The Structural Logic of Dynamic Signaling in theEscherichia coliSerine Chemoreceptor

Abstract

ABSTRACTThe experimental challenges posed by integral membrane proteins hinder molecular understanding of transmembrane signaling mechanisms. Here, we exploited protein crosslinking assays in living cells to follow conformational and dynamic stimulus signals in Tsr, theEscherichia coliserine chemoreceptor. Tsr mediates serine chemotaxis by integrating transmembrane serine-binding inputs with adaptational modifications of a methylation helix bundle to regulate a signaling kinase at the cytoplasmic tip of the receptor molecule. We created a series of cysteine replacements at Tsr residues adjacent to hydrophobic packing faces of the bundle helices and crosslinked them with a cell-permeable, bifunctional thiol-reagent. We identified an extensively crosslinked dynamic junction midway through the methylation helix bundle that seemed uniquely poised to respond to serine signals. We explored its role in mediating signaling shifts between different packing arrangements of the bundle helices by measuring crosslinking in receptor molecules with apposed pairs of cysteine reporters in each subunit and assessing their signaling behaviors with an in vivo kinase assay. In the absence of serine, the bundle helices evinced compact kinase-ON packing arrangements; in the presence of serine, the dynamic junction destabilized adjacent bundle segments and shifted the bundle to an expanded, less stable kinase-OFF helix-packing arrangement. An AlphaFold 3 model of kinase-active Tsr showed a prominent bulge and kink at the dynamic junction that might antagonize stable structure at the receptor tip. Serine stimuli probably inhibit kinase activity by shifting the bundle to a less stably-packed conformation that relaxes structural strain at the receptor tip, thereby freezing kinase activity.SIGNIFICANCEThis study used in vivo protein crosslinking to follow stimulus-induced signals through Tsr, a bacterial transmembrane receptor for chemotaxis to serine. Our experiments distinguished Tsr conformations that activate or inhibit its signaling kinase partner and showed how those signals reach the receptor’s kinase-controlling cytoplasmic tip. A dynamic junction in the Tsr molecule triggers stimulus responses by propagating a less stable helix-packing arrangement to flanking structural elements, thereby reducing structural stresses at the receptor tip. An AlphaFold model indicated that the dynamic junction might cause a structural distortion that destabilizes the receptor tip in the absence of a serine stimulus. This model and experimental approach could help to elucidate the signaling logic and mechanisms in other transmembrane chemoreceptor proteins.