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

Abstract

The transmembrane aspartate receptor of bacterial chemotaxis regulates an associated kinase protein in response to both attractant binding to the receptor periplasmic domain and covalent modification of four adaptation sites on the receptor cytoplasmic domain. The existence of at least 16 covalent modification states raises the question of how many stable signaling conformations exist. In the simplest case, the receptor could have just two stable conformations ("on" and "off") yielding the two-state behavior of a toggle-switch. Alternatively, covalent modification could incrementally shift the receptor between many more than two stable conformations, thereby allowing the receptor to function as a rheostatic switch. An important distinction between these models is that the observed functional parameters of a toggle-switch receptor could strongly covary as covalent modification shifts the equilibrium between the on- and off-states, due to population-weighted averaging of the intrinsic on- and off-state parameters. By contrast, covalent modification of a rheostatic receptor would create new conformational states with completely independent parameters. To resolve the toggle-switch and rheostat models, the present study has generated all 16 homogeneous covalent modification states of the receptor adaptation sites, and has compared their effects on the attractant affinity and kinase activity of the reconstituted receptor-kinase signaling complex. This approach reveals that receptor covalent modification modulates both attractant affinity and kinase activity up to 100-fold, respectively. The regulatory effects of individual adaptation sites are not perfectly additive, indicating synergistic interactions between sites. The three adaptation sites at positions 295, 302, and 309 are more important than the site at position 491 in regulating attractant affinity and kinase activity, thereby explaining the previously observed dominance of the former three sites in in vivo studies. The most notable finding is that covalent modification of the adaptation sites alters the receptor attractant affinity and the receptor-regulated kinase activity in a highly correlated fashion, strongly supporting the toggle-switch model. Similarly, certain mutations that drive the receptor into the kinase activating state are found to have correlated effects on attractant affinity. Together these results provide strong evidence that chemotaxis receptors possess just two stable signaling conformations and that the equilibrium between these pure on- and off-states is modulated by both attractant binding and covalent adaptation. It follows that the attractant and adaptation signals drive the same conformational change between the two settings of a toggle. An approach that quantifies the fractional occupancy of the on- and off-states is illustrated.

Figure 1

Schematic model of the full-length homodimeric membrane bound receptor in the associated signaling complex (Falke and Hazelbauer 2001). Cylinders represent helical domains determined by crystallographic or cysteine and disulfide scanning methods. The two 60-kD receptor subunits are differentially shaded. The receptor provides a scaffold for the formation of a large super-molecular signaling complex. Proposed docking sites for the methyltransferase CheR, methylesterase CheB, histidine kinase CheA, coupling protein CheW and motor response regulator CheY are shown. The core ternary signaling complex consisting of the dimeric receptor, dimeric CheA, and CheW molecules is stable both in the presence and absence of attractant. Filled circles represent the four adaptation sites on each receptor subunit. Binding of attractant at the periplasmic domain of the receptor and modification of the cytoplasmic adaptation sites serve to modulate the kinase activity of the signaling complex.

Figure 2

(A) In vivo activity of the engineered receptors. Engineered receptors representing all possible homogeneous modification states of the adaptation sites were expressed in an E. coli strain lacking major chemoreceptors but containing other pathway components, including the adaptation enzymes CheB and CheR. The ability of each engineered receptor to restore cellular taxis up a self-generated aspartate gradient in soft agar was measured by the chemotactic swarm assay (materials and methods). All swarm rates have been normalized to the swarm rate of the wild-type receptor (QEQE). (B) In vitro activity of the engineered receptors. Receptor-coupled CheA kinase activity was measured for each modified receptor in the reconstituted phosphorylation pathway. Native E. coli membranes containing a given receptor were mixed with purified CheA, CheW, and CheY to reconstitute the ternary signaling complex in the presence of excess response regulator CheY. Radiolabeled ATP was added to initiate the phosphotransfer cascade resulting in phosphorylation of CheY. After quenching of the reaction after 10 s, the amount of phospho-CheY was determined and used to quantitate the initial rate of receptor-coupled CheA autophosphorylation. The resulting initial rate was normalized to that of the reconstituted wild-type receptor (QEQE) complex, providing the relative CheA kinase activity. Filled bars indicate activity in the absence of attractant and open bars indicate the activity in the presence of 1 mM l-aspartate. The data shown represents the average activity determined by three independent measurements of two different membrane preparations and is summarized in Table . (C) The effects of increasing attractant concentration on the receptor-coupled kinase activities of the QEEE, QEQE (wild type), QQEQ, and QQQQ receptor modification states. For each engineered receptor the relative CheA kinase activity was measured at different α-methyl-aspartate concentrations. The resulting activities were fit with a multisite Hill model, which provided K_1/2 and Hill coefficient values summarized in Table . Each point is the average of three independent measurements.

Figure 3

(A) Comparison of the α-methyl-aspartate K_1/2 values and receptor-coupled CheA kinase activities measured for different receptor modification states. Shown are the K_1/2 value (closed circle) and relative kinase activity (open circle) for each modification state, both measured using the standard in vitro receptor–coupled kinase assay described in the legend to Fig. 2B and Fig. C. (B) Correlation between the receptor-coupled CheA kinase activities and α-methyl-aspartate K_1/2 values measured for different receptor modification states. Shown are the data of Fig. 3 A and the best-fit straight line, as well as the Pearson product moment correlation coefficient. (C) Effects of specific mutations on the correlation. Plotted is the kinase activity against the α-methyl-aspartate K_1/2 for the indicated mutants, as well as the same correlation line defined by different receptor modification states in Fig. 3 B. The parameters of partial lock-on mutants I415C QEQE and I227C QEQE fall far from the line. Given their measured kinase activities, the attractant affinities of these lock-on mutants are considerably lower than predicted by the correlation of Fig. 3 B.

Figure 4

(A) CheA kinase activities stimulated by superactivating receptor mutations in a QQQQ modification background. Seven receptor mutations, selected for their ability to stimulate a high degree of CheA kinase activity in the QEQE modification background, were each incorporated into the QQQQ background. The CheA kinase activity stimulated by each mutant receptor was subsequently measured in the in vitro receptor–coupled kinase assay and normalized to the kinase activity generated by the wild-type receptor (QEQE). Filled bars indicate activity in the absence of attractant and open bars indicate the activity in the presence of 1 mM l-aspartate. The average kinase activity generated by the seven superactivating mutants in the absence of attractant is 5.5 ± 0.7-fold greater than that stimulated by the wild-type receptor (QEQE). (B) Time course of phospho-CheY production stimulated by superactivating and wild-type receptors in the reconstituted receptor–kinase complex. The level of radiolabeled phospho-CheY generated in the standard in vitro receptor–coupled kinase assay was monitored over time for reconstituted complexes containing the either the wild-type receptor (QEQE) or one of the two most superactivating receptors (A387C QQQQ or G278V QQQQ). Shown are the phospho-CheY levels measured at specific time points, as well as the best-fit initial rate lines defined by the 0–15 s time points. Even for the two most superactivating mutants, the initial reactions were linear to at least 15 s. Since the standard in vitro receptor–coupled kinase assay utilizes 10-s time points to define CheA kinase activities, it follows that all kinase activities measured in the present study are valid initial rates measured within the linear range of the kinase reaction.

Figure 5

(A) CheA kinase activities stimulated by the lock-on G278V mutation in different modification backgrounds. Shown are receptor-stimulated CheA kinase activities measured in the standard in vitro receptor–coupled kinase assay for the indicated receptor mutants. The G278V mutation is observed to superactivate the kinase and to block the normal attractant and adaptation signals. The normal inhibitory effects of both saturating attractant (1 mM l-aspartate) and glutamate residues at the adaptation sites are largely abolished, except when G287V is moved into the EEEE background where partial attractant sensitivity is restored (see discussion). (B) CheA kinase activities stimulated by partial lock-on receptors in different modification backgrounds. Three representative receptor mutations (I227C, S356C, and I415C) previously found to have partial lock-on character in the QEQE modification background were moved into the EEEE and QQQQ modification states and, using the standard in vitro receptor–coupled kinase assay, tested for their ability to stimulate CheA kinase activity in the presence and absence of 1 mM l-aspartate. All three partial-lock mutations partially block the normal attractant and adaptation signals. However, all three mutations still allow partial inhibition of kinase activity by saturating attractant (1 mM l-aspartate) and by the incorporation of glutamate residues at the adaptation sites. Thus, the lock-on character of these three mutations is weaker than that of the G278V mutation (compare with Fig. 5 A).

Figure 6

(A) Schematic toggle-switch model for aspartate receptor signaling, illustrating the two-state equilibrium proposed by the model. Shown are the two conformational states of the receptor with their different attractant binding and kinase activating propensities. The inactivated receptor conformation has high attractant affinity and low receptor-coupled kinase activity, whereas the activated receptor conformation exhibits low attractant affinity and high kinase activity. Modification of the adaptation sites by either methylation or amidation shifts the equilibrium toward the activated conformation, whereas attractant binding shifts the equilibrium toward the inactivated conformation. (B) Method to estimate the fractional populations of the two conformational states. Shown are the receptor-coupled CheA kinase activities of the standard receptor modification states plotted against their α-methyl-aspartate K_1/2 values (closed circles), as well as the best-fit straight line defined by these points (diagonal line). These data are the same as those of Fig. 3 B. Added to the figure is a scale used to quantitate the fraction of the receptor population in the activated conformation. This scale, which can be applied to any receptor population described by the standard correlation of Fig. 3 B, is used to interpolate the fraction activated receptor from the relative CheA kinase activity measured for a given receptor population in the in vitro receptor–coupled kinase assay. Note that to carry out this interpolation, the relative CheA kinase activity is the only parameter that need be measured. The method assumes that the inactivated state exhibits an α-methyl-aspartate K_1/2 in the micromolar to submicromolar range and little or no activation of CheA autophosphorylation, whereas the activated state exhibits an α-methyl-aspartate K_1/2 of 180 μM and a relative CheA kinase activity of 5.5 (see discussion).