Evidence that both ligand binding and covalent adaptation drive a two-state equilibrium in the aspartate receptor signaling complex

The transmembrane aspartate receptor of bacterial chemotaxis regulates all associated kinase protein in response to both attractant binding to the rec eptor periplasmic domain and covalent modification of four adaptation site' s on the receptor cytoplasmic domain. The existence of at least 16 covalent modification states raises the question of how many stable signaling confo rmations exist. In the simplest case, the receptor could have just two stab le conformations ("on" and "off") fielding the two-state behavior of a togg le-switch. Alternatively, covalent modification could incrementally shift t he receptor between many more than two stable conformations, thereby allowi ng the receptor to function as a rheostatic switch. An important distinctio n between these models is that the observed functional parameters of a togg le-switch receptor could strongly covary as covalent modification shifts th e equilibrium between the on- and off-states, due to population-weighted av eraging of the intrinsic on- and off-state parameters. By contrast, covalen t modification of a rheostatic receptor would create new conformational sta tes with completely independent parameters. To resolve the toggle-switch an d rheostat models, the present study has generated all 16 homogeneous coval ent modification states of file receptor adaptation sites. and has compared their effects oil the attractant affinity and kinase activity of the recon stituted receptor-kinase signaling complex. This approach reveals that rece ptor, covalent modification modulates both attractant affinity and kinase a ctivity up to 100-fold, respectively. The regulatory effects of individual adaptation sites are not perfectly additive, indicating synergistic interac tions between sites. The three adaptation sites at positions 295, 302, and 309 are more important than the site at position 491 in regulating attracta nt affinity and kinase activity, thereby explaining the previously observed dominance of the former three sites in in vivo studies. The most notable f inding is that covalent modification of the adaptation sites alters the rec eptor attractant affinity and the receptor-regulated kinase activity in a h ighly correlated fashion, strongly supporting the toggle-switch model. Simi larly,, certain mutations that drive the receptor into the kinase activatin g state are found to have correlated effects oil attractant affinity. Toget her these results provide strong evidence that chemotaxis receptors possess just two stable signaling conformations and that the equilibrium between t hese pure on- and off-states is modulated by both attractant, binding and c ovalent adaptation. It follows that the attractant and adaptation signals d rive the same conformational change between the two settings of a toggle. A ll approach that quantifies the fractional occupancy of the on- and off-sta tes is illustrated.