Correspondence to: Joseph J. Falke, Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309-0215. Fax:(303) 492-5894 E-mail:falke{at}colorado.edu.
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Abstract |
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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 receptorkinase 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.
Key Words: bacterial chemotaxis, two-component signaling pathway, CheA, adaptation site, transmembrane signal
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INTRODUCTION |
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In motile bacteria, chemotaxis toward or away from attractants or repellents is mediated by the chemotaxis signaling pathway in which transmembrane receptors serve to modulate the activity of an associated histidine kinase (
The structure of the chemotaxis receptors has been extensively characterized by biochemical and crystallographic studies (
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The chemotaxis receptors transmit two opposing signals to the bound kinase: (1) attractant binding to the periplasmic domain generates a transmembrane signal that inhibits cytoplasmic kinase activity; and (2) covalent modification of specific adaptation sites on the receptor cytoplasmic domain stimulates kinase activity. These two opposing signals can be termed the attractant and adaptation signals. The adaptation sites of the aspartate receptor are four glutamate side chains located at positions 295, 302, 309, and 491 on the surface of the cytoplasmic 4-helix bundle (
A fundamental question in the study of transmembrane surface receptors is how ligand binding and covalent modification of a receptor shifts the receptor signaling state between active and inactive states. Two extreme models can be proposed. At one extreme, a given receptor could possess just two stable conformations, such that an individual receptor molecule is a simple two-state, on-off toggle-switch (
For the bacterial chemotaxis receptors, a two-state equilibrium model was first proposed by
To resolve the toggle-switch and rheostat models of aspartate receptor conformational states, it is necessary to generate homogeneous populations of receptors with well-defined modifications of their adaptation sites. The different methylation states of the receptor can be mimicked by mutating the adaptation Glu residues to Gln, since amidation yields a functional response similar to that of the methyl ester (
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MATERIALS AND METHODS |
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Materials
The plasmid pSCF6 used to express the S. typhimurium aspartate receptor under control of its native promoter has been described previously ((cheA-cheZ)DE2209 tsr-1 leuB6 his-4 eda-50 rpsL136 [thi-1
(gal-attl)DE99 ara-14 lacY1 mtl-1 xyl-5 tonA31 tsx-78]/mks/) expressing the aspartate receptor from plasmid pSCF6 or its engineered variants. Tests of receptor function in vivo were performed in the E. coli strain RP8611 (tsr-7028
(tar-tap)DE5201 zbd::Tn5
(trg)DE100 leuB6 his-4 rpsL136 thi-1 ara-14 lacY1 mtl-1 xyl-5 tonA31 tsx-78) containing the plasmid pSCF6 or its variants. Both of these strains of E. coli were provided by Dr. John S. Parkinson (University of Utah, Salt Lake City, UT;
-methyl-D,L-aspartate (>99% purity and containing no detectable aspartic acid as assayed by TLC) was purchased from Sigma-Aldrich. The enzyme substrate
-[32P] ATP (6,000 Ci/mmol) was obtained from NEN Lifesciences.
Protein Engineering
Receptors with modified adaptation sites were generated by oligonucleotide directed site specific mutagenesis of the plasmid pSCF6 according to the method of Kunkel et al. with modifications as previously described (
In Vivo Activity Assays
Chemotaxis swarm assays were performed as described previously (24 h after spotting. Swarm rates were determined by least-squares linear best-fit to the slope of diameter as a function of time. The aspartate specific swarm rates were determined by subtracting the (-) aspartate swarm rate from the (+) aspartate swarm rate to correct for pseudotaxis, and any other nonaspartate-specific taxis. The resulting rate was normalized to the wild-type rate for comparison. Cells containing vector alone swarmed at an
10-fold lower rate than cells containing wild-type receptor and exhibited no aspartate specific swarming.
Purification of the Engineered Receptors and Cytoplasmic Chemotaxis Components
Plasmids encoding engineered receptors were transformed into the E. coli strain RP3808 that lacks functional major chemotaxis receptors and cytoplasmic chemotaxis components. Subsequent expression and preparation of the engineered receptors in native E. coli membranes was performed as previously described (10% of total protein in the native membrane preparations. The soluble chemotaxis components CheA, CheW, and CheY were overexpressed and purified in E. coli as described previously (
In Vitro Activity Assays
The in vitro receptorcoupled kinase assay was performed essentially as described previously (-[32P]ATP to a the reaction mixture and quenched after a 10-s incubation period. Receptor-regulated CheA autophosphorylation is the rate determining step in this reaction so that the rate of phosphorylated CheY production is linearly proportional to the receptor-regulated CheA autophosphorylation rate (see Fig 4 B and DISCUSSION;
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The Attractant Dependence of Receptor-coupled Kinase Activity
Quantification of the K1/2 and Hill coefficient associated with attractant regulation of receptor-coupled kinase activity was performed using the in vitro receptorcoupled kinase assay (-methyl-aspartate was used. This attractant was originally used to quantitate the sensitivity of the chemotaxis pathway at low attractant concentrations (
-methyl-aspartate is that its aspartate receptor binding affinity (Kd
10100 µM; see below) is significantly less than that of aspartate (Kd
1 µM;
-methyl-aspartate than for aspartate when titrations are performed using receptor concentrations in the low micromolar range. Relative receptor-coupled CheA activities were determined over a range of
-methyl-aspartate concentrations for each engineered receptor. A cooperative, multi-site Hill equation (Equation 1) was used to fit the averaged data obtained for each attractant concentration, yielding a best-fit titration curve:
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(1) |
where R is the normalized receptor-regulated CheA kinase rate, M is the maximal rate, m is the background CheA kinase rate at saturating attractant concentration, [A] is the free attractant concentration, K1/2 is the attractant concentration at which the receptor-coupled CheA kinase activity is one-half maximal, and H is the Hill coefficient.
The best fit titration curve to the Hill equation was used to determine both the Hill coefficient and the K1/2 for the attractant response of each engineered receptorkinase complex. Titration curves were plotted and fit using KaleidaGraph 3.0 software for Macintosh (Synergy Software).
Determination of Correlation Coefficients
All indicated linear correlation coefficients represent the Pearson product moment correlation coefficient, r, as determined using Excel 2001 (Microsoft).
Error Determination
All indicated error bars represent the SD for n 3.
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RESULTS |
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Generation and Expression of All Possible Modification States
A set of aspartate receptor mutants representing all 16 homogeneous modification states of the receptor was created by generating all possible combinations of glutamate or glutamine at the four receptor adaptation sites. The glutamine residues at the adaptation sites closely mimic the regulatory effects of the native methyl esterification of glutamate as catalyzed by CheR (
Effect of Modification State on Receptor Function In Vivo
As a positive control, receptors expressed from plasmids encoding all 16 possible modification states were assayed for the ability to stimulate in vivo swarming activity in response to a self-generated radial aspartate gradient in semisolid agar plates. The E. coli RP8611 strain used in this assay lacks functional major chemotactic receptors, but contains functional soluble components of the chemotactic signaling pathway including the methyl esterase enzyme CheB, which hydrolyzes either glutamine or methyl-esterified glutamate at an adaptation site to yield the free glutamate side chain, and CheR which methyl-esterifies this free glutamate side chain. When plasmids expressing different combinations of glutamines and glutamates at the adaptation sites were expressed in RP8611, all of the modified receptors yielded chemotactic swarm rates similar to the wild-type receptor as shown in Fig 2 A. It has been shown previously that swarm rates are significantly slowed by mutations preventing the normal methylation and demethylation of any adaptation site (
Effect of Receptor Modification State on Kinase Activation In Vitro
To compare the receptor-coupled CheA kinase activities associated with different receptor modification states, the in vitro receptorcoupled kinase assay was used to measure the kinase activity of the reconstituted signaling complex in the absence of attractant. Since the apo receptor stimulates CheA autophosphorylation whereas the attractant-occupied receptor inhibits CheA, kinase activity measurements performed in the absence of attractant provided quantitation of the maximal kinase activity generated by each receptor modification state. To perform the receptor-coupled kinase assay, engineered receptors in native E. coli membranes were added to purified CheA, CheW, and CheY proteins to reconstitute the active receptorkinase signaling complex. Upon addition of radiolabeled ATP, the formation of phospho-CheY was followed as a measure of receptor-coupled kinase activity. Excess CheY was used to ensure that the rate-determining step in the overall reaction was the autophosphorylation of CheA, which is the step regulated by the receptor. Table 1 and Fig 2 B summarize the relative levels of receptor-coupled CheA kinase activity for all 16 possible modification states, measured in the absence of attractant. As has been demonstrated previously by the well-characterized EEEE, QEQE, and QQQQ modification states and their methyl-esterified analogues, increasing modification of the adaptation sites by either amidation or methylation stimulates the autophosphorylation activity of receptor-bound CheA (
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The different adaptation sites are observed to have different propensities to regulate kinase activity (Table 1 and Fig 2 B). When a single Q is placed in the EEEE background or a single E is placed in the QQQQ background, modification of the fourth site has the smallest effect on kinase activity, whereas modification of the third site has the largest effect on kinase activity. However, the effects of multiple modifications are not always additive. In general, when multiple Q residues are introduced into the EEEE background, the observed kinase activities are greater than the sum of the single Q modifications. For example, if the degree of kinase activation resulting from a single Q modification at each individual adaptation site were additive, the total QQQQ receptor-mediated activity would be 1.6 ± 0.3 times that of QEQE (wild type) receptor-mediated kinase activity. In contrast, the experimentally observed kinase activity for the QQQQ modification state is 3.1 ± 0.4 times that of the QEQE (wild type) receptor. Thus, there are significant synergistic effects between adaptation sites in the regulation of kinase activity. Yet, the data support the general conclusion that in receptors with multiple modifications, the degree of kinase activation depends primarily on the number of modifications at the first three adaptation sites on signaling helix CD1, whereas the effects of modification at the fourth adaptation site on helix CD2 are relatively minor.
Effect of Receptor Modification State on Attractant Affinity In Vitro
To quantitate the relative attractant affinities of modified receptors, the in vitro receptorcoupled kinase assay was used to measure the inhibitory effect of increasing attractant concentrations on receptor-coupled kinase activity as illustrated in Fig 2 C. The attractant -methyl aspartate was chosen for titrations because it has been used in cellular behavioral studies and possesses a receptor affinity favorable for quantitation (MATERIALS AND METHODS;
The best-fit -methyl aspartate K1/2 and Hill coefficient values obtained for the 16 receptor modification states are summarized in Table 1. No values were determined for the EEEE receptor, which had too little receptor-mediated CheA kinase activity to be accurately quantitated. The observed K1/2 values increased
10-fold as the modification level increased from a single glutamine (QEEE, EQEE, EEQE, and EEEQ) to four glutamines (QQQQ). The 10-fold effect of adaptation state on the attractant K1/2 parameter is in agreement with direct in vitro binding measurements of attractant Kd values and with in vivo measurements of attractant K1/2 values for stimulation of chemotactic behavior (
1.5 to
3.0 (Table 1), which is consistent with limited positive cooperativity between receptor signaling units believed to be individual receptor dimers (
Correlation Between Effects of Receptor Modification on Kinase Activation and Attractant Affinity
When the effects of adaptation site modification on kinase activation and attractant affinity (both measured in the in vitro receptorcoupled kinase assay as described above and summarized in Table 1) were compared, a strong correlation emerged. Fig 3 A summarizes the maximal kinase activities and attractant K1/2 values observed for the 16 modification states excepting EEEE (see above). Fig 3 B tests the correlation by plotting the K1/2 value for each modified receptor against its maximal kinase activity, revealing a linear relationship with a correlation coefficient of 0.97. Thus, a highly modified receptor such as QQQQ exhibits a high K1/2 value (low attractant affinity) and high kinase activity, whereas, at the other extreme, the QEEE, EQEE, EEQE and EEEQ receptors exhibit low K1/2 values (high attractant affinity) and low kinase activity. This is the behavior predicted for a toggle-switch model in which modification of the adaptation sites shifts an equilibrium between a low attractant affinity, high kinase activity "on" state and a high attractant affinity, low kinase activity "off" state (see DISCUSSION).
Some mutations outside of the adaptation sites yield K1/2 values and relative kinase activities that fall near the same correlation line exhibited by the adaptation site modifications (Fig 3 B), as illustrated in Fig 3 C for mutations G278M QEQE, G278M QQQQ, A387C QEQE, and A387C QQQQ. By contrast, certain other mutations yield points further from the correlation line as illustrated by I415C QEQE and I227C QEQE. Both behaviors are predicted by the toggle-switch model: some mutations shift the equilibrium between the two fundamental states without altering the characteristics of either state, yielding points falling on the standard correlation line. The remaining mutations significantly alter the characteristics of one or both fundamental states or add additional states, and thus yield points that differ from the standard correlation (see DISCUSSION).
Estimating the Upper Limit of Receptor-coupled Kinase Activation
To determine the upper limit of receptor-coupled kinase activation in the absence of attractant, seven mutations previously observed to increase the kinase activity of the QEQE receptor above wild-type levels were moved into the QQQQ receptor background (5.5-fold that stimulated by the WT (QEQE) receptor as shown in Fig 4 A. Time courses of the kinase reaction revealed that these maximal rates were measured well within the linear, initial-rate range of the assay, illustrated in Fig 4 B for mutants G278V QQQQ and A387C QQQQ, indicating that the super-normal rate was not being limited by the assay conditions, even when the receptor mutations that had the largest enhancing effects on receptor-mediated CheA kinase activity were tested. Instead, these superactivating mutations appear to fully drive the receptor into the functional state that yields the maximal turnover rate of the bound CheA kinase. In the toggle-switch model, such behavior is observed when the entire receptor population is driven into the kinase-activating conformation. For a toggle-switch system knowledge of this maximal rate is quite significant, since it can be used to calculate the fraction of the receptor population in each of the two fundamental states for any receptor described by the correlation in Fig 6 B (DISCUSSION).
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Identification of a Mutant Locked in the Kinase-activating State
The present study revealed one mutant receptor, G278V, whose characteristics indicate it is strongly locked in the kinase-activating conformation in a range of modification state backgrounds. The G278 residue is located at a buried regulatory hotspot in the cytoplasmic 4-helix bundle where helix packing is tightly linked to receptor activation (
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DISCUSSION |
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The present study used an in vitro receptorcoupled kinase assay in which native E. coli membranes containing native or engineered aspartate receptors were reconstituted with purified CheW (coupling protein), CheA (His kinase), and CheY (response regulator). The resulting receptorkinase signaling complex was used to examine the attractant affinities and kinase stimulating propensities of all possible homogeneous covalent modification states of the receptor adaptation sites. Systematic mutagenesis of the adaptation site glutamates to glutamines, which mimics the native methyl esterification of these regulatory side chains, was found to alter both the attractant K1/2 and receptor-coupled kinase activity. Comparison of the four adaptation sites revealed that modification of the third site usually triggered the largest changes in attractant K1/2 and kinase activity, whereas modification of the fourth site yielded the smallest changes. Notably, previous in vivo studies have shown that the third site is methylated most rapidly and extensively under cellular conditions, whereas the fourth site is methylated most slowly and the least extensively (
Although the adaptation sites exhibited consistent differences between their regulatory potencies and modifications at multiple adaptation sites were largely additive, analysis of all 16 homogeneous modification states revealed subtle deviations from additivity. For example, modification of all four sites from glutamate to glutamine yielded a twofold greater increase in kinase stimulation than predicted by adding the effects of individual site modifications (Fig 2 B). Thus, synergistic interactions occur when multiple sites are modified. Synergistic interactions are presumably facilitated by the close proximity between the first, second, and third adaptation sites at positions 295, 302, and 309 on the surface of cytoplasmic helix CD1, where they are separated by consecutive heptad repeats of the coiled-coil, whereas the fourth site at position 491 on the surface of cytoplasmic helix CD2 lies near the other three due to the hairpin arrangement of the CD1 and CD2 helices (
An important mechanistic question is whether the different covalent modification states of the receptor adaptation sites yield multiple, stable receptor conformations that each exhibits independent functional parameters. Alternatively, covalent modification could simply modulate an equilibrium between two fundamental conformations. To resolve these possibilities, the present study has asked whether a correlation exists between the effects of modification state changes on two key receptor parameters: attractant affinity and the level of kinase activation, both of which were measured using the in vitro receptorcoupled kinase activity assay. When the modification state was changed from the singly modified state (QEEE, EQEE, EEQE, and EEEQ) to the fully modified state (QQQQ), the measured K1/2 for the attractant -methyl aspartate increased
10-fold (Table 1), indicating a significant decrease in attractant affinity as previously noted by more limited studies of receptors in isolated native membranes or chemotaxing cells (
10-fold (Table 1). A quantitative analysis of the correlation between modification effects on K1/2 and kinase activity yielded a linear correlation coefficient of 0.97 (Fig 3A and Fig B), indicating a strong, highly significant linear relationship between the attractant concentration required for half-maximal kinase inhibition and the maximal kinase activity of the receptorkinase complex. Extrapolation of the correlation to the unmeasurable EEEE state suggests that both attractant affinity and kinase activity vary over 100-fold ranges between the EEEE and QQQQ states.
The observation of strongly correlated effects of adaptation site modifications on two different functional parameters, attractant K1/2 and kinase activation, is consistent with the predictions of the toggle-switch model for receptor regulation but is difficult to reconcile with the rheostat model. The toggle-switch model proposes that attractant binding and adaptation site modification simply shift an equilibrium between two fundamental receptor conformations: one exhibiting high attractant affinity and low kinase activation, the other exhibiting low attractant affinity and high kinase activation as illustrated in Fig 6 A. Intermediate states of attractant occupancy or covalent modification yield a mixed receptor population containing both types of fundamental conformations. In such a system, the apparent K1/2 and kinase activation parameters of a given receptor modification state are a weighted average of the individual parameters of the two fundamental conformations, and thus these parameters vary in a correlated way as modification state changes shift the proportions of the population in the two fundamental states. Tight linkage between attractant affinity and receptor-mediated kinase activity is a characteristic feature of an equilibrium between two fundamental states and can be simulated (
The dramatic difference between the toggle and rheostat models is emphasized by an analysis of their predictions for the piston-type displacement of the 4/TM2/CD1 signaling helix during transmembrane signaling (
The observed linear relationship between the attractant K1/2 and kinase activation parameters reveals the characteristic parameters for both fundamental conformations of the toggle-switch model, and allows calculation of the fraction of the receptor population in each conformation. This analysis assumes that the attractant K1/2 and kinase activation parameters (Fig 3 B) vary linearly with the fractional populations of receptors in the two fundamental conformational states. The off-state is found to possess an -methyl aspartate K1/2 in the submicromolar to micromolar range and a kinase activity approaching zero, as extrapolated from the correlation (Fig 3 B). The modification state EEEE drives the receptor population almost completely into this off-state. The on-state is found to have an
-methyl aspartate K1/2 of 180 µM and a kinase activity 5.5-fold that of the wild-type (QEQE) receptor, where this upper limit is defined by the maximal kinase activity observed for multiple superactivating receptor mutants (Fig 4 A). As Fig 6 B illustrates, the line connecting these points for the pure on- and off-states allows calculation of the fractional occupancies of the fundamental on- and off-states for a native receptor population exposed to arbitrary attractant and modification conditions. To carry out such a calculation, the receptor-coupled kinase activity of the receptor population is measured via the standard in vitro assay, then the scale on Fig 6 B is used to interpolate the fractional occupancy of the on-state. Any mutant receptor adequately described by the standard correlation can also be analyzed in this manner (although it should be noted that not all mutant receptors satisfy this requirement; see below).
The fractional populations in the on- and off-conformations depend on both the receptor modification level and the attractant concentration. Notably, in the absence of attractant the EEEE modification state shifts the equilibrium nearly completely toward the off-position of the toggle-switch but the QQQQ state shifts the equilibrium only 60% toward the on-position. This observation leaves open the possibility of further kinase activation (e.g., by repellent binding) in the fully modified state (Fig 6 B). Alternatively, the native modification of the adaptation sites by methyl esterification may shift the equilibrium more strongly toward the on-position than amidation, such that the EmEmEmEm state might be fully activated. Overall, the conversion of the EEEE state to the fully activated state increases kinase activity at least 140-fold, indicating a corresponding increase in the population of the on-state. It follows that the free energy difference between the EEEE and fully activated states is 3 kcal mol-1 at physiological temperature. Binding of the native attractant L-aspartate provides a free energy driving force exceeding 8 kcal mol-1 toward the off-state (given a binding constant of 106 M-1 for the QQQQ receptor; unpublished data), thereby explaining the observation that saturating aspartate is able to reduce the kinase activities of even the most highly activated receptors to unmeasurable levels.
Mutations at sites other than the adaptation sites can also shift the equilibrium between the on- and off-conformations, or can alter the intrinsic parameters of one or both fundamental states. Mutations of the former type yield attractant K1/2 and kinase activation parameters that fall on the standard correlation, whereas mutations of the latter type yield parameters distant from the standard correlation (Fig 3 C). Findings to date indicate that lock-on mutations typically shift parameters away from the standard correlation (examples are illustrated in Fig 3 C). However, even these mutations may still be described as toggle-switches biased toward the on-state, since the EEEE modification state typically restores at least partial attractant sensitivity as expected for a modification that biases the toggle-switch back toward the off-state (Fig 5A and Fig B). The most dramatic lock-on mutation discovered thus far is G278V, which drives the toggle-switch toward the on-state so strongly that both the attractant and adaptation signals are unable to inactivate the kinase except in the EEEE background where saturating attractant provides partial inactivation (Fig 5 A). Similarly, the sensitivity of weaker lock-on mutants to attractant increases as the modification level is decreased from QQQQ to EEEE (Fig 5 B). The greater sensitivity of the EEEE background to attractant is expected since this background exhibits the highest attractant affinity, such that attractant binding provides the maximal driving force toward the off-state. Overall, the results indicate that lock-on mutations stabilize the on-state by as much as 8 kcal mol-1, but also modify the native parameters of the on- or off-state so the standard correlation between attractant K1/2 and kinase activation no longer applies.
The design of the toggle-switch equilibrium has important implications for the biology of chemotaxis. As a cell chemotaxes toward increasing attractant concentrations, the average modification state of the receptor population gradually increases (corresponding to a shift toward the activated conformation in Fig 6 A). Concomitantly, this modification change causes the apparent K1/2 for attractant binding to increase, thereby allowing the sensitivity of the chemotaxis receptor to be appropriately adjusted for the ambient attractant concentration. Such tuning of the attractant affinity allows the cell to chemotax in a much larger range of attractant concentrations than one would expect for a receptor population with a fixed K1/2 value. Moreover, computational studies have suggested that a two-state equilibrium-type receptor can successfully reproduce various experimentally observed characteristics of the chemotaxis signaling system (
The impressive sensitivity of the chemotaxis pathway at low attractant concentrations (1.5 and 3, and generally increased as the aspartate receptor modification level increased from EEEE to QQQQ (Table 1). A parallel study of the serine receptor also observed that the Hill coefficient of the attractant regulation increased with the receptor modification level (
More broadly, two-state behavior is observed in other components of the chemotaxis pathway and is widespread in signaling biology. NMR studies have revealed that periplasmic binding proteins of the chemotaxis system exhibit on- and off-conformations modulated by ligand binding, whereas the response regulator CheY displays an equilibrium between on- and off-states modulated by phosphorylation and proteinprotein interactions (
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Acknowledgements |
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We thank Sandy Parkinson and his laboratory for bacterial strains, helpful conversations, and comments on the manuscript. We also thank Chris Miller and Gary Yellen for helpful discussions. Finally, we are grateful to past and current members of the Falke lab, including Randal Bass, Eric Nalefski, Susy Kohout, Ryan Mehan, Matt Trammell, Gina Westhoff, and Noah White, for helpful advice and assistance.
Support for this study was provided by National Institutes of Health Grant GM R01-40731 (to J.J. Falke) and by NIH Training Grant T32-GM08759 (to J.A. Bornhorst).
Submitted: 10 September 2001
Revised: 2 November 2001
Accepted: 5 November 2001
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