Bacterial Chemoreceptors Transmit Stimulus Signals Through Coupled Entropic Switches.

Tracking ligand-induced conformational changes through transmembrane sensory proteins remains a challenge in signal transduction research. Our study followed propagation of stimulus signals through a bacterial transmembrane chemoreceptor (Tsr) that controls a cytoplasmic signaling kinase (CheA). We marked relay elements in the ∼200-Å long cytoplasmic four-helix bundle signaling domain with amino acid replacements that locked Tsr in kinase-ON or -OFF output and characterized the mutant receptors with in vivo assays that monitored Tsr structure (crosslinking) and function (kinase control). We found that conformational changes emanating from the mutant lesions propagated bidirectionally throughout the Tsr signaling domain and did not dissipate appreciably with distance, implying conformational coupling between subdomains. These behaviors proved intrinsic to Tsr homodimers and independent of higher order signaling complexes. AlphaFold 3 atomic models of the mutant receptors exhibited structural changes that would likely affect local helix packing interactions at the mutant sites. One member of each ON-OFF mutant pair appeared to reduce packing stability, whereas the other enhanced packing stability. This analysis pinpointed two sites of structural logic inversion ("entropic switches") in the Tsr transmission path. Kinase-OFF lesions appeared to be stabilizing at the input and output segments of the cytoplasmic domain but destabilizing in the intervening signaling region that contains the modification sites for sensory adaptation. These findings support the notion of chemoreceptor signaling through opposed dynamic switches and provide new insights into the mechanism of kinase output control. This work also highlights the powerful interplay possible between cellular signaling readouts and AF3-generated models of mutant proteins.