Ligand-Specific Activation of Escherichia coli Chemoreceptor Transmethylation

Adaptation in the chemosensory pathways of bacteria like Escherichiacoli is mediated by the enzyme-catalyzed methylation (and demethylation)of glutamate residues in the signaling domains of methyl-acceptingchemotaxis proteins (MCPs). MCPs can be methylated in trans,where the methyltransferase (CheR) molecule catalyzing methylgroup transfer is tethered to the C terminus of a neighboringreceptor. Here, it was shown that E. coli cells exhibited adaptationto attractant stimuli mediated through either engineered ornaturally occurring MCPs that were unable to tether CheR aslong as another MCP capable of tethering CheR was also present,e.g., either the full-length aspartate or serine receptor (Taror Tsr). Methylation of isolated membrane samples in which engineeredtethering and substrate receptors were coexpressed demonstratedthat the truncated substrate receptors (trTsr) were efficientlymethylated in the presence of tethering receptors (Tar withmethylation sites blocked) relative to samples in which noneof the MCPs had tethering sites. The effects of ligand bindingon methylation were investigated, and an increase in rate wasproduced only with serine (the ligand specific for the substratereceptor trTsr); no significant change in rate was producedby aspartate (the ligand specific for the tethering receptorTar). Although the overall efficiency of methylation was lower,receptor-specific effects were also observed in trTar- and trTsr-containingsamples, where neither Tar nor Tsr possessed the CheR bindingsite at the C terminus. Altogether, the results are consistentwith a ligand-induced conformational change that is limitedto the methylated receptor dimer and does not spread to adjacentreceptor dimers.

INTRODUCTION

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Introduction

Bacterial chemotaxis signaling pathways are two-component systemsin which the sensors are composed of noncovalent ternary complexesof transmembrane receptor proteins, the cytoplasmic adaptorprotein CheW and the histidine kinase CheA (9, 35). The transmembranereceptors, which are also known as methyl-accepting chemotaxisproteins (MCPs), are organized as homodimers to bind ligand(28), where each subunit of the dimer consists of a periplasmicligand-binding domain (27, 49), two ${alpha}$ -helical transmembrane segments(31), and a helical cytoplasmic region that contains the highlyconserved signaling domain flanked by the methylation helices(16, 20). Receptor dimers of different ligand specificity clusterin the membrane in synergy with CheW and CheA, frequently atthe poles of the cell (26). The available evidence suggeststhat the receptors in these signaling patches possess the trimer-of-dimersorganization, first identified in the crystal structure of thecytoplasmic domain (1, 16, 41). The complexity of the organizationis further heightened by the possible involvement of interdigitatingcytoplasmic domains, which have been observed by an electronmicroscope study of receptor arrays (46). Evidence consistentwith extensive interactions among receptor dimers in the excitationand adaptation phases of signaling has been obtained from bothbiochemical analyses of reconstituted systems (21-23) and invivo analyses of chemotactic ability, signaling, and protein-proteininteractions (1, 10, 14, 38, 41). Taken together, the resultsindicate that interdimer interactions are manifested at a numberof levels, i.e., from two dimers to possibly very large arraysof dimers.

Adaptation to stimuli is mediated by reversible methylationof the MCPs through a feedback loop that involves a methyltransferase(CheR) (39), which catalyzes methyl group transfer to specificglutamyl residues in the cytoplasmic domain (33, 43), and amethylesterase (CheB), which catalyzes methyl ester hydrolysis(40). It is now appreciated that methylation can occur in trans(21, 23) because CheR tethers to serine and aspartate receptors(Tsr and Tar) through a conserved motif (NWETF) at the C terminus.From this location, CheR can more effectively catalyze methylgroup transfer from S-adenosyl-L-methionine (SAM) to the variousmethylation sites of neighboring dimers (48). In vitro studiesof receptor methylation have utilized mixtures of full-lengthand truncated receptors with the same ligand specificity (21,23), which demonstrated that efficient methylation of truncatedreceptors depended on a full-length receptor for a CheR tetheringinteraction. Methylation may occur by both intradimer and interdimerprocesses with receptors of the same ligand specificity (21),but interdimer methylation is the only plausible process availablefor the ribose-galactose and dipeptide receptors in Escherichiacoli (Trg and Tap, respectively) because receptor heterodimersdo not form (28) and Trg and Tap lack the CheR-docking siteat the C terminus (5, 19). These properties of Trg and Tap areconsistent with their low methyl-accepting activity and poorability to adapt to stimuli when they are the only receptorspresent in the cell (2, 8, 45) and also with the functionalrescue of Trg produced by genetically fusing the NWETF bindingmotif from Tsr to the C terminus of Trg (8).

In spite of the evidence for extensive interactions among receptors,it must be the case, at least to a certain extent, that ligand-specificresponses during excitation and adaptation are retained withinthe heterogeneous array of receptor signaling complexes. Thus,we designed a set of experiments to test the extent to whichthe activation of transmethylation was ligand specific by usingengineered forms of the aspartate and serine receptors (Tarand Tsr), because both of these receptors bind their respectiveattractants directly and with high specificity (4, 6, 24). Theresults provided clear evidence that CheR tethering interactionsbetween receptors of different ligand specificities were essentialfor adaptation and efficient methylation, yet the increasesin the methylation rate that resulted from attractant bindingwere communicated exclusively through receptor dimers acceptingthe methyl groups and not by the receptor dimers involved intethering the transferase. The results of these in vitro experimentsalso provided evidence that attractant binding produced a muchlarger activation factor than has been observed previously.Taken together, these results contribute to an understandingof the process of adaptation to disparate stimuli experiencedby the bacterial cell.

MATERIALS AND METHODS

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Materials and Methods

Cell strains. The E. coli strains used in this study (followed by their relevantchemotaxis genotypes) were as follows: RP437 was the wild typefor chemotaxis (32); HCB433 [ ${Delta}$ tsr(7021) ${Delta}$ trg(100) zbd::Tn5] haddeletions in the genes for the serine and ribose-galactose receptors(tsr and trg, respectively) (47); HCB316 [ ${Delta}$ tsr(7021) ${Delta}$ tar-tap(5201)]carried deletions in tsr and in the genes for the aspartateand dipeptide receptors (tar and tap, respectively) (47); HCB721[ ${Delta}$ tsr(7021) trg::Tn10 ${Delta}$ (cheA-cheY)::XhoI(Tn5)] was devoid of tar,tap, tsr, and trg as well as genes required for phosphotransferactivity in the chemotaxis system (cheA and cheW), receptormethylation (cheR), and receptor demethylation-deamidation (cheB)(7).

Expression vectors. Table 1 lists the plasmids used to express and/or coexpressTsr and Tar. The pFA plasmids shown in Table 1 were derivedfrom pHSe5 (29) or pBAD (12) to have compatible origins of replicationand independently inducible promoters. A combination of standardsubcloning (34) and site-directed mutagenesis protocols (Quickchange;Stratagene Corp.) were used to introduce tar (from pAK101R1)(18) into pFA23 and pFA24 and modify it to produce glutaminesat the four major methylation sites (295, 302, 309, and 491)(42). The resulting plasmids expressed full-length Tar (Tar_QQQQ;pFA23) or Tar with 39 amino acids truncated from the C terminus(trTar_QQQQ; pFA24). A plasmid that produced the C-terminal truncatedform of Tsr (trTsr) with the third methylation site available(trTsr_QQEQ; pFA32) was constructed from pJL31 (trTsr_QQQQ) bysite-directed mutagenesis. The tar and tsr genes in these vectorswere verified by sequencing. All plasmids were monitored forprotein production and showed bands with the expected molecularmasses on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) gels. For the behavioral studies, pBR322 was usedas a control plasmid that did not express MCP.

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TABLE 1. Plasmids used in behavioral experiments and the production of membrane samples

Swarm and swimming assays. Swarm assays were conducted in semisolid (0.3%) agar platesmade with M9 medium [0.05 M Na₂HPO₄ · 7H₂O, 0.02 M KH₂PO₄,1.0 mM NaCl, 1.0 mM MgSO₄, 5.0 mM (NH₄)₂SO₄] supplemented with0.4% glycerol, thiamine (10 µg/ml), ampicillin (150 µg/ml)and histidine, methionine, leucine, and threonine (each at 50µg/ml). The attractants aspartate, serine, and ribosewere incorporated at 100 µM. Swarm plates were inoculatedwith log-phase M9 cultures and incubated for 12 to 14 h at 30°C,at which time dark-field observation was used to measure theswarm ring radius every 2 h over a 10-h period. The averageswarm rates were determined from nine data sets.

To analyze ligand-stimulated adaptation by free-swimming bacteria,fresh overnight cultures grown in M9 medium were diluted 1:10into 5 ml of medium and were grown at 30°C until an opticaldensity at 600 nm of $~$ 0.7 was reached. The culture was preparedfor microscope observation by first diluting an aliquot withan equal volume of motility medium (0.67 M NaCl, 0.01 M KH₂PO₄,0.01 M Na lactate, 1.0 mM EDTA, 10 µM methionine, 10 µMleucine) and was then mixed with an equal amount of attractant-supplementedmotility medium. A 30-µl aliquot of the mixture was immediatelyapplied to a microscope slide within a ring of vacuum grease,which was then sealed beneath a cover glass, at which pointthe cells were videotaped under the microscope. Videotapes weresubsequently analyzed to assess adaptation from the time dependenceof tumble frequencies. Tumble events were identified as abruptcell turnings ( $~$ 0.1-s duration) followed by a smooth swimmingepisode ( $~$ 1-s duration). Tumble frequencies (per cell per second)at each time point were determined from an average over a 10-speriod for at least 10 different cells. Adaptation times wereestimated as the period of time required to achieve 50% adaptation,i.e., halfway between the tumble frequency observed immediatelyafter stimulus application and the fully adapted state. Baselinetumble frequencies were determined in control experiments involvingeach cell strain-plasmid combination used in which the dilutedculture aliquot was mixed with an equal volume of motility mediumwithout attractant. Swarm rates and adaptation times were measuredfor cell strain-plasmid combinations without added IPTG (isopropyl-ß-D-thiogalactopyranoside).

Membrane preparations. HCB721 was used to isolate membrane samples expressing and/orcoexpressing Tsr and Tar, which ensured that the receptors didnot undergo the posttranslational modifications catalyzed byCheR and CheB. Receptor-containing inner membrane fractionswere isolated on sucrose gradients as previously described (9,24). The Luria broth cultures of HCB721 containing compatibleplasmids were supplemented with ampicillin (150 µg/ml)and/or chloramphenicol (10 µg/ml) and were grown to anoptical density at 600 nm of 0.6, at which time expression wasinduced by the addition of 1 mM IPTG and/or 0.2% arabinose.Estimates of the receptor concentrations in the membrane sampleswere conducted by comparisons to an affinity-purified trTsrstandard on SDS-PAGE gels by using scanning densitometry (GS-700densitometer with Molecular Analyst version 1.4 software; Bio-Rad).

Methylation assays. Salmonella enterica serovar Typhimurium CheR was purified accordingto a published procedure (37). Samples were typically composedof a solution containing 7 µM methylatable receptor, 14µM [³H-methyl]SAM (15 Ci/mmol; Amersham Biosciences, Piscataway,N.J.), 1 µM CheR, and, as needed, a 1 mM concentrationof the attractant in 100 µl of buffer (50 mM sodium phosphate[pH 7.5], 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonylfluoride). Methylation reactions were initiated by the additionof CheR. At various times, 14-µl aliquots were removedand quenched by the addition of 3x SDS (7 µl) reducingsample buffer followed by 4 min in a boiling water bath. Aliquotsof 14 µl were resolved on SDS-PAGE gels (10% [wt/vol]acrylamide), stained for 10 min with Gel Blue Code stain (PierceBiotechnology, Inc.), and washed with water. Receptor-containingbands were excised and placed in scintillation vials containing1 ml of 1 M NaOH followed by the addition of 2 ml of scintillationfluid. The extent of tritiated methyl group incorporation wasestimated based on the assumption of 100% sample recovery. Thedependence of methylation on receptor concentration was measuredin samples containing fixed amounts of [³H]SAM and CheR (14and 1 µM, respectively) but a range of concentrationsof methylatable receptor (1.5 to 20 µM).

RESULTS

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Results

The CheR-docking segment contributes to efficient cellular adaptation and chemotaxis. Figure 1A shows the C-terminal sequences of the natural andengineered MCPs used to assess the requirement for the CheR-dockingsegment (NWETF) in adaptation and to test for the ligand specificityin the activation of methylation. The C-terminal deletions inTar and Tsr resulted in engineered forms (trTar and trTsr, respectively)that were unable to bind CheR at the docking site (23, 48),analogous to the naturally occurring form of Trg (Fig. 1A).Interdimer methylation was thus the only expected means of efficientmethylation (Fig. 1B), since the formation of receptor heterodimerswith two subunits of different ligand binding specificity hasnot been observed (28). Comparison of the swarm rates in Fig.2 supported the idea that the presence of a receptor with aCheR-docking site was necessary for efficient chemotaxis andcould provide assistance to receptors lacking the CheR-dockingsite since (i) the introduction of a receptor with the CheR-dockingsite improved the overall chemotactic ability (swarm rate ofTrg plus Tsr > Trg plus trTsr $~$ Trg [Fig. 2A]) and (ii) theswarm rate of the truncated form of Tsr (trTsr) was assistedsignificantly by the presence of full-length Tar. (The swarmrate of Trg plus trTsr in Fig. 2A was significantly less thanthe swarm rate of Tar & Tap plus trTsr in Fig. 2B.)

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FIG. 1. (A) The aligned C termini of native (Tar, Tsr, and Trg) and engineered (trTar and trTsr) MCPs, with the CheR-docking site (NWETF) shown in boldface type. (B) Cartoon that depicts engineered interdimer methylation between homodimers of trTsr and Tar (left and right, respectively). CheR is bound to the Tar dimer at the docking site (depicted as the C-terminal filled rectangle), which is fully amidated (Tar_QQQQ) at the sites of methylation (depicted as filled circles). The trTsr dimer has one site available for methylation (site 3, depicted as an open circle) and is unable to bind CheR.

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FIG. 2. Swarm rates of cells expressing either full-length or truncated serine receptor (Tsr or trTsr) in combination with Trg (A) or Tar (B) on semisolid agar plates. Panel A (left to right): swarm rates of plasmid-containing HCB316 expressing Trg (HCB316/pBR322), Trg and trTsr (HCB316/pJL21), Trg and Tsr (HCB316/pHSe5.tsr_QEQE), and also RP437/pBR322 (wild type for chemotaxis). Open bars, without attractant; striped bars, 100 µM ribose; filled bars, 100 µM serine. Panel B (left to right): swarm rates of plasmid-containing HCB433 expressing Tar and Tap (HCB433/pBR322); Tar, Tap, and trTsr (HCB433/pJL21); Tar, Tap, and Tsr (HCB433/pHSe5.tsr_QEQE); and RP437/pBR322. Open bars, without attractant; striped bars, 100 µM aspartate; filled bars, 100 µM serine. Uncertainties are standard deviations.

Adaptation to attractants by free-swimming cells expressingvarious combinations of Trg, trTsr, Tsr, and Tar provided evidencefor the universal nature of the assistance by receptors witha CheR-docking segment (Fig. 3 and Table 2). Adaptation timeswere used as indicators of the relative efficiency of receptormethylation (Fig. 3). The Tar⁺ HCB433 cells exhibited full adaptationto aspartate, whether the cells were Tsr^– or also expressedtrTsr or Tsr (Fig. 3A). HCB433 also responded and adapted partiallyto serine when trTsr (or Tsr) was present (Fig. 3B), which wasconsistent with functional assistance from Tar. It was notedthat the extents of adaptation to aspartate and serine weresignificantly different; full adaptation to aspartate was observed,while adaptation to serine was partial. Partial adaptation toserine has been observed in a previous study with wild-typechemotaxis strain AW405 (3). We also observed partial adaptationwith wild-type strain RP437 (data not shown). Thus, partialadaptation seems to be a normal feature of the serine responseand not an artifact of either chromosome-versus-plasmid-basedtsr expression or the C-terminal truncation. Consequently, thesimilar adaptation times in cells expressing either trTsr orTsr with Tar were interpreted as evidence of functional interactionwith Tar in the interdimer methylation of trTsr.

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FIG. 3. Tumble frequencies as a function of time after the introduction of 50 µM aspartate (A) or 50 µM serine (B). HCB433/pBR322, ${square}$ (Tap and Tar); HCB433/pHSe5.tsr_QEQE, ${circ}$ (Tap, Tar, and Tsr); HCB433/pJL21, ${triangleup}$ (Tap, Tar, and trTsr); HCB433/pBR322 (no attractant control), ${blacksquare}$ (A). The curves drawn through the data are either least-square lines or sigmoid functions to help guide the eyes.

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TABLE 2. Adaptation times of swimming cells

Table 2 summarizes the adaptation times of swimming cells toaspartate, ribose, and serine stimuli. The adaptation timeswere readily divided into two groups, which correlated withthe expected efficiencies of methyl group addition to the receptorsconveying the attractant stimuli. Receptors were regarded asefficiently methylated when the adaptation times were comparableto those of wild-type cells (Table 2, samples 10 to 12). Incontrast, adaptation was not observed with inefficiently methylatedreceptors. In every situation where adaptation was observed,the receptor conveying the attractant stimulus either had aCheR-docking site or was coexpressed with a receptor with thedocking site. Conversely, adaptation was not observed when receptorswithout the CheR-docking segment were the only receptors presentin the cell (Trg and trTsr [Table 2, samples 7 and 9]). Specifically,adaptation to the ribose stimulus sensed through Trg was assistedby the presence of Tsr but not trTsr (Table 2, sample 8 versussample 9), a result that was in agreement with previous observationsof adaptation to ribose stimuli, where the presence of Tar and/orTsr was required for efficient adaptation (13). The data inTable 2 extended the generality of the result, since adaptationto serine could be conveyed through trTsr when Tar was alsopresent (Table 2, sample 5 versus sample 7). Overall, the observationsmade in these swarm and adaptation assay experiments were consistentwith promiscuous interdimer methylation; i.e., any receptorconveying the signal generated by ligand binding could be assistedby any receptor with a CheR-docking site irrespective of ligandbinding specificities of the two receptor dimers involved. Themost important factor contributing to efficient adaptation (andthus methylation) was the presence of at least one receptorwith a CheR-docking segment.

Ligand specificity of transmethylation. In vitro methylation was used to assess the extent of the interactionsamong receptor subunits by examining the influence of attractantsin inner membrane samples with coexpressed Tar_QQQQ and trTsr_QQEQ(trTsr_QQEQ/Tar_QQQQ). These inner membrane samples, preparedfrom HCB721, contained MCPs that were in defined levels of covalentmodification (Fig. 4) and in comparable amounts when two MCPswere coexpressed (Fig. 4, lanes 3 and 5). Methylation of thesesamples was expected to proceed by the interdimer process depictedin Fig. 1B, since Tar_QQQQ provided the transferase-docking sitebut lacked an available major methylation site and trTsr_QQEQhad one available major methylation site (E311) but lacked thetransferase-docking site. To test for ligand-specific responses,the methylation of trTsr_QQEQ/Tar_QQQQ-containing membranes wasmeasured in the absence of attractant and in the presence ofserine, aspartate, or both serine and aspartate (Table 3). Themethylation rate increased significantly when serine was presentand to a similar extent when both serine and aspartate werepresent, but it did not increase significantly when aspartatewas the only ligand added (Fig. 5A; Table 3, samples 1 to 4).This increase was evident in ratios of the serine receptor methylationrates with attractant(s) to the rate without attractant (2.0and 1.9 versus 1.1, respectively, in Table 3). A control sampleconsisting of coexpressed trTsr_QQQQ/Tar_QQQQ was used to measureincorporation at the minor methylation sites, which are knownto contribute to Tsr methylation (33). The structural similarityof Tsr cytoplasmic fragments in the QEQE and QQQQ modificationstates determined by X-ray crystallography (17) suggests thattrTsr_QQQQ is well suited as a control for trTsr_QQEQ in theseexperiments, although it is possible that the cytoplasmic domainsin the intact receptor molecules adopt different structures.The methylation rate of the trTsr_QQQQ/Tar_QQQQ sample was notaffected by either serine or aspartate (data not shown); thus,the rate reported in Table 3 (0.31 pmol/min), which representedan average of several experiments conducted with and withoutligand, was subtracted as background. The background-subtractedrates indicated that the contribution to methylation from theminor sites was significant. Thus, in spite of the greater uncertaintyin ratios calculated with the background-subtracted data (dueto the small value for the background-corrected rate in theabsence of serine), it seemed that the rate increase at site3 produced by serine binding was likely to be larger than theratios calculated with the uncorrected rates suggested.

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FIG. 4. SDS-PAGE (12.5% gels) of inner membrane samples expressing Tsr and/or Tar. Lanes: 1, molecular weight markers (in thousands); 2, Tar_QQQQ; 3, coexpressed Tar_QQQQ and trTsr_QQEQ; 4, trTsr_QQEQ; 5, coexpressed trTar_QQQQ and trTsr_QQEQ.

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TABLE 3. Initial rates of serine receptor methylation

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FIG. 5. Methylation of inner membranes coexpressing Tar and Tsr. (A) Coexpressed Tar_QQQQ and trTsr_QQEQ (filled symbols) and a control sample (coexpressed Tar_QQQQ and trTsr_QQQQ [ ${star}$ ]). (B) Coexpressed trTar_QQQQ and trTsr_QQEQ (open symbols). ${blacksquare}$ and ${square}$ , no ligand; • and ${circ}$ , 1 mM serine; ${blacktriangleup}$ and ${triangleup}$ , 1 mM aspartate; ${blacktriangledown}$ and ${triangledown}$ , both serine and aspartate. Reaction conditions included a solution containing 7 µM methylatable receptor (trTsr_QQEQ), 1 µM CheR, and 14 µM [³H]SAM.

The third site (E311) was chosen at the outset of these studiesto be the only major site available for methylation, based onprevious studies of Tar in which site 3 was found to be themost rapidly methylated site by far (44) and on the significanthomology between Tar and Tsr in the methylation helices (33,43). However, rate measurements at the major sites in full-lengthTsr have demonstrated that site 4 is methylated over 10-foldmore rapidly than either site 2 or site 3 (in Tsr_QQQE, Tsr_QEQQ,and Tsr_QQEQ samples, respectively) (A. Chalah and R. M. Weis,unpublished data). Not only were comparable methylation ratesobserved for trTsr_QQEQ/Tar_QQQQ (Table 3, sample 1) and Tsr_QQEQsamples (data not shown) under similar assay conditions, themethylation rates of trTsr_QEQE/Tar_QQQQ and trTsr_QQEQ/trTar_QQQQdiffered by $~$ 8-fold (Table 3, samples 1 and 9), which was qualitativelyconsistent with the rate difference in the full-length Tsr molecules.Also, the lack of a significant increase in the methylationrate upon the addition of serine in the trTsr_QEQE/Tar_QQQQ samples(Table 3, samples 9 to 12) mirrored the behavior of (full-length)Tsr_QQQE, which showed no demonstrable increase in the methylationrate with serine (Chalah and Weis, unpublished). Thus, the methylationrate of trTsr_QEQE/Tar_QQQQ could be plausibly attributed to rapidmethylation at site 4, which was unaffected by serine. Fortuitously,serine produced a more pronounced effect on the methylationrate at site 3 (E311) in these transmethylation experiments.

Ligand-specific stimulation does not require CheR tethering. To test the requirement of the docking segment for efficientmethylation and ligand-specific stimulation, methylation assayswere performed on coexpressed Tar and Tsr samples in which neitherMCP possessed the CheR-docking site (trTsr_QQEQ/trTar_QQQQ) (Fig.5B; Table 3, samples 5 to 8). The addition of serine producedthe same proportional rate increase in the trTsr_QQEQ/trTar_QQQQsamples as in trTsr_QQEQ/Tar_QQQQ, although the absolute ratewas 10-fold lower with the doubly truncated samples (note thedifferent y axis scales in Fig. 5A and B). Overall, the doublytruncated samples displayed the same ligand-specific responseas the trTsr_QQEQ/Tar_QQQQ-containing membranes. The lower methylationrates of the doubly truncated receptor samples (Table 3, samples5 to 8) compared to the control sample for the minor methylationsites (trTsr_QQQQ/Tar_QQQQ) (Table 3) underscored the requirementof a CheR-docking segment for efficient receptor methylation.In any event, the corresponding background (trTsr_QQQQ/trTar_QQQQ)sample was not prepared or tested due to the already low initialrate observed with trTsr_QQEQ/trTar_QQQQ.

Further evidence for the significant difference in the methylationefficiency of trTsr_QQEQ/trTar_QQQQ from that of trTsr_QQEQ/Tar_QQQQwas obtained from initial rate measurements conducted as a functionof the trTsr_QQEQ concentration, which was produced by varyingthe total amount of membrane (Fig. 6). The trTsr_QQEQ/Tar_QQQQdata resulted in similar K_m values in the absence and presenceof serine (4 and 3 µM, respectively), and the V_max increasedby $~$ 2-fold. trTsr_QQEQ/trTar_QQQQ samples could not be preparedat sufficiently large concentrations to estimate V_max and K_mvalues independently, yet the low rates of methylation in thesesamples were plausibly explained by fits in which the V_max wasfixed to the values obtained with coexpressed trTsr_QQEQ/Tar_QQQQ,which led to larger K_m values (>100 µM). Altogether,the properties of trTsr_QQEQ/Tar_QQQQ and trTsr_QQEQ/trTar_QQQQsamples were consistent with a ligand-specific activation ofreceptor methylation within the receptor dimer accepting themethyl groups. These properties were consistent with the hypothesisthat the binding of CheR to the receptor via NWETF served toincrease the effective CheR concentration near the sites ofmethylation.

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FIG. 6. Methylation rates as a function of the trTsr_QQEQ concentration in trTsr_QQEQ/Tar_QQQQ samples in the absence of ligand ( ${blacksquare}$ ) and in the presence of 1 mM serine (•) and in trTsr_QQEQ/trTar_QQQQ samples in the absence of ligand ( ${square}$ ) and in the presence of 1 mM serine ( ${circ}$ ) are shown. Samples at the different trTsr_QQEQ concentrations also included 1 µM CheR and 14 µM [³H]SAM.

DISCUSSION

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Discussion

The chemoreceptor clusters localized in the inner membrane ofE. coli are probably heterogeneous with respect to ligand bindingspecificity (41). Within this heterogeneous environment, adaptationis achieved by modulating the rates of receptor methylationand demethylation in response to changes in the concentrationof attractant. An increase in the attractant concentration speedsmethylation (11) and also slows demethylation transiently byinhibiting CheA-mediated phosphorylation (and activation) ofCheB until adaptation is reestablished (15, 25). Thus, ligand-specificadaptation may be achieved by ligand-specific increases in methylation,ligand-specific decreases in demethylation, or both. Such ligand-specificeffects must be at work in addition to the global influencesthat have been proposed to contribute to adaptation, e.g., aglobal activation of methylesterase activity brought about byCheA-mediated phosphorylation of CheB (15).

Here, evidence that defines the features of ligand-specificreceptor methylation is presented. The present results, togetherwith those of previous studies (8, 21, 23), indicate that efficientreceptor methylation can occur as long as a receptor that possessesthe CheR-docking segment (NWETF) (30, 48) is also present. Ithas been shown previously that methylation rates of truncatedreceptors, when coexpressed with full-length receptors of thesame type, e.g., trTar/Tar and trTsr/Tsr, are similar to therates measured with their full-length counterparts (21, 23).Moreover, the methylation of Tsr in trTsr/Tar_QQQQ samples (Table3) and in full-length Tsr samples (Chalah and Weis, unpublished)occurs at similar rates. The CheR-docking segment is requiredfor efficient methylation and adaptation. When all the MCPsin a membrane sample lack the CheR-docking segment, methylationrates are decreased more than 10-fold compared to samples inwhich at least one of the MCPs has the docking segment (Table3 and Fig. 6) (2, 21). Also, cells fail to adapt to an attractantstimulus in behavioral assays whenever all the MCPs lack theCheR-docking segment (Table 2) (13). Altogether, the resultsof these studies reinforce the notion that interdimer methylationcan occur between receptor dimers of any ligand specificity;e.g., Tsr can be assisted by Tar, Trg can be assisted by eitherTsr or Tar, etc. The molecular basis for this interaction isinterpretable within the framework of interdimer methylation(21, 23), the trimer-of-dimer organization (36) that was firstidentified in crystals of the Tsr cytoplasmic domain (16), andin vivo evidence which has been obtained through site-directedmutagenesis and site-specific cross-linking (1, 41).

By utilizing an engineered form of Tsr with one available methylationsite, but without the CheR-docking segment (trTsr_QQEQ), andan engineered form of Tar with the CheR-docking segment, butwithout a major methylation site available (Tar_QQQQ), the individualand collective influences of attractant binding to the substrate(trTsr_QQEQ) and tethering (Tar_QQQQ) receptors could be tested.Two possible effects of attractant binding are depicted in Fig.7, which we imagine could be at work within the context of acluster of receptors and interdimer methylation. First, a ligand-inducedchange in proximity could lead to an increase in the methylationrate by reducing distances between receptor dimers, therebygiving tethered CheR molecules access to a larger number ofmethylation sites (Fig. 7A). Second, a ligand-induced changein receptor conformation could improve the ability of the receptorto function as a substrate (Fig. 7B). The experimental resultssupport a ligand-induced change in conformation, akin to theprocess depicted in Fig. 7B.

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FIG. 7. Illustrations of ligand-induced changes in methylation based on (A) changes in proximity and (B) changes in receptor dimer conformation. Each pair of circles represents a receptor dimer cytoplasmic domain in cross section (Tar_QQQQ, light gray; trTsr_QQEQ, dark gray), where the sites of methylation are depicted as circles on the surface of the domain (available, open circles; blocked, filled circles). Serine (Ser, ${diamond}$ ) and aspartate (Asp, ) are shown bound to the dimer interface.

Figure 7A serves to illustrate that ligand-induced changes inreceptor proximity might be somewhat ligand specific withinthe heterogeneous receptor (dimer) environment, where bindingaffects the proximity in the immediate neighborhood of receptorsbinding attractant. Yet, according to this line of reasoning,aspartate as well as serine should alter the proximity betweenTar_QQQQ and Tsr_QQEQ subunits and consequently should also influencethe methylation rate. This was not observed, as only serineproduced an increase in methylation (Fig. 5 and Table 3). Aligand-specific effect can be more strictly maintained whena propagated conformational change is confined to the dimerthat binds ligand, which improves the methylation rate onlyon that dimer. Figure 7B illustrates one possible way in whichligand binding can influence the receptor dimer as a changein the relative orientation of the methylation helices withinthe cytoplasmic domain, which positions the methyl-acceptingglutamyl residues more favorably for methylation. A mechanismof this kind is consistent with the following experimental observations.(i) Only the attractant that binds to the methyl-accepting dimer(trTsr_QQEQ) increases the methylation rate, whereas the attractantthat binds to the tethering dimer (Tar_QQQQ) does not. (ii) TheCheR tethering interaction is not required to observe ligand-specificincreases in the methylation rate. Although the overall efficiencyof methylation is lower when no tethering subunits are present,as in the trTsr_QQEQ/trTar_QQQQ samples, ligand-specific increasesin the methylation rate are still observed.

A consistent mechanism of receptor methylation emerges in whichreceptor dimers are near enough to mediate efficient interdimermethylation via the CheR tethering interaction but in whichligand-specific effects are confined to the receptor dimer thatbinds ligand. Thus, the process of receptor methylation representsa ligand-specific process not regulated at a global level; incontrast, the regulation of the kinase CheA seems to be distributedthroughout a receptor array, one that is sensitive to both theextent of ligand binding and the level of covalent modification.

ACKNOWLEDGMENTS

We thank Anthony Shrout for purified CheR. We also appreciatehelpful discussions with Abdalin Asinas, Tatiana Besschetnova,Anas Chalah, David Montefusco, Anthony Shrout, and Li Zhi.

This work was supported by the U.S. Public Health Service NIHgrant GM53210 to R.M.W.

FOOTNOTES

* Corresponding author. Mailing address: Department of Chemistry, LGRT 701, 710 North Pleasant St., University of Massachusetts, Amherst, MA 01003-9336. Phone: (413) 545-0464. Fax: (413) 545-4490. E-mail: rmweis{at}chem.umass.edu.

Present address: Pfizer Central Research, Candidate EnhancementGroup, Groton, CT 06340.

REFERENCES

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