Proc. Nati. Acad. Sci. USA
Vol. 88, pp. 750-754, February 1991 Biochemistry
Signal transduction in bacteria: CheW forms a reversible complex with the protein kinase CheA (bacterial chemotaxis/protein-protein interaction)
JULIE A. GEGNER AND FREDERICK W. DAHLQUIST Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403
Communicated by Peter H. von Hippel, October 19, 1990
response to attractant stimuli, the methylesterase is transiently inhibited. The mechanism responsible for methylesterase inhibition is not fully understood, but the adaptation response to positive stimuli appears to require only CheW (11). This observation suggests that parts of a CheWdependent signaling pathway may be independent of CheA. We have initiated studies of the physical interactions between CheW and the other components of the signal transduction and adaptation pathways of the chemotaxis system. Through such experiments, we hope to obtain a more detailed picture of the molecular pathway involved in chemotaxis signal transduction. It is not known how the kinase activity of CheA is coupled to the receptor by CheW, but the lack of any known CheW covalent modification suggests that complex formation between CheW and CheA may be involved. Here we report the results of our investigation that demonstrate an interaction between monomeric Mr 18,000 CheW and dimeric Mr 142,000 CheA. The ultimate goal of this work is to understand the molecular events that control swimming behavior in response to chemotactic stimuli. It appears that the formation of reversible complexes among various chemotaxis proteins may be an important feature of these molecular events.
An essential step in the signal transduction ABSTRACT pathway of Escherichia coli is the control of the protein kinase activity of CheA by the chemotaxis receptor proteins. This control requires the participation of the CheW protein. Although the biochemical nature of the coupling between the receptors and the kinase is unknown, it is likely that CheW interacts with the receptors and with CheA. In this communication, we report direct measurement of a physical interaction between CheW and CheA. We utilized the equilibrium column chromatography method of Hummel and Dreyer to show that CheW and CheA exhibit reversible binding with the stoichiometry of two CheW monomers per CheA dimer. CheW was found to exist as monomers and CheA was found to exist as dimers by equilibrium analytical ultracentrifugation. The dissociation constant for the CheW-CheA interaction (in 160 mM KCI/5 mM MgCl2, pH 7.4 at 40C) was determined to be in the physiologically relevant range of 17 FAM. No evidence for cooperativity in the association of CheW with CheA was found.
The bacteria Escherichia coli and Salmonella typhimurium control their swimming behavior in response to changes in the environment by alternating between smooth and tumbly swimming (for reviews of bacterial chemotaxis, see refs. 1 and 2). Transmembrane receptor/transducer proteins detect the presence of increased concentrations of repellent substances and signal the flagellar motor to promote tumbling by causing clockwise flagellar rotation. The same receptor/ transducer proteins suppress the tumble signal and promote smooth swimming in response to increases in attractant concentrations. The work of Hess et al. (3) and Borkovich et al. (4) showed that CheA is a protein kinase and that control ofits phosphorylation activity is coupled to the signaling state of the receptor/transducer via CheW. CheA phosphorylates CheY to generate tumbly behavior. The tumble signal is thought to occur by the direct interaction of phospho-CheY with the flagellar switch (5). The attractant response appears to be the result of the inhibition of the CheA phosphorylation cascade. CheW and CheA may act at some level in the attractant response, since overproduction of either protein results in suppression of the tumble signal (6-8). CheW also plays a role in coordinating the kinase activity of CheA in the adaptation response. The cell adapts to stimuli by adjusting the methylation state of its receptors (9). Adaptation to attractant results in an increase in the methylation state of the receptor, whereas adaptation to a repellent results in a decrease. In the repellent adaptation response, CheA phosphorylates CheB (the methylesterase), which is then activated to demethylate the receptor and restore the cell to its prestimulus swimming state of random tumbles and smooth swims (10, 11). Demethylation in response to repellent requires both CheA and CheW. During the adaptation
MATERIALS AND METHODS Bacterial Strains and Plasmids. The cheW overexpression plasmid pCW was created by inserting the cheW gene from pJL63 (7) into pHSe 5 (12). This was done by introducing an Nde I site immediately upstream of the cheW start codon and an Xba I site immediately downstream of the cheW stop codon by using standard oligonucleotide-directed mutagenesis procedures (13). In pCW, expression is under the control of the tac promoter. The cheA expression vector is similar to that described above for cheW (11). Growth of Cells and Protein Purification. The cheW and cheA plasmids were expressed in E. coli mutant strain RP3098 (a AflhA-flhD mutant), which was provided by J. S. Parkinson (University of Utah). Cells were grown at 300C in L broth (1% tryptone/0.5% yeast extract/0.5% NaCl) containing 0.1 mg of ampicillin per ml to a density of ~1 x 108 cells per ml. Expression of cheW and cheA was then induced with 1 mM isopropyl /-D-thiogalactoside. Cells expressing cheA were harvested after 3 hr of induction; those cells expressing cheW were harvested after 6 hr. CheW purification is based on the procedure described by Stock et al. (14) with the following modifications. Cells were harvested by centrifugation at 5000 rpm (Beckman JA 10 rotor) for 5 min, resuspended in a small volume of buffer containing 10 mM Mes (pH 6.0), 100 mM NaCl, 0.5 mM EDTA, and 50 ;LM phenylmethylsulfonyl fluoride, and then broken by French press. The lysate was ultracentrifuged at 50,000 rpm (Beckman Ti 60 rotor) for 1 hr to remove cellular debris. Protein was precipitated from the supernatant by adding (NH4)2SO4 to 40%o saturation and pelleted by centrifugation. The pellet was resuspended, dialyzed against the
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Biochemistry: Gegner and Dahlquist Mes buffer and loaded onto a Whatman DE-52 column. Protein was eluted from the column with a linear gradient of 100-250 mM NaCI. Fractions containing CheW were concentrated by precipitation with 50% (NH4)2SO4 and finally purified by using a Sephadex G-100 column (2.5 x 160 cm) equilibrated in the Mes buffer containing 0.02% NaN3. CheW was >99% pure as determined by Coomassie Blue staining. The identity of the protein was confirmed by sequencing the N-terminal nine amino acids. CheA was purified as described by Hess et al. (3) with the additional use of a Whatman DE-52 column. More than 90%o of the purified CheA was the long-form protein (15) as determined by Coomassie Bluestained gels. The functional activities of purified CheA and CheW were evaluated in the reconstitution phosphorylation assay described by Borkovich et al. (4). The concentrations of CheW and CheA were determined by measuring UV absorbance of 280 nm on a Beckman DU-7 spectrophotometer. The molar extinction coefficients of CheW and CheA were calculated to be 5120 and 16,290 M-l cm-l (±5%), respectively, by the procedure of Gill and von Hippel (16). Ultracentrifugation. The self-association states of CheW and CheA were determined by the equilibrium sedimentation meniscus depletion method of Yphantis (17). The buffer used contained 50 mM Tris (pH 7.2), 160 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol, and 5% glycerol. Rotor (Beckman An-D) speeds were 36,000 and 44,000 rpm for CheW and 17,000 and 22,000 rpm for CheA. The procedure detailed by Gill et al. (18) was used. Temperature was held constant at 200C. The amino acid composition was used to calculate the partial specific volume of CheA (v2 = 0.739) and CheW (v2 = 0.749) using the amino acid partial specific volumes of Zamyatnin (19). Chromatography. The equilibrium method of Hummel and Dreyer (20) was used to observe the protein-protein interactions of CheA and CheW. A Superose 12-prepacked HR 10/30 gel filtration column of 18 ml included volume connected to a Waters 650 pump system and was equilibrated with various concentrations of purified CheW. A 100-,ul injection of CheA plus concentrations of CheW equal to or greater than the column concentration was pumped through the column at the rate of 0.2 ml/min. Absorbance of the eluant was monitored at 280 nm with a Pharmacia UV-1 control unit with an HR flow cell and an Altex C-R1A integrator. Relative trough and peak volumes were determined by photocopying the recorder trace and cutting out and weighing the area of the trough or peak. Binding experiments were done at 40C. The Hummel-Dreyer binding buffer contained 50 mM Tris (pH 7.2), 160 mM KCI, 5 mM MgCI2, 0.5 mM EDTA, 1 mM dithiothreitol, and 0.02% NaN3 (HD buffer).
Proc. Natl. Acad. Sci. USA 88 (1991)
751
4
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50
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FIG. 1. Equilibrium sedimentation of CheW. CheW at an initial concentration of 0.5 mg/ml was sedimented at 36,000 rpm (Beckman An-D) in 50 mM Tris, pH 7.2/160 mM KCI/5 mM MgCl2/0.5 mM EDTA/1 mM dithiothreitol/5% glycerol at 20°C. Shown is the plot of logarithm of CheW concentration measured in fringe displacement (arbitrary units) versus r2 (square of the distance to the center of rotation), which yields a molecular weight of 17,500 ± 200 for CheW.
duced from the E. coli CheW DNA sequence (21). A molecular weight of 18,300 is obtained from the 44,000 rpm CheW run (data not shown). The equilibrium plot of CheA at 17,000 rpm is shown in Fig. 2. Again, the linearity of the plot suggests a homogeneous population of protein molecules. A molecular weight of 146,000 is obtained from the slope. The monomeric molecular weight of the long form of CheA as calculated from the amino acid composition deduced from the E. coli CheA DNA sequence (24) is 71,300. The result of 146,000 from the equilibrium sedimentation run is indicative of a CheA dimer because it is within 2% of the theoretical value of the CheA dimer. The equilibrium plot of CheA at 22,000 rpm gives a molecular weight of 145,000 (data not shown). The stability of the CheA dimer was investigated by frontal gel filtration at 4°C (22). No change in the elution profile was observed for CheA over a 15-fold change in concentration ranging from 3 ,uM to 45 ,uM (data not shown). The sensitivity of the CheA oligomeric structure to temperature was investigated by subjecting CheA to gel filtration chromatography at 4°C and 20°C. CheA was eluted from a Superose 12 column 2% later at 20°C than at 4°C (data not shown). The elution times of CheA cannot be correlated with molecular weight because CheA is eluted anomalously offgel filtration columns (personal observations). It is not known if the slight increase in retention time with temperature is due to a change in the .
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RESULTS Self-Association States of CheW and CheA. A prerequisite for the analysis of binding interactions is an understanding of the self-association states of the interacting species. Equilibrium analytical sedimentation was performed to unambiguously establish the self-association states of CheW and CheA. Fig. 1 shows a plot of the logarithm of CheW concentration in displacement of interference fringes (arbitrary units) versus r2 (square of the distance from the center of rotation) obtained from the equilibrium run at 36,000 rpm. The linear plot strongly indicates that CheW exists as a homogeneous species. The slope of the line in Fig. 1 provides a measure of the weight-average molecular weight of the protein in solution that is shape independent. A molecular weight of 17,500 is obtained, which lies within 3% of the expected molecular weight of the CheW monomer (Mr 18,000) as calculated from the amino acid composition de-
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FIG. 2. Equilibrium sedimentation of CheA. CheA at an initial concentration of 0.5 mg/ml was sedimented at 17,000 rpm (Beckman An-D) in 50 mM Tris, pH 7.2/160 mM KCI/5 mM MgCl2/0.5 mM EDTA/1 mM dithiothreitol/5% glycerol at 20°C. Shown is the plot of the logarithm of CheA concentration measured in fringe displacement (arbitrary units) versus r2 (square of the distance to the center of rotation), which yields a molecular weight of 146,000 + 1500 for CheA.
Biochemistry: Gegner and Dahlquist
752
Proc. NatL Acad. Sci. USA 88 (1991)
oligomeric state of CheA; however, the equilibrium sedimentation data in combination with the results of frontal gel filtration strongly suggest that CheA exists as a stable species that does not dissociate into monomers over these ranges of concentration and temperature. Binding of CheW to CheA. Purified CheA and CheW give well-resolved peaks when subjected to gel filtration chromatography on a number of supports, including Sephadex G-100, Sephacryl S-200, Sephacryl S-300, Ultrogel AcA 22, and Superose 12. We chose the Superose 12 gel filtration column for binding studies. The elution times of CheW and CheA when coapplied onto the column were the same (within experimental error) as when the proteins were applied separately to the column, and no complex between CheA and CheW was observed (Fig. 3A). In addition, when fractions collected from the run were subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Coomassie blue staining, none of the fractions were found to contain both CheA and CheW (data not shown). These results suggest that if CheA and CheW complex formation occurs, the complex is dynamic and equilibrates on a time scale that is comparable to or faster than the separation time of about 15 min. We were able to detect reversible binding between CheA and CheW by the equilibrium chromatography method of Hummel and Dreyer (20). In a typical experiment, the gel filtration column was equilibrated in buffer containing a uniform concentration of CheW such as 20 uM. Samples containing various concentrations of CheA plus the buffer concentration of 20.uM CheW were applied onto the column. 0.5 -A A CheA
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When CheW binding to CheA occurs, the amount of free CheW in the applied sample is reduced by the amount bound to CheA. The CheW bound to CheA has increased mobility and is eluted from the column before the free CheW. Since the remaining amount of CheW is now less than the column concentration, a trough is observed at the normal elution time of CheW (Fig. 3B). The size of the trough increases as the concentration of CheA in the applied sample increases. No trough is observed when there is no CheA in the applied sample. Similarly, the addition of a noninteracting protein such as hen lysozyme results in no trough at the elution position of CheW. To determine the dissociation constant and stoichiometry for the CheW interaction with CheA, the free CheW concentration must be known. In the above experiment, band spreading in the column makes it difficult to determine the free CheW concentration. Instead, the free CheW concentration can be determined by adding additional CheW to the CheA-containing sample applied to the column equilibrated with CheW-containing buffer. The concentration of CheW in the sample will be reduced by the amount bound to CheA. When the remaining free CheW concentration is less than that in the column buffer, a trough will be observed; when the free concentration is greater than that in the column buffer, a peak will be observed. When no trough or peak is observed, then the free CheW concentration equals the CheW concentration in the column buffer. This experiment was done at several concentrations of CheW in the column buffer, while the CheA dimer concentration was held constant. The point at which no peak or trough occurred was determined by plotting the weight area of CheW peaks (or troughs) versus ,umol of CheW added. A typical result is shown in Fig. 4 as a plot of the amplitude of the CheW peak versus the CheW concentration in the applied sample when the CheA dimer concentration in the applied sample was 20 uM. As can be seen, there is an approximately linear relationship between the amplitude of the CheW peak and the CheW concentration in the applied sample. The plot crosses zero amplitude at a total CheW concentration of 44 1.M. The column buffer was equilibrated with 20MuM CheW; thus, 20M&M CheW is free and 24 ,uM CheW is bound to 20,uM CheA dimer or 40MLM CheA monomer subunits. When one assumes two independent CheW binding sites per CheA dimer, the dissociation constant corresponds to Kd = [CheA][CheW]/[CheA-CheW] = (40 AM - 24 uM)(20 IMM)/(24 MuM) or 13 MuM. To determine the stoichiometry of the CheW and CheA interaction, the above experiment was repeated with different column concentrations of CheW. A Scatchard plot of 0.02
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Time, min FIG. 3. Gel filtration chromatography of CheA and CheW binding. A Superose 12 gel filtration column was used as described. (A) Approximately 10 MM CheA dimer and 20 ,uM CheW were injected onto the column equilibrated in HD buffer. The elution times of CheA and CheW were the same as when the proteins were applied separately to the column. No CheA-CheW complex was observed. (B) A sample of 7.5 MM CheA dimer and 20 A&M CheW was applied to a column equilibrated in HD buffer plus 20 AtM CheW. A trough at the normal elution time of CheW is observed. The trough is the result of the CheW bound to CheA being eluted faster than free CheW.
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FIG. 4. The areas of the CheW peaks and troughs following application of various amounts of micromolar CheW and 20 ,uM CheA dimer to a column equilibrated with buffer containing 20 AM CheW versus the added CheW concentration. At 44 MM, the line crosses zero, and the free CheW concentration equals the CheW column buffer concentration of 20 MM, with the bound CheW concentration as the difference, 24 MM. arb, Arbitrary.
Biochemistry: Gegner and Dahlquist
Proc. NatL Acad. Sci. USA 88 (1991)
bound CheW concentration divided by free CheW versus the concentration of bound CheW gives a slope of -1/Kd, the negative reciprocal of the dissociation constant. The abscissa intercept of the Scatchard plot gives the concentration of potential binding sites. The potential binding sites per CheA dimer can be calculated by dividing the potential sites by the CheA dimer concentration of 20 ,uM. A Scatchard plot of our experimental results is shown in Fig. 5. The dissociation constant obtained from the plot is 17 ,uM with the number of potential CheW binding sites per CheA dimer of two. This is in agreement with the binding constant of 13 ,uM calculated above. The linearity of the Scatchard plot strongly suggests that the two sites per CheA dimer are identical and independent. To further confirm the well-behaved nature of the CheA and CheW interaction, we examined the binding of CheW to CheA at a single free CheW concentration (5 AuM) and two CheA dimer concentrations (10 ,M and 20 tiM). The calculated affinity of CheW for CheA was the same for these different CheA concentrations. Thus, it appears that the affinity does not depend on the CheA concentration. This observation appears to rule out changes in the CheA association state upon CheW binding.
DISCUSSION The results presented here provide the first physical evidence for the association of CheW and CheA. Previous work by Borkovich et al. (4) demonstrates that CheW is needed to provide coupling between the signaling state of the receptor/ transducer proteins and the phosphorylation events catalyzed by CheA. Our results suggest that the mechanism of coupling involves the direct binding of CheW to CheA. In such a mechanism, the extent of complex formation between CheW and CheA would need to be modulated in some way. We estimate CheW and CheA to be about 20% complexed in vivo. This was calculated from the dissociation constant of 17 AuM and the intracellular CheW monomer and CheA dimer concentrations (as determined from immunoblots) of 5 p.M and 2.5 A.M, respectively. Under these conditions, relatively small changes in the signaling properties of CheA (e.g., ATP binding or phosphorylation of CheA) could cause substantial changes in the concentration of the CheA-CheW complex. In the work presented here, we show that equilibration is rapid or comparable to the gel filtration separation time of about 15 4a)
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30
40
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FIG. 5. A Scatchard plot of bound CheW micromolar concentration divided by free CheW micromolar concentration versus the concentration of bound CheW. The dissociation constant for the CheW-CheA interaction obtained from the negative reciprocal of slope is 17 uM with an estimated error of ±70%6. The abscissa intercept gives the total number of CheW binding sites per 20 IsM CheA dimer as "40 tiM, or two CheW binding sites per CheA dimer. The lack of curvature in the Scatchard plot over the concentration range of 5-100 .uM CheW suggests that the CheW binding sites are independent and identical.
753
min. If such changes are to be an important step in the signal transduction pathway, the complex must form and dissociate on a time scale of 0.2 sec or less (23). In addition to the CheW-CheA interaction, we have observed specific binding of CheW to Tsr-containing membranes, using the gel filtration assay described for the CheWCheA interaction (unpublished observations). No binding was observed with membrane preparations that lack the chemotaxis receptors/transducers. This direct and specific interaction between CheW and Tsr supports the physiological experiments of Liu and Parkinson (7), which show an apparent compensation of high levels of CheW by corresponding high levels of Tsr in intact cells. Although these observations do not establish a mechanistic role for these complexes in the transduction pathway, they do provide._a framework to consider how stimulus binding to the receptor might be coupled to regulation of CheA kinase activity. In one view, CheW could shuttle between the receptor/ transducer proteins and CheA. In this model, the amount of CheW-CheA complex would be determined by the competition between the receptor/transducer and CheA for CheW. Attractant or repellent binding to the receptor/transducer could then modulate the affinity of CheW for receptor/ transducer. This in turn would cause the amount of CheACheW complex to vary. For this model to provide coupling, it is necessary that binding of CheW to CheA results in a change in the kinase activity of CheA. The results of Borkovich et al. (4) suggest about a 60%6 increase in the kinase activity of CheA and CheY in the presence of 2 ,uM CheW. It is unlikely that this small change can account for the several-hundred-fold increase in CheA kinase activity observed when both receptor/transducer membranes and CheW are present. Thus, although a "shuttle" mechanism cannot be strictly ruled out, it seems an unlikely alternative. The more likely explanation for the coupling between CheA and the receptor/transducer by CheW is that ternary complexes are formed involving CheA, CheW, and the receptors/transducers. In this view, the coupling of binding events at the receptor/transducer could be transmitted to CheA via direct physical contact. The role of CheW could be either as a physical link in the contact between CheA and the receptor/transducer or CheW could serve to modulate the affinity of CheA for the receptor/transducer. It is possible that CheW serves to control the specificity of the CheA kinase activity. It appears that CheA can either phosphorylate CheY, which then directly interacts with the switch that regulates the sense of the flagellar rotation, or phosphorylate CheB, which is then activated to remove methyl esters from the receptors/transducers. In a wild-type cell, there appears to be a competition between CheB and CheY for the CheA kinase activity. We have isolated two mutants of CheW that appear to modify the partitioning of phosphoryltransfer between CheY and CheB. In one, containing a C-terminal deletion, bacteria with the mutant CheW have relatively normal flagellar response asjudged by chemotaxis on swarm plates but have very defective modulation of CheB methylesterase activity. In the other, a CheW point mutant, flagellar response appears to be quite defective as judged by poor swarm behavior, but modulation of CheB methylesterase activity is essentially the same as wild type. Initial experiments suggest that some CheW mutants have substantially altered affinity for CheA. In addition to providing insights into potential mechanisms of sensory transduction during bacterial chemotaxis, the interactions between CheW and CheA may provide a good model for understanding how reversible binding between a specific protein kinase and a potential regulatory protein that is not the substrate of the kinase can modulate the kinase output. Bacterial chemotaxis also offers the obvious advantages offacile biochemistry and genetics for studying general
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problems involving protein-protein interactions in sensory transduction and other regulatory systems. We thank A. F. Roth for the CheW mutant work, D. R. Graham for helpful advice, and H. Geiselmann and M. C. Young for help with the analytical sedimentation experiments and analysis. This work was supported by a grant from the National Institutes of Health (GM33677). J.A.G. is a National Institutes of Health predoctoral trainee (GM07759). 1. Stewart, R. C. & Dahlquist, F. W. (1987) Chem. Rev. 87, 997-1025. 2. Macnab, R. M. (1987) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, ed. Neidhardt, F. C. (Am. Soc. Microbiol., Washington, DC), pp. 732-759. 3. Hess, J. F., Oosawa, K., Kaplan, N. & Simon, M. I. (1988) Cell 53, 79-87. 4. Borkovich, K. A., Kaplan, N., Hess, J. F. & Simon, M. I. (1989) Proc. Natl. Acad. Sci. USA 86, 1208-1212. 5. Bourret, R. B., Hess, J. F. & Simon, M. I. (1990) Proc. Natl. Acad. Sci. USA 87, 41-45. 6. Stewart, R. C., Russell, C. B., Roth, A. F. & Dahlquist, F. W. (1988) Cold Spring Harbor Symp. Quant. Biol. 59, 27-40. 7. Liu, J. & Parkinson, J. S. (1989) Proc. Natl. Acad. Sci. USA 86, 8703-8707. 8. Sanders, D. A., Mendez, B. & Koshland, D. E. (1989) J. Bacteriol. 171, 6271-6278.
Proc. NatL Acad Sci. USA 88 (1991) 9. Kehry, M. R., Doak, T. G. & Dahlquist, F. W. (1984) J. Biol. Chem. 259, 11828-11835. 10. Lupas, A. & Stock, J. (1989) J. Biol. Chem. 264, 17337-17342. 11. Stewart, R. C., Roth, A. F. & Dahlquist, F. W. (1990) J. Bacteriol. 172, 3388-3399. 12. Muchmore, D. C., McIntosh, L. P., Russell, C. B., Anderson, D. E. & Dahlquist, F. W. (1989) Methods Enzymol. 177,44-73. 13. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492. 14. Stock, A., Mottonen, J., Chen, T. & Stock, J. (1987) J. Biol. Chem. 262, 535-537. 15. Smith, R. A. & Parkinson, J. S. (1980) Proc. Natl. Acad. Sci. USA 77, 5370-5374. 16. Gill, S. C. & von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326. 17. Yphantis, D. (1964) Biochemistry 3, 297-317. 18. Gill, S. C., Yager, T. D. & von Hippel, P. H. (1991) J. Mol. Biol., in press. 19. Zamyatnin, A. A. (1972) Prog. Biophys. Mol. Biol. 24,109-123. 20. Hummel, J. P. & Dreyer, W. J. (1962) Biochim. Biophys. Acta 63, 530-532. 21. Mutoh, N. & Simon, M. I. (1986) J. Bacteriol. 165, 161-166. 22. Winzor, D. J., Loke, J. P. & Nichol, L. W. (1967) J. Phys. Chem. 71, 4492-4498. 23. Sesall, J. E., Manson, M. D. & Berg, H. C. (1982) Nature (London) 296, 855-857. 24. Kofoid, E. C. & Parkinson, J. S. (1991) J. Bacteriol., in press.
Vol. 88, pp. 750-754, February 1991 Biochemistry
Signal transduction in bacteria: CheW forms a reversible complex with the protein kinase CheA (bacterial chemotaxis/protein-protein interaction)
JULIE A. GEGNER AND FREDERICK W. DAHLQUIST Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403
Communicated by Peter H. von Hippel, October 19, 1990
response to attractant stimuli, the methylesterase is transiently inhibited. The mechanism responsible for methylesterase inhibition is not fully understood, but the adaptation response to positive stimuli appears to require only CheW (11). This observation suggests that parts of a CheWdependent signaling pathway may be independent of CheA. We have initiated studies of the physical interactions between CheW and the other components of the signal transduction and adaptation pathways of the chemotaxis system. Through such experiments, we hope to obtain a more detailed picture of the molecular pathway involved in chemotaxis signal transduction. It is not known how the kinase activity of CheA is coupled to the receptor by CheW, but the lack of any known CheW covalent modification suggests that complex formation between CheW and CheA may be involved. Here we report the results of our investigation that demonstrate an interaction between monomeric Mr 18,000 CheW and dimeric Mr 142,000 CheA. The ultimate goal of this work is to understand the molecular events that control swimming behavior in response to chemotactic stimuli. It appears that the formation of reversible complexes among various chemotaxis proteins may be an important feature of these molecular events.
An essential step in the signal transduction ABSTRACT pathway of Escherichia coli is the control of the protein kinase activity of CheA by the chemotaxis receptor proteins. This control requires the participation of the CheW protein. Although the biochemical nature of the coupling between the receptors and the kinase is unknown, it is likely that CheW interacts with the receptors and with CheA. In this communication, we report direct measurement of a physical interaction between CheW and CheA. We utilized the equilibrium column chromatography method of Hummel and Dreyer to show that CheW and CheA exhibit reversible binding with the stoichiometry of two CheW monomers per CheA dimer. CheW was found to exist as monomers and CheA was found to exist as dimers by equilibrium analytical ultracentrifugation. The dissociation constant for the CheW-CheA interaction (in 160 mM KCI/5 mM MgCl2, pH 7.4 at 40C) was determined to be in the physiologically relevant range of 17 FAM. No evidence for cooperativity in the association of CheW with CheA was found.
The bacteria Escherichia coli and Salmonella typhimurium control their swimming behavior in response to changes in the environment by alternating between smooth and tumbly swimming (for reviews of bacterial chemotaxis, see refs. 1 and 2). Transmembrane receptor/transducer proteins detect the presence of increased concentrations of repellent substances and signal the flagellar motor to promote tumbling by causing clockwise flagellar rotation. The same receptor/ transducer proteins suppress the tumble signal and promote smooth swimming in response to increases in attractant concentrations. The work of Hess et al. (3) and Borkovich et al. (4) showed that CheA is a protein kinase and that control ofits phosphorylation activity is coupled to the signaling state of the receptor/transducer via CheW. CheA phosphorylates CheY to generate tumbly behavior. The tumble signal is thought to occur by the direct interaction of phospho-CheY with the flagellar switch (5). The attractant response appears to be the result of the inhibition of the CheA phosphorylation cascade. CheW and CheA may act at some level in the attractant response, since overproduction of either protein results in suppression of the tumble signal (6-8). CheW also plays a role in coordinating the kinase activity of CheA in the adaptation response. The cell adapts to stimuli by adjusting the methylation state of its receptors (9). Adaptation to attractant results in an increase in the methylation state of the receptor, whereas adaptation to a repellent results in a decrease. In the repellent adaptation response, CheA phosphorylates CheB (the methylesterase), which is then activated to demethylate the receptor and restore the cell to its prestimulus swimming state of random tumbles and smooth swims (10, 11). Demethylation in response to repellent requires both CheA and CheW. During the adaptation
MATERIALS AND METHODS Bacterial Strains and Plasmids. The cheW overexpression plasmid pCW was created by inserting the cheW gene from pJL63 (7) into pHSe 5 (12). This was done by introducing an Nde I site immediately upstream of the cheW start codon and an Xba I site immediately downstream of the cheW stop codon by using standard oligonucleotide-directed mutagenesis procedures (13). In pCW, expression is under the control of the tac promoter. The cheA expression vector is similar to that described above for cheW (11). Growth of Cells and Protein Purification. The cheW and cheA plasmids were expressed in E. coli mutant strain RP3098 (a AflhA-flhD mutant), which was provided by J. S. Parkinson (University of Utah). Cells were grown at 300C in L broth (1% tryptone/0.5% yeast extract/0.5% NaCl) containing 0.1 mg of ampicillin per ml to a density of ~1 x 108 cells per ml. Expression of cheW and cheA was then induced with 1 mM isopropyl /-D-thiogalactoside. Cells expressing cheA were harvested after 3 hr of induction; those cells expressing cheW were harvested after 6 hr. CheW purification is based on the procedure described by Stock et al. (14) with the following modifications. Cells were harvested by centrifugation at 5000 rpm (Beckman JA 10 rotor) for 5 min, resuspended in a small volume of buffer containing 10 mM Mes (pH 6.0), 100 mM NaCl, 0.5 mM EDTA, and 50 ;LM phenylmethylsulfonyl fluoride, and then broken by French press. The lysate was ultracentrifuged at 50,000 rpm (Beckman Ti 60 rotor) for 1 hr to remove cellular debris. Protein was precipitated from the supernatant by adding (NH4)2SO4 to 40%o saturation and pelleted by centrifugation. The pellet was resuspended, dialyzed against the
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Biochemistry: Gegner and Dahlquist Mes buffer and loaded onto a Whatman DE-52 column. Protein was eluted from the column with a linear gradient of 100-250 mM NaCI. Fractions containing CheW were concentrated by precipitation with 50% (NH4)2SO4 and finally purified by using a Sephadex G-100 column (2.5 x 160 cm) equilibrated in the Mes buffer containing 0.02% NaN3. CheW was >99% pure as determined by Coomassie Blue staining. The identity of the protein was confirmed by sequencing the N-terminal nine amino acids. CheA was purified as described by Hess et al. (3) with the additional use of a Whatman DE-52 column. More than 90%o of the purified CheA was the long-form protein (15) as determined by Coomassie Bluestained gels. The functional activities of purified CheA and CheW were evaluated in the reconstitution phosphorylation assay described by Borkovich et al. (4). The concentrations of CheW and CheA were determined by measuring UV absorbance of 280 nm on a Beckman DU-7 spectrophotometer. The molar extinction coefficients of CheW and CheA were calculated to be 5120 and 16,290 M-l cm-l (±5%), respectively, by the procedure of Gill and von Hippel (16). Ultracentrifugation. The self-association states of CheW and CheA were determined by the equilibrium sedimentation meniscus depletion method of Yphantis (17). The buffer used contained 50 mM Tris (pH 7.2), 160 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol, and 5% glycerol. Rotor (Beckman An-D) speeds were 36,000 and 44,000 rpm for CheW and 17,000 and 22,000 rpm for CheA. The procedure detailed by Gill et al. (18) was used. Temperature was held constant at 200C. The amino acid composition was used to calculate the partial specific volume of CheA (v2 = 0.739) and CheW (v2 = 0.749) using the amino acid partial specific volumes of Zamyatnin (19). Chromatography. The equilibrium method of Hummel and Dreyer (20) was used to observe the protein-protein interactions of CheA and CheW. A Superose 12-prepacked HR 10/30 gel filtration column of 18 ml included volume connected to a Waters 650 pump system and was equilibrated with various concentrations of purified CheW. A 100-,ul injection of CheA plus concentrations of CheW equal to or greater than the column concentration was pumped through the column at the rate of 0.2 ml/min. Absorbance of the eluant was monitored at 280 nm with a Pharmacia UV-1 control unit with an HR flow cell and an Altex C-R1A integrator. Relative trough and peak volumes were determined by photocopying the recorder trace and cutting out and weighing the area of the trough or peak. Binding experiments were done at 40C. The Hummel-Dreyer binding buffer contained 50 mM Tris (pH 7.2), 160 mM KCI, 5 mM MgCI2, 0.5 mM EDTA, 1 mM dithiothreitol, and 0.02% NaN3 (HD buffer).
Proc. Natl. Acad. Sci. USA 88 (1991)
751
4
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49
48
50
51
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FIG. 1. Equilibrium sedimentation of CheW. CheW at an initial concentration of 0.5 mg/ml was sedimented at 36,000 rpm (Beckman An-D) in 50 mM Tris, pH 7.2/160 mM KCI/5 mM MgCl2/0.5 mM EDTA/1 mM dithiothreitol/5% glycerol at 20°C. Shown is the plot of logarithm of CheW concentration measured in fringe displacement (arbitrary units) versus r2 (square of the distance to the center of rotation), which yields a molecular weight of 17,500 ± 200 for CheW.
duced from the E. coli CheW DNA sequence (21). A molecular weight of 18,300 is obtained from the 44,000 rpm CheW run (data not shown). The equilibrium plot of CheA at 17,000 rpm is shown in Fig. 2. Again, the linearity of the plot suggests a homogeneous population of protein molecules. A molecular weight of 146,000 is obtained from the slope. The monomeric molecular weight of the long form of CheA as calculated from the amino acid composition deduced from the E. coli CheA DNA sequence (24) is 71,300. The result of 146,000 from the equilibrium sedimentation run is indicative of a CheA dimer because it is within 2% of the theoretical value of the CheA dimer. The equilibrium plot of CheA at 22,000 rpm gives a molecular weight of 145,000 (data not shown). The stability of the CheA dimer was investigated by frontal gel filtration at 4°C (22). No change in the elution profile was observed for CheA over a 15-fold change in concentration ranging from 3 ,uM to 45 ,uM (data not shown). The sensitivity of the CheA oligomeric structure to temperature was investigated by subjecting CheA to gel filtration chromatography at 4°C and 20°C. CheA was eluted from a Superose 12 column 2% later at 20°C than at 4°C (data not shown). The elution times of CheA cannot be correlated with molecular weight because CheA is eluted anomalously offgel filtration columns (personal observations). It is not known if the slight increase in retention time with temperature is due to a change in the .
a)
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RESULTS Self-Association States of CheW and CheA. A prerequisite for the analysis of binding interactions is an understanding of the self-association states of the interacting species. Equilibrium analytical sedimentation was performed to unambiguously establish the self-association states of CheW and CheA. Fig. 1 shows a plot of the logarithm of CheW concentration in displacement of interference fringes (arbitrary units) versus r2 (square of the distance from the center of rotation) obtained from the equilibrium run at 36,000 rpm. The linear plot strongly indicates that CheW exists as a homogeneous species. The slope of the line in Fig. 1 provides a measure of the weight-average molecular weight of the protein in solution that is shape independent. A molecular weight of 17,500 is obtained, which lies within 3% of the expected molecular weight of the CheW monomer (Mr 18,000) as calculated from the amino acid composition de-
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FIG. 2. Equilibrium sedimentation of CheA. CheA at an initial concentration of 0.5 mg/ml was sedimented at 17,000 rpm (Beckman An-D) in 50 mM Tris, pH 7.2/160 mM KCI/5 mM MgCl2/0.5 mM EDTA/1 mM dithiothreitol/5% glycerol at 20°C. Shown is the plot of the logarithm of CheA concentration measured in fringe displacement (arbitrary units) versus r2 (square of the distance to the center of rotation), which yields a molecular weight of 146,000 + 1500 for CheA.
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752
Proc. NatL Acad. Sci. USA 88 (1991)
oligomeric state of CheA; however, the equilibrium sedimentation data in combination with the results of frontal gel filtration strongly suggest that CheA exists as a stable species that does not dissociate into monomers over these ranges of concentration and temperature. Binding of CheW to CheA. Purified CheA and CheW give well-resolved peaks when subjected to gel filtration chromatography on a number of supports, including Sephadex G-100, Sephacryl S-200, Sephacryl S-300, Ultrogel AcA 22, and Superose 12. We chose the Superose 12 gel filtration column for binding studies. The elution times of CheW and CheA when coapplied onto the column were the same (within experimental error) as when the proteins were applied separately to the column, and no complex between CheA and CheW was observed (Fig. 3A). In addition, when fractions collected from the run were subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Coomassie blue staining, none of the fractions were found to contain both CheA and CheW (data not shown). These results suggest that if CheA and CheW complex formation occurs, the complex is dynamic and equilibrates on a time scale that is comparable to or faster than the separation time of about 15 min. We were able to detect reversible binding between CheA and CheW by the equilibrium chromatography method of Hummel and Dreyer (20). In a typical experiment, the gel filtration column was equilibrated in buffer containing a uniform concentration of CheW such as 20 uM. Samples containing various concentrations of CheA plus the buffer concentration of 20.uM CheW were applied onto the column. 0.5 -A A CheA
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When CheW binding to CheA occurs, the amount of free CheW in the applied sample is reduced by the amount bound to CheA. The CheW bound to CheA has increased mobility and is eluted from the column before the free CheW. Since the remaining amount of CheW is now less than the column concentration, a trough is observed at the normal elution time of CheW (Fig. 3B). The size of the trough increases as the concentration of CheA in the applied sample increases. No trough is observed when there is no CheA in the applied sample. Similarly, the addition of a noninteracting protein such as hen lysozyme results in no trough at the elution position of CheW. To determine the dissociation constant and stoichiometry for the CheW interaction with CheA, the free CheW concentration must be known. In the above experiment, band spreading in the column makes it difficult to determine the free CheW concentration. Instead, the free CheW concentration can be determined by adding additional CheW to the CheA-containing sample applied to the column equilibrated with CheW-containing buffer. The concentration of CheW in the sample will be reduced by the amount bound to CheA. When the remaining free CheW concentration is less than that in the column buffer, a trough will be observed; when the free concentration is greater than that in the column buffer, a peak will be observed. When no trough or peak is observed, then the free CheW concentration equals the CheW concentration in the column buffer. This experiment was done at several concentrations of CheW in the column buffer, while the CheA dimer concentration was held constant. The point at which no peak or trough occurred was determined by plotting the weight area of CheW peaks (or troughs) versus ,umol of CheW added. A typical result is shown in Fig. 4 as a plot of the amplitude of the CheW peak versus the CheW concentration in the applied sample when the CheA dimer concentration in the applied sample was 20 uM. As can be seen, there is an approximately linear relationship between the amplitude of the CheW peak and the CheW concentration in the applied sample. The plot crosses zero amplitude at a total CheW concentration of 44 1.M. The column buffer was equilibrated with 20MuM CheW; thus, 20M&M CheW is free and 24 ,uM CheW is bound to 20,uM CheA dimer or 40MLM CheA monomer subunits. When one assumes two independent CheW binding sites per CheA dimer, the dissociation constant corresponds to Kd = [CheA][CheW]/[CheA-CheW] = (40 AM - 24 uM)(20 IMM)/(24 MuM) or 13 MuM. To determine the stoichiometry of the CheW and CheA interaction, the above experiment was repeated with different column concentrations of CheW. A Scatchard plot of 0.02
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Time, min FIG. 3. Gel filtration chromatography of CheA and CheW binding. A Superose 12 gel filtration column was used as described. (A) Approximately 10 MM CheA dimer and 20 ,uM CheW were injected onto the column equilibrated in HD buffer. The elution times of CheA and CheW were the same as when the proteins were applied separately to the column. No CheA-CheW complex was observed. (B) A sample of 7.5 MM CheA dimer and 20 A&M CheW was applied to a column equilibrated in HD buffer plus 20 AtM CheW. A trough at the normal elution time of CheW is observed. The trough is the result of the CheW bound to CheA being eluted faster than free CheW.
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FIG. 4. The areas of the CheW peaks and troughs following application of various amounts of micromolar CheW and 20 ,uM CheA dimer to a column equilibrated with buffer containing 20 AM CheW versus the added CheW concentration. At 44 MM, the line crosses zero, and the free CheW concentration equals the CheW column buffer concentration of 20 MM, with the bound CheW concentration as the difference, 24 MM. arb, Arbitrary.
Biochemistry: Gegner and Dahlquist
Proc. NatL Acad. Sci. USA 88 (1991)
bound CheW concentration divided by free CheW versus the concentration of bound CheW gives a slope of -1/Kd, the negative reciprocal of the dissociation constant. The abscissa intercept of the Scatchard plot gives the concentration of potential binding sites. The potential binding sites per CheA dimer can be calculated by dividing the potential sites by the CheA dimer concentration of 20 ,uM. A Scatchard plot of our experimental results is shown in Fig. 5. The dissociation constant obtained from the plot is 17 ,uM with the number of potential CheW binding sites per CheA dimer of two. This is in agreement with the binding constant of 13 ,uM calculated above. The linearity of the Scatchard plot strongly suggests that the two sites per CheA dimer are identical and independent. To further confirm the well-behaved nature of the CheA and CheW interaction, we examined the binding of CheW to CheA at a single free CheW concentration (5 AuM) and two CheA dimer concentrations (10 ,M and 20 tiM). The calculated affinity of CheW for CheA was the same for these different CheA concentrations. Thus, it appears that the affinity does not depend on the CheA concentration. This observation appears to rule out changes in the CheA association state upon CheW binding.
DISCUSSION The results presented here provide the first physical evidence for the association of CheW and CheA. Previous work by Borkovich et al. (4) demonstrates that CheW is needed to provide coupling between the signaling state of the receptor/ transducer proteins and the phosphorylation events catalyzed by CheA. Our results suggest that the mechanism of coupling involves the direct binding of CheW to CheA. In such a mechanism, the extent of complex formation between CheW and CheA would need to be modulated in some way. We estimate CheW and CheA to be about 20% complexed in vivo. This was calculated from the dissociation constant of 17 AuM and the intracellular CheW monomer and CheA dimer concentrations (as determined from immunoblots) of 5 p.M and 2.5 A.M, respectively. Under these conditions, relatively small changes in the signaling properties of CheA (e.g., ATP binding or phosphorylation of CheA) could cause substantial changes in the concentration of the CheA-CheW complex. In the work presented here, we show that equilibration is rapid or comparable to the gel filtration separation time of about 15 4a)
L-
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0
10 20 CheW bound,
30
40
AiM
FIG. 5. A Scatchard plot of bound CheW micromolar concentration divided by free CheW micromolar concentration versus the concentration of bound CheW. The dissociation constant for the CheW-CheA interaction obtained from the negative reciprocal of slope is 17 uM with an estimated error of ±70%6. The abscissa intercept gives the total number of CheW binding sites per 20 IsM CheA dimer as "40 tiM, or two CheW binding sites per CheA dimer. The lack of curvature in the Scatchard plot over the concentration range of 5-100 .uM CheW suggests that the CheW binding sites are independent and identical.
753
min. If such changes are to be an important step in the signal transduction pathway, the complex must form and dissociate on a time scale of 0.2 sec or less (23). In addition to the CheW-CheA interaction, we have observed specific binding of CheW to Tsr-containing membranes, using the gel filtration assay described for the CheWCheA interaction (unpublished observations). No binding was observed with membrane preparations that lack the chemotaxis receptors/transducers. This direct and specific interaction between CheW and Tsr supports the physiological experiments of Liu and Parkinson (7), which show an apparent compensation of high levels of CheW by corresponding high levels of Tsr in intact cells. Although these observations do not establish a mechanistic role for these complexes in the transduction pathway, they do provide._a framework to consider how stimulus binding to the receptor might be coupled to regulation of CheA kinase activity. In one view, CheW could shuttle between the receptor/ transducer proteins and CheA. In this model, the amount of CheW-CheA complex would be determined by the competition between the receptor/transducer and CheA for CheW. Attractant or repellent binding to the receptor/transducer could then modulate the affinity of CheW for receptor/ transducer. This in turn would cause the amount of CheACheW complex to vary. For this model to provide coupling, it is necessary that binding of CheW to CheA results in a change in the kinase activity of CheA. The results of Borkovich et al. (4) suggest about a 60%6 increase in the kinase activity of CheA and CheY in the presence of 2 ,uM CheW. It is unlikely that this small change can account for the several-hundred-fold increase in CheA kinase activity observed when both receptor/transducer membranes and CheW are present. Thus, although a "shuttle" mechanism cannot be strictly ruled out, it seems an unlikely alternative. The more likely explanation for the coupling between CheA and the receptor/transducer by CheW is that ternary complexes are formed involving CheA, CheW, and the receptors/transducers. In this view, the coupling of binding events at the receptor/transducer could be transmitted to CheA via direct physical contact. The role of CheW could be either as a physical link in the contact between CheA and the receptor/transducer or CheW could serve to modulate the affinity of CheA for the receptor/transducer. It is possible that CheW serves to control the specificity of the CheA kinase activity. It appears that CheA can either phosphorylate CheY, which then directly interacts with the switch that regulates the sense of the flagellar rotation, or phosphorylate CheB, which is then activated to remove methyl esters from the receptors/transducers. In a wild-type cell, there appears to be a competition between CheB and CheY for the CheA kinase activity. We have isolated two mutants of CheW that appear to modify the partitioning of phosphoryltransfer between CheY and CheB. In one, containing a C-terminal deletion, bacteria with the mutant CheW have relatively normal flagellar response asjudged by chemotaxis on swarm plates but have very defective modulation of CheB methylesterase activity. In the other, a CheW point mutant, flagellar response appears to be quite defective as judged by poor swarm behavior, but modulation of CheB methylesterase activity is essentially the same as wild type. Initial experiments suggest that some CheW mutants have substantially altered affinity for CheA. In addition to providing insights into potential mechanisms of sensory transduction during bacterial chemotaxis, the interactions between CheW and CheA may provide a good model for understanding how reversible binding between a specific protein kinase and a potential regulatory protein that is not the substrate of the kinase can modulate the kinase output. Bacterial chemotaxis also offers the obvious advantages offacile biochemistry and genetics for studying general
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problems involving protein-protein interactions in sensory transduction and other regulatory systems. We thank A. F. Roth for the CheW mutant work, D. R. Graham for helpful advice, and H. Geiselmann and M. C. Young for help with the analytical sedimentation experiments and analysis. This work was supported by a grant from the National Institutes of Health (GM33677). J.A.G. is a National Institutes of Health predoctoral trainee (GM07759). 1. Stewart, R. C. & Dahlquist, F. W. (1987) Chem. Rev. 87, 997-1025. 2. Macnab, R. M. (1987) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, ed. Neidhardt, F. C. (Am. Soc. Microbiol., Washington, DC), pp. 732-759. 3. Hess, J. F., Oosawa, K., Kaplan, N. & Simon, M. I. (1988) Cell 53, 79-87. 4. Borkovich, K. A., Kaplan, N., Hess, J. F. & Simon, M. I. (1989) Proc. Natl. Acad. Sci. USA 86, 1208-1212. 5. Bourret, R. B., Hess, J. F. & Simon, M. I. (1990) Proc. Natl. Acad. Sci. USA 87, 41-45. 6. Stewart, R. C., Russell, C. B., Roth, A. F. & Dahlquist, F. W. (1988) Cold Spring Harbor Symp. Quant. Biol. 59, 27-40. 7. Liu, J. & Parkinson, J. S. (1989) Proc. Natl. Acad. Sci. USA 86, 8703-8707. 8. Sanders, D. A., Mendez, B. & Koshland, D. E. (1989) J. Bacteriol. 171, 6271-6278.
Proc. NatL Acad Sci. USA 88 (1991) 9. Kehry, M. R., Doak, T. G. & Dahlquist, F. W. (1984) J. Biol. Chem. 259, 11828-11835. 10. Lupas, A. & Stock, J. (1989) J. Biol. Chem. 264, 17337-17342. 11. Stewart, R. C., Roth, A. F. & Dahlquist, F. W. (1990) J. Bacteriol. 172, 3388-3399. 12. Muchmore, D. C., McIntosh, L. P., Russell, C. B., Anderson, D. E. & Dahlquist, F. W. (1989) Methods Enzymol. 177,44-73. 13. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492. 14. Stock, A., Mottonen, J., Chen, T. & Stock, J. (1987) J. Biol. Chem. 262, 535-537. 15. Smith, R. A. & Parkinson, J. S. (1980) Proc. Natl. Acad. Sci. USA 77, 5370-5374. 16. Gill, S. C. & von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326. 17. Yphantis, D. (1964) Biochemistry 3, 297-317. 18. Gill, S. C., Yager, T. D. & von Hippel, P. H. (1991) J. Mol. Biol., in press. 19. Zamyatnin, A. A. (1972) Prog. Biophys. Mol. Biol. 24,109-123. 20. Hummel, J. P. & Dreyer, W. J. (1962) Biochim. Biophys. Acta 63, 530-532. 21. Mutoh, N. & Simon, M. I. (1986) J. Bacteriol. 165, 161-166. 22. Winzor, D. J., Loke, J. P. & Nichol, L. W. (1967) J. Phys. Chem. 71, 4492-4498. 23. Sesall, J. E., Manson, M. D. & Berg, H. C. (1982) Nature (London) 296, 855-857. 24. Kofoid, E. C. & Parkinson, J. S. (1991) J. Bacteriol., in press.
