inserted Tar receptors [49] at different methylation levels would help to clarify this picture.…”