Chemotaxis Receptors: A Progress Report on Structure and Function

Chemotaxis Receptors: A Progress Report on Structure and Function

Sherry L. Mowbray*,1 and Mats O. J. Sandgren†

Department of Molecular Biology, *Swedish Agricultural University, and †Uppsala University, Box 590,Uppsala Biomedical Centre, S-751 24 Uppsala, Sweden

Received July 17, 1998, and in revised form September 30, 1998

Recent biochemical and structural studies haveprovided many new insights into the structure andfunction of bacterial chemoreceptors. Aspects oftheir ligand binding, conformational changes, andinteractions with other members of the signalingpathway are being defined at the structural level. Itis anticipated that the combined effort will soonprovide a detailed, unified view of an entire re-sponse system. r 1998 Academic Press

Key Words: chemotaxis; receptor; periplasmic bind-ing proteins; bacterial proteins.

INTRODUCTION

Chemotaxis, the movement of cells in response tochemical attractants and repellents, is an importantsurvival strategy for many bacterial species and afrequent feature in bacterial pathogenesis. The pro-cess requires the cooperative function of proteinsthat receive chemical signals and use the informa-tion to direct appropriate swimming behavior. Thepresent review will concentrate on the gram-nega-tive organisms Escherichia coli and Salmonella typhi-murium, because the most is known for these sys-tems, and because the principles are applicable tomost others. Excellent general reviews include Blair(1995), Stock and Mowbray (1995), Stock and Surette(1996), and Falke et al. (1997). Particular emphasiswill be focused here on the receptors and theirrelationships to other components of the signalingsystem.

Flagella can rotate either clockwise or counterclock-wise; the former generates nonproductive tumbles,and the latter, smooth swimming. Control of thedirection of flagellar rotation turns simple motilityinto chemotaxis. In the absence of stimuli, a series ofsmooth swims separated by short tumbles take abacterium on a random walk through its surround-ings. When cells detect an increasing concentration

of an attractant (or decreasing concentration ofrepellent), they are more likely to swim smoothly.Gradients in the opposite direction cause a greaterfrequency of tumbling. Thus the bacteria spend moretime moving in directions they perceive as favorable.

The proteins required for chemotaxis in E. coli areshown in Fig. 1. Initial recognition of an effectormolecule requires its binding to a specific receptor onthe outside of the cell. Some attractants bind first toa soluble primary receptor, or binding protein, re-cruited from the ABC transport system (Higgins,1992; Tam and Saier, 1993; Boos and Lucht, 1996).These ligand-loaded binding proteins can interactwith, and activate, a particular membrane-boundreceptor. Other attractants and repellents are able tobind directly to specific membrane receptors. Ineither case, it is the membrane-bound protein thatactually carries the signal to the cytoplasm andconveys the proper instructions to the rest of thesystem. All membrane receptors trigger the sameintracellular events.

Signal processing and output involve a phosphore-lay system. The activity of CheA, a cytoplasmichistidine kinase, is modulated by its interactionswith the membrane receptor. CheA is inhibited whenan attractant is bound to the receptor and stimu-lated when a repellent signal is given. When acti-vated, CheA can autophosphorylate (using ATP) andthen pass the phosphate groups to either of twoaspartate autokinases involved in signal output(CheY) and adaptation (CheB). CheW couples CheAto chemoreceptor control by physically linking theproteins in a complex. CheY, the tumble or responseregulator, catalyzes its own acceptance of a phos-phate group from CheA on an aspartic acid residue.The default state of a flagellum is smooth swimmingin the absence of bound CheY. Only phosphorylatedCheY interacts with a flagellum and generates atumble. CheZ controls the level of phosphorylatedCheY by accelerating its rate of autodephosphoryla-tion; thus tumbles rapidly cease when CheA isturned off.

Adaptation resets the system by changing the1 To whom correspondence should be addressed. Fax: (46)-18–

536971. E-mail: [email protected].

JOURNAL OF STRUCTURAL BIOLOGY 124, 257–275 (1998)ARTICLE NO. SB984043

257 1047-8477/98 $25.00Copyright r 1998 by Academic Press

All rights of reproduction in any form reserved.

signaling (kinase modulating) properties of the recep-tor through reversible methylation. Methylated re-ceptor is a more efficient activator of the kinase. Twoproteins work in tandem to determine receptor meth-ylation under particular conditions. CheR, a methyl-transferase, continuously transfers methyl groupsfrom S-adenosylmethionine molecules to specific siteson the membrane receptor. CheB, the methylester-ase, specifically removes methyl groups from thereceptor. CheB becomes activated by accepting aphosphate group from CheA; this control largelydetermines the observed level of methylation at anytime. The cooperative function of these proteins inchemotaxis is illustrated in Fig. 1.

Chemotaxis includes the first-discovered and best-characterized two-component regulatory system.Similar transmitter (histidine kinase, CheA-like)and receiver (aspartate kinase, CheY-like) modulesare found with many different signaling roles inprokaryotes and eukaryotes (Stock et al., 1995; Falkeet al., 1997; Wurgler-Murphy and Saito, 1997). Thesesystems are indeed so closely related that cross-talkwith the chemotaxis proteins can be demonstrated insome cases, as with the nitrogen regulation system(Ntr; Ninfa et al., 1988).

PERIPLASMIC RECEPTOR STRUCTURE

Only some of the soluble receptors from the ABCtransport systems can participate in chemotaxis.The chemoreceptors of E. coli include the periplas-mic binding proteins for the attractants ribose (RBP),glucose/galactose (GBP), maltose/maltodextrins(MBP), and dipeptides (DBP); a recent report impli-cates the nickel-binding protein (NiBP) in repellentresponses through Tar (De Pina et al., 1995). Theirinteractions with particular transmembrane recep-tors are summarized in Fig. 2. The soluble receptorsare inducible, with final concentrations of 1 mM ormore in the periplasm (Manson et al., 1985). MBPhas been shown to be concentrated near the poles ofthe cell after induction (Dietzel et al., 1978).

A great deal is known about the structures of theperiplasmic binding proteins (for a recent review, seeQuiocho and Ledvina, 1996). Three general classeshave been characterized, with each including one ormore chemoreceptors. RBP and GBP are similar instructure, while MBP (Fig. 3) has a different foldingtopology; DBP and NiBP resemble MBP with anextra domain introduced at the N-terminus of each.Only one small, approximately 60-residue segment

FIG. 1. A response cycle. In the absence of any stimulus, receptors (MCPs) activate the CheA kinase slightly, so that some tumblesoccur and some methylated MCP is present. Binding of attractant (either directly or via a binding protein, BP) causes the MCP to inhibitCheA; the resulting reduction in the level of phosphorylated CheY (assisted by CheZ) allows the flagella to return to their defaultsmooth-swimming state. CheB also loses its phosphate group, reducing methylesterase activity. MCPs eventually become more highlymethylated (by CheR), which allows them to regain their kinase-stimulating activity. The slow time scale of methylation permits smoothswimming to persist for a short time. Removal of the attractant results in MCP that efficiently stimulates CheA kinase activity, and morefrequent tumbles occur. The CheB esterase eventually reduces the methylation of the receptor to the original, prestimulus state. Repellentswork in the opposite manner. The system is extremely sensitive; the binding of a single chemical effector at the surface of the cell can alterof the behavior of the entire cell (Segall et al., 1986).

258 MOWBRAY AND SANDGREN

(corresponding to the N-terminal residues of MBP) issignificantly conserved in sequence and structureamong the three classes; this portion of the periplas-mic receptors has been implicated in transport ratherthan chemotaxis. Despite differences in folding, allhave two globular domains connected by a flexible,multistranded hinge; the structure of MBP illus-trates this in Fig. 3. Monomers are observed insolution studies, even at high protein concentrations(Shilton et al., 1996).

Conformational changes of the binding proteinsare essential for their function. Structures of RBP(Mowbray and Cole, 1992; Bjorkman and Mowbray,1998), MBP (Spurlino et al., 1991; Sharff et al., 1992)and DBP (Dunten and Mowbray, 1995; Nickitenko etal., 1995) have been solved in both closed, ligand-bound and open, ligand-free forms. The two statesdiffer by a large, almost rigid-body rotation of thedomains relative to each other. Comparison of theknown structures of open and closed binding pro-

FIG. 2. Periplasmic and membrane chemoreceptors of E. coli. The membrane-bound MCPs, Tar (aspartate receptor), Tsr (serinereceptor), Trg (ribose/glucose/galactose receptor),and Tap (dipeptide receptor), can interact with some ligands directly, and other responsesrequire the periplasmic binding proteins, MBP (maltose-binding protein), GBP, (glucose/galactose-binding protein), RBP (ribose-bindingprotein), or DBP (dipeptide-binding protein). The repellent response to nickel has been reported to work through NiBP (nickel-bindingprotein) and Tar.

259CHEMOTAXIS RECEPTORS

teins using the small conserved portion has shownthat each of the opening motions involves a similarrotation axis passing near the hinge (Flocco andMowbray, 1998). Each open form is stabilized bycontacts between the two domains on the outside ofthe protein, just as closed forms are stabilized byinteractions of each domain with the ligand (Floccoand Mowbray, 1998).

The primary function of these large conforma-tional changes is to provide distinct species thathave distinguishable interactions with chemotaxisand transport partners. The population of forms insolution, however, includes closed ligand-free recep-tors (Flocco and Mowbray, 1994), open ligand-boundreceptors (Sack et al., 1989), and probably manyintermediate (Bjorkman and Mowbray, 1998) and

FIG. 3. Conformational changes of MBP. Open, ligand-free (Sharff et al., 1992) and closed, ligand-bound (Spurlino et al., 1991) forms ofMBP (N-terminal domain at right) with the regions implicated in chemotaxis marked in gray.


less-defined species (Butler and Falke, 1996; Care-aga and Falke, 1992). Ligand should probably beviewed as increasing the population of similar closedforms, rather than causing one all-or-nothing confor-mational change. The exact nature of the open formis probably unimportant, as its function is mostly toreduce the competition of ‘‘inappropriate’’ forms forthe attention of membrane components of chemo-taxis and transport.

MEMBRANE RECEPTOR STRUCTURE

Most transmembrane signaling is carried out by aset of homologous proteins often called MCPs2

(methyl-accepting chemotaxis proteins) because oftheir adaptational methylation. Four closely relatedMCPs are found in E. coli. The responses they directand their specific interactions with soluble receptorsare summarized in Fig. 2. Salmonella has a slightlydifferent set, with Tap replaced by Tcp, a citrate/phenol sensor (Yamamoto and Imae, 1993).

Tar, the Aspartate Receptor

The aspartate receptor, Tar, serves as the primaryexample here. This 60-kDa protein can generatesignals in response to direct binding of the at-tractants aspartate and glutamate (Clarke and Kosh-land, 1979). Although Tar from E. coli and Salmo-nella have 79% sequence identity, only E. coli Tarcan respond to MBP-mal as an attractant (Dahl andManson, 1985; Mizuno et al., 1986) and the ions Ni21

and Co21 as repellents (Reader et al., 1979). A role inthermosensing is shared with the serine receptor,Tsr (Mizuno and Imae, 1984). There are about 2500copies of Tar per cell (Clarke and Koshland, 1979).The absence of cysteine residues in wild-type Tar hasenabled the use of cysteine mutants to test functionand proximity of particular residues.

Homodimers

That dimers are the smallest functional unit of Tarstructure was first established by disulfide cross-linking (with cysteines introduced by mutation) andsedimentation measurements. Neither the bindingof aspartate and nor the methylation altered theobserved size, although aspartate did prevent ex-change of subunits between dimers (Milligan andKoshland, 1988). Tetramers had been previouslyreported as a major species in the membrane usingmore promiscuous cross-linking techniques (Chelskyand Dahlquist, 1980) and may represent a validmechanism for subunit exchange in the absence ofligand. There is no evidence that heterodimers of Tar

can form with other MCPs; even heterodimers ofSalmonella and E. coli Tar are not observed (Milli-gan and Koshland, 1988).

Basic Structure

That the MCPs are remarkably flexible was sug-gested by disulfide trapping experiments with theperiplasmic region of Tar (Falke and Koshland,1987), which were later extended to the transmem-brane and cytoplasmic regions (Danielson et al.,1997). To a first approximation, any introducedcysteine can eventually cross-link with any other inthe vicinity. This flexibility has made structuralwork with these proteins extremely difficult.

No x-ray structure exists for the intact molecule,and electron microscopic studies have met withlimited success. Two-dimensional crystals of Trg(Barnakov et al., 1994) exhibited a P4 symmetry,which is difficult to consolidate with a dimeric struc-ture. It was proposed that the two-dimensionalcrystals might represent an antiparallel arrange-ment of two symmetrical dimers that shows appar-ent fourfold symmetry in the averaged projection.The 150-A spacing in multilamellar stacks indicatesthat the maximum length for any single solubleportion of the receptor is 120 A (assuming a30-A-thick membrane).

Early circular dichroic measurements showed thatthe helical content of Tar is very high (,80%; Fosteret al., 1985), a theme that has persisted in all laterstudies. Most available structural information hasresulted from a determined divide-and-conquer strat-egy utilizing a variety of methods. The compositeview of the Tar dimer in Fig. 4 thus represents thesynthesis of many results. In the descriptions of theindividual regions, a prime (8) notation will be usedto designate the second subunit of a dimer.

N-terminus. The N-terminal methionine of Taris blocked with a formyl group (Milligan and Kosh-land, 1990). The small N-terminal cytoplasmic seg-ment of Tar (residues 1–6) is believed to be helical,with residues 4 and 48 closest together near thedimer interface (Stoddard et al., 1992). Although the4/48 distance is reduced slightly on binding aspartate(Stoddard et al., 1992), this portion of the receptorcan be altered greatly without major functionaleffects (Chen and Koshland, 1995).

Periplasmic region. The role of the periplasmicregion in ligand-binding (Tar residues 31–188) isreflected in the relatively low level of sequenceconservation in this part of the MCPs; bindingdifferent chemoeffectors requires variations in se-quence, although the same basic structure is main-tained.

This portion of the Tar structure is well character-ized. An 18-kDa soluble fragment of the Salmonella

2 Abbreviations used: MCP, methyl-accepting chemotaxis pro-tein; TM1, transmembrane segment 1; TM2, transmembranesegment 2.


protein was produced and shown to bind aspartatewith high affinity (Milligan and Koshland, 1993).Although monomers predominated at low proteinconcentrations in the absence of aspartate, highprotein concentrations or the presence of aspartatefavored the formation of dimers. X-ray structures ofperiplasmic fragments of Salmonella and E. colihave been solved (Milburn et al., 1991; Bowie et al.,1995; Yeh et al., 1996). This fragment of Tar is asymmetric dimer in the absence of ligand. Eachsubunit is composed of an antiparallel four-helixbundle roughly 20 A in diameter, with a1 and a4making extensive contacts throughout their 70-Alength (Milburn et al., 1991; Yeh et al., 1993; Bowie etal., 1995). NMR studies of a periplasmic piece of Tarhave confirmed that the x-ray structure is a goodmodel for the MCP in solution (Danielson et al.,1994).

These structures have provided the basis for mod-eling the structure of Tsr (Jeffery and Koshland,1993). Studies of intact, membrane-bound Tsr bysolid-state NMR validate its use (Wang et al., 1997).

Transmembrane segments. Two segments of Tarcross the membrane: residues 7–30 (TM1; immedi-ately before a1 of the periplasmic region) and resi-dues 189–212 (TM2; directly following a4). By ex-trapolation from the structure of the periplasmicdomain, the membrane segments of the dimer wereproposed to form a pseudo four-helix bundle (Mil-burn et al., 1991). This model is consistent with theresults of cross-linking studies with introduced cyste-ine residues (Lynch and Koshland, 1991; Pakula andSimon, 1992; Chervitz et al., 1995; Chervitz andFalke, 1995). Both transmembrane segments show aclear helical pattern. TM1s provide the closest asso-ciation on the dimer interface, and clear contactsexist between TM1 and TM2 within each subunit.Similar data have been obtained for Trg, both in vivoand in vitro (Lee et al., 1994; Lee and Hazelbauer,1995; Hughson et al., 1997).

TM1s and the cytoplasmic residues immediatelyfollowing them are thought to form a continuouscoiled coil with a1/a18 (Scott and Stoddard, 1994).TM2s appear to be normal a-helices (Chervitz andFalke, 1996) that make contact with the TM1s oftheir respective domains, but not with each other.

Cytoplasmic region. The cytoplasmic region isabout 37 kDa in size and includes residues 213–553

of Tar. This is the most conserved part of the MCPs(sequence identity ,70% between Tar and Tsr),reflecting the fact that all receptors function withinthe same context of intracellular signaling/adapta-tion. It is also the part of the receptor that is leastunderstood.

No X-ray or NMR structure has been obtained forany portion of the cytoplasmic region, apparentlybecause it is highly dynamic (Seeley et al., 1996). Thebroad picture available at present has therefore beenderived from a number of different types of studies.Circular dichroic measurements again showed alarge helical component (Mowbray et al., 1985).Previous success in predicting the general arrange-ment of the periplasmic region (Moe and Koshland,1986) led to a similar attempt for the cytoplasmicpart of the receptors. Sequence comparisons com-bined with secondary structure prediction producedthe helical assignments that are shown in Fig. 4 (LeMoual and Koshland, 1996; Danielson et al., 1997)and used as navigational aids in the following discus-sion. These helices are illustrated in proportion totheir predicted length (on the same scale as theperiplasmic fragment) and in the ‘‘dimeric’’ arrange-ment suggested by experimental data, although noparticular model is implied for the disposition of a7and a8. The available data on size (Long and Weis,1992a; Barnakov et al., 1994; Liu et al., 1997) hintthat a7 and a8 are not fully extended and may foldback in some way onto a6 and a9.

Residues corresponding to 213–259 of Tar arecommonly referred to as the linker. This region issomewhat conserved (Le Moual and Koshland, 1996),and its integrity is critical to receptor function(Danielson et al., 1997; Ames et al., 1988; Ames andParkinson, 1988; Gardina and Manson, 1996). Cyste-ine and disulfide scanning (Butler and Falke, 1998)indicated that it includes two amphiphilic helices,one of which is an extension of a4, while the other(a5) is at the dimer interface. Helix a4 includes aconserved proline that may encourage a kink. Theregion between a4 and a5 (229–242) is apparentlymore ordered and less exposed than either of thenearby helices; other studies indicate that this is ahot spot for mutations that disrupt signaling (Ameset al., 1988).

A protease-accessible site at residue 259 marksthe end of a5 (Mowbray et al., 1985). Although such

FIG. 4. Composite structure of a Tar dimer. Various experimental information was combined with the x-ray structure of theaspartate-bound periplasmic region (Brookhaven Protein Data Bank entry 1WAT) to give an overall schematic. The helical regionsobserved in the x-ray structure, combined with those suggested for TM1 and TM2, are a1 (4–76), a2 (86–109), a3 (116–144), and a4(146–212). A single bound aspartate is shown as a space-filling model. The additional helical regions proposed by Le Moual and Koshland(1996) and Danielson et al. (1997) are shown at approximately the same scale as the periplasmic part, assuming a rise/residue of 1.5 A: a4(212–228), a5 (247–261), a6 (265–337), a7 (345–387), a8 (400–426), and a9 (438–516). Helices a6 and a68 are shown coiled around eachother with a pitch of ,140 A. Methylation sites at residues 295, 302, 309, and 491 are indicated by stars.


protease cleavage is abolished by conditions thatfavor helix formation (such as high concentrations ofglycerol), analysis of sequences (Le Moual and Kosh-land, 1996; Danielson et al., 1997) suggests that a5 isdistinct from the following helix, a6. The division ofTar into two stable structural units with limitedproteolysis led to studies utilizing a convenientnearby restriction site to prepare C-terminal frag-ments consisting of residues 258–553 (Kaplan andSimon, 1988). This portion of the receptor includestwo regions involved in methylation, linked by ahighly conserved region implicated in interactionswith CheA and CheW; it terminates in a distinctC-terminal ‘‘tail.’’ Such fragments appear to be highlyhelical in CD measurements (Mowbray et al., 1985;Wu et al., 1995) and move as much larger species ongel filtration chromatography (Mowbray et al., 1985;Kaplan and Simon, 1988; Long and Weis, 1992a,b). Amonomer–dimer equilibrium is observed, with mono-mers having an apparent Stokes radius of about 30A, and dimers, of about 40 A; the relative proportionsof monomer and dimer are greatly altered by solu-tion conditions as well as by mutation (Long andWeis, 1992a). Dissociation of the dimers seems torequire some unfolding (Wu et al., 1995), as wouldnot be surprising if they intertwine in the mannershown in Fig. 4. Structural work with these frag-ments has been hampered by their extreme flexibil-ity. They are highly sensitive to protease (Mowbrayet al., 1985), even in dimeric form (Seeley et al.,1996), and unusually mobile according to severalspectroscopic criteria (Seeley et al., 1996). The iso-lated fragments crystallize, but the crystals havepoor diffraction characteristics (M. O. J. Sandgren, B. H.Shilton, and S. L. Mowbray, unpublished results).

Reversible methylation of MCPs occurs at four tosix glutamate residues (Springer and Koshland,1977; Terwilliger and Koshland, 1984; Nowlin et al.,1987; Rice and Dahlquist, 1991), corresponding toGln295, Glu302, Gln309, and Glu491 in Tar. Thesites originally coded for as glutamine residues aremade available for methylation by a secondary deami-dating activity of the chemotaxis methylesterase(Rollins and Dahlquist, 1981; Sherris and Parkin-son, 1981). The methylation regions were earlyproposed to be helical, based on sequence patternsnear the modification sites (Kehry and Dahlquist,1982; Terwilliger et al., 1983), a model that issupported by sequence comparisons and disulfidescanning (Danielson et al., 1997). The methylationregions are currently thought to be part of the longerhelices a6 and a9. Patterns consistent with theformation of coiled coils are observed for MCP resi-dues from approximately 290 to 330 and 475 to 520(Lupas et al., 1991). Helices a6 and a9 are suggestedto be antiparallel to each other, forming a four-helix

bundle that includes the corresponding helices of theother subunit (Stock et al., 1991; Surette and Stock,1996). Helices a6 and a68 have been shown to make aparallel contact (Danielson et al., 1997), while a9 anda98 have yet to be examined in detail. An intra-subunit coiled coil should be stabilized by free gluta-mates (Lupas et al., 1991), while the bundle may befavored by neutralization of these charges withmethyl or amide groups (Stock et al., 1991). Such anarrangement would be expected to ‘‘wrap around’’itself with a left-handed pitch of about 140 A (Fig. 4).

Various fragments containing a highly conserved‘‘core’’ (approximately residues 350–425, that is, a7and a8 and the loop connecting them) have beencalled the ‘‘signaling domain’’ with reference to rolesin binding CheA and CheW as well as kinase modula-tion. An independently folding fragment of Tsr (resi-dues 350–470) has been shown to stimulate CheA ina CheW-dependent manner (Ames et al., 1996);combined with similar observations for an overlap-ping section of Tar (Surette and Stock, 1996), itappears that residues from ,350 to 455 actuallyform the minimum folding unit.

A model where a7 and a8 are extended in directlines from a6 and a9, respectively, seems incompat-ible with the available data on the length of thereceptor, and so more compact arrangements shouldbe considered. A coil pattern analysis (see, e.g., Stocket al., 1991) shows similar amphiphilic patterns inthis region of the sequence and in the periplasmicdomain, suggesting the possibility a four-helixbundle, with individual helices in the regions of340–365 (start of a7), 365–385 (end of a7), 390–430(a8), and 430–470 (start of a9). The beginning and/orending helices could be continuous with a6/a9. An-other possibility involves the simple folding up of a7and a8 onto a6 and a9. An intrasubunit bundle ofthis type would merge into a dimer bundle in the‘‘upper’’ reaches of a6 and a9, since a7 and a8 areshorter (Fig. 4 illustrates this possibility); again, thesimilarity between this arrangement and that ofthe periplasmic domain is clear. Such folding mightbe affected by methylation, since the modificationsites could be buried in the folded form. It is quitepossible that more than one type of helical arrange-ment will ultimately be found; the accessibility ofmultiple states seems to be a common, and impor-tant, feature of proteins containing coiled coils(Lupas, 1996).

A short region of indeterminate structure (resi-dues 515–553) follows a9. This C-terminal ‘‘tail’’ iseasily removed with protease (M. O. J. Sandgren,unpublished results) and appears highly mobile inspectroscopic measurements (Seeley et al., 1996).The last five residues of Tar have now been shown to


be essential for methyltransferase tethering (seebelow).

Other receptors. Similar MCPs are found inmany bacterial species (Dahl and Manson, 1985;Alam and Hazelbauer, 1991), including nonmotile(Dahl et al., 1989) as well as gliding bacteria (Mor-gan et al., 1993). The general topology places theseMCPs in the type I family of transmembrane recep-tors (Heldin, 1995), a relationship that has beensupported by the construction of functional chimericreceptors of Tar with the insulin receptor (Ellis et al.,1986; Moe et al., 1989; Biemann et al., 1996). To-gether with the discovery that a dimer of CheA is theactive form (Surette et al., 1996), this idea hasprovoked much discussion of transmembrane signal-ing by monomer–dimer mechanisms, as is appar-ently the case for many eukaryotic type I receptors.However, it appears that as for the insulin receptor,allosteric mechanisms predominate for the MCPs.

A fifth, somewhat different, MCP of E. coli hasrecently been characterized (Rebbapragada et al.,1997; Zhulin et al., 1997; Bibikov et al., 1997; Grisha-nin and Bibikov, 1997). The Aer receptor works insynergy with Tsr in aerotaxis and energy sensing.Aer has a cytoplasmic signal-transducing regionsimilar to the more classical MCPs, but a cytoplas-mic sensing region of a completely distinct design(containing a noncovalently associated FAD). SomeMCPs in other organisms have no periplasmic por-tions and either one or two membrane-spanningsegments (Krah et al., 1994; Spudich, 1994; LeMoual and Koshland, 1996), while others are solubletransducers including only the cytoplasmic signalingand methylation regions (Zhang et al., 1996; Broounet al., 1997). It is presumed that the cytoplasmicportions of all of these signaling systems will followthe same general model.

The phosphotransferase system also uses CheAand CheY to send intracellular signals, effectivelyusing enzymes I and II as receptors (Lux et al., 1995).

Receptors Are Concentrated at Poles of Cells

Associations of large numbers of receptor dimersare likely to play an important role in their function.About 80% of the Tar in wild-type E. coli is foundclustered in large patches at the poles of the cell.Formation of the patches has been shown to bedependent on the presence of CheW and, to a lesserextent, CheA; CheY and CheZ are apparently notnecessary (Maddock and Shapiro, 1993). Althoughpolar clustering requires the intracellular (C-termi-nal) portions of the MCP (Alley et al., 1992, 1993),the origins of the phenomenon are not yet clear.Flagella are not localized in this way, nor are MCPsspecifically clustered near the flagella (Nathan et al.,

1986). A correlation has, however, been noted with asimilar disposition of MBP (Maddock et al., 1993);expression of MBP had been linked earlier withpolar cap formation (Boos and Staehelin, 1981; Diet-zel et al., 1978).

Smaller, regular arrays were observed in electronmicroscopic studies of a cytoplasmic construct of Tarwhere oligomerization was induced with leucinezippers (Liu et al., 1997). This association occurredin the absence of CheW and CheA, a long thin shapeof the cytoplasmic fragments allowing their packinginto compact bundles. Each individual receptor re-gion appeared to account for approximately 140 A inlength, similar to the maximum of 120 A suggestedby electron microscopy of Trg. This would correspondroughly to the length of cytoplasmic portions of a4,a5 and a6 arranged in extended form. A ‘‘nonex-tended’’ arrangement in the cytoplasmic regionsmight be expected to ease packing of intact MCPsinto such arrays, by making the periplasmic andcytoplasmic regions more similar in size and shape.CheA and CheW bound at the ends of the bundles,with approximately 14 receptor dimers being associ-ated with six CheW molecules and two CheA dimers.Clustering was clearly linked to kinase activation,and both kinase activity and clustering were in-creased by higher levels of methylation (Liu et al.,1997). Methylation may act to promote bundle forma-tion by neutralizing the negative charges of themodified residues, as suggested above for dimers.

LIGAND BINDING ALTERS AN MCP

The measured dissociation constant for aspartatebinding to Tar is approximately 1 µM in membrane-bound and solubilized intact receptors, as well asperiplasmic fragments (Aksamit et al., 1975; Clarkeand Koshland, 1979; Foster et al., 1985; Mowbray etal., 1985; Milligan and Koshland, 1993; Biemannand Koshland, 1994; Danielson et al., 1994). Thisrepresents binding to the first of two possible sites onthe dimer; a second aspartate binds with loweraffinity (Milligan and Koshland, 1993; Biemann andKoshland, 1994). The relative affinity of the two sitesfor aspartate is not constant. While binding at thesecond site occurs fairly readily in the SalmonellaTar, the negative cooperativity is more absolute in E.coli Tar (Biemann and Koshland, 1994), as well as inTsr (Lin et al., 1994), where only a single ligandmolecule binds to each dimer.

These results can be understood in terms ofchanges observed in the x-ray structures on aspar-tate binding. The binding site has contributions fromboth subunits (Fig. 5; Milburn et al., 1991). Whenaspartate binds to one site between the two initiallyequivalent subunits, they rearrange to a nonsym-


metric dimer (Milburn et al., 1991; Yeh et al., 1996).Local alterations at the second site, and more globalchanges within the dimer, dictate that affinity at thesecond site will be different (Scott and Stoddard,1994). Binding of a second aspartate to wild-typeSalmonella Tar does not appear to cause any addi-tional changes in the overall structure compared tothe single-site binding (Yeh et al., 1996), althoughthe form observed could be biased by crystal packing.That this situation is ‘‘adjustable’’ is indicated notonly by the E. coli/Salmonella differences, but by thefact that mutation of Salmonella Tar at Ser68 in thedimer interface can give rise to positive, negative, orno cooperativity (Kolodziej et al., 1996).

A simple monomer–dimer equilibrium cannot ex-plain transmembrane signaling; many cross-linkeddimers can signal (Falke and Koshland, 1987). In-stead, changes within an individual subunit appearto be critical (Scott and Stoddard, 1994; Danielson etal., 1994; Chervitz and Falke, 1996). Main- andside-chain groups on a1 and a4 of one subunitinteract almost exclusively with main-chain groups

of the aspartate ligand. Complementary groups ona18 of the other subunit make most contacts with theligand side chain. When aspartate enters the site, a4moves about 1.6 A toward the membrane relative toa1, and is tilted by about 5°, in a ‘‘swinging piston’’motion (Fig. 5; Chervitz and Falke, 1996). In thisway, a4 moves about 1 A with respect to the helices ofthe other subunit, which itself is unchanged. Thedimer interface, mainly a function of a1/a18 interac-tions, remains relatively static. Inspection of thex-ray structures of periplasmic fragments of E. coliTar with and without bound aspartate (Bowie et al.,1995) suggests a similar situation. NMR studiesdemonstrate further that although aspartate andglutamate generate somewhat different conforma-tions locally at the binding site, they cause a similarintrasubunit conformational signal to be sent throughthe receptor (Danielson et al., 1994). Electron para-magnetic resonance measurements with intact, mem-brane-bound Salmonella Tar also indicate a relativemovement of a4, which may be accompanied by aslightly closer approach of a1 and a18 (Ottemann etal., 1998).

Motion of an a4 is transmitted to the membranesegment (TM2) to which it is attached and thus cancarry a signal across the membrane. Cross-linkingstudies confirm that the interface between TM1/TM18 plays a mainly static, structural role, while themotion of TM2 relative to TM1 is most critical inkinase modulation (Lynch and Koshland, 1991;Danielson et al., 1994; Scott and Stoddard, 1994;Chervitz et al., 1995; Chervitz and Falke, 1995).These changes are similar to the helix-on-helix slid-ing motions observed to affect form and function inother proteins (Chothia and Lesk, 1985). Modeling oflock-off disulfides suggests that they generate thesame membrane-ward motion of a4 as the binding ofaspartate, while lock-on disulfides appear to cause amotion in the opposite direction (Chervitz and Falke,1996).

Such motions can carry binding signals to thecytoplasm, but can information from the cytoplasmbe sent back to the periplasmic regions? Most studies(Springer et al., 1979; Clarke and Koshland, 1979;Dunten and Koshland, 1991; Borkovich et al., 1992;Lin et al., 1994; Iwama et al., 1997) suggest thatmethylation of the receptors has only a small effecton the affinity for ligand, at least at the first site.Differences would also be expected in the ligandaffinity of MCPs with varying kinase-modulationproperties, but have been similarly hard to pinpoint.Although clustering may allow small effects withinany individual receptor to combine synergistically(Liu et al., 1997), the impact of parameters specific tothe in vivo system is very hard to assess.

FIG. 5. The aspartate-binding site of Tar. Arg64 (on a1) formshydrogen bonds with the a-carboxylate of the aspartate ligand,while the a-amino group is bound by Thr154 and a nitrocationhole made up of the backbone carbonyl oxygens of residues 149and 152 (all on a4 or the loop just before it). Arg698 and Arg738 arelocated on a18 of the other subunit and make contact with both a-and g-carboxylates. The position of a4 and the preceding loopprior to aspartate binding is indicated by the lighter backbone.Not shown are the side chain of Tyr149, which makes van derWaals contacts as well as hydrogen bonds to both a- and g-carbox-ylates indirectly through water molecules, and Ser68 whichinteracts indirectly via water. The figure was made using ProteinData Bank entries 1LIH and 2LIG.


INTERACTIONS BETWEEN MEMBRANEAND PERIPLASMIC RECEPTORS

Trg and Tap recognize only binding proteins, andTsr only small molecules, while E. coli Tar is uniquelycapable of recognizing both small molecule andbinding protein ligands (Fig. 2). The available stud-ies indicate that the mechanism of transmembranesignaling is similar for the two types of attractant(Lee et al., 1994, 1995a,b; Lee and Hazelbauer, 1995;Hughson and Hazelbauer, 1996; Baumgartner andHazelbauer, 1996; Hughson et al., 1997; Gardina etal., 1997). Mutants of Trg can be found, for example,that continuously send kinase-modulating signals,as well as ones that are insensitive to bindingprotein (Yaghmai and Hazelbauer, 1992). Bindingprotein-dependent responses are inherently weaker(e.g., Mowbray and Koshland, 1987) even when theMCP is apparently saturated (Manson et al., 1985),implying that binding protein-induced conforma-tional changes are either smaller or otherwise lesseffective. Since the primary effect of attractant bind-ing is to inactivate the kinase, there may indeed bemultiple ways to accomplish this task and variationsin their efficiency.

The dissociation constant for the MBP–Tar interac-tion been suggested by in vivo expression studies tobe of the order of 250 mM (Manson et al., 1985),although it is not clear how polar localization of MBPwithin the periplasm might affect this estimate. Acombination of mutational studies of MBP and Tar(Manson and Kossman, 1986; Kossman et al., 1988;Zhang et al., 1992; Gardina et al., 1997) and com-puter modeling gave rise to the view of the complexpresented in Fig. 6 (Zhang et al., 1998). As with asimilar earlier model (Stoddard and Koshland, 1992,1993), a single molecule of MBP is proposed tointeract with a dimer of E. coli Tar through the loopsat the ‘‘top’’ of the MCP; the present model, however,agrees better with the effects of known mutations.The surface of MBP that is implicated in chemotaxisis similar in size and disposition relative to the sitesimportant in transport, although not in structure, tothat proposed for the RBP/Trg complex (Binnie et al.,1992). Interactions of MBP with the a3–a4 loop ofTar could easily give rise to conformational changeswithin one subunit of the MCP that are analogous tothose caused by aspartate binding (Fig. 6).

This model also suggests an explanation for theobservation that maltose and aspartate responses ofthe E. coli receptor are quite independent: maltosecan generate a signal even in the presence of saturat-ing aspartate and vice versa (Wolff, 1983; Mowbrayand Koshland, 1987). Previous experimental datasuggested that the two signals are carried in parallelby different subunits of the same receptor dimer(Gardina et al., 1997). If the relationship of a18 and

a48 is unchanged in the second subunit of an singlyaspartate-bound dimer, MBP should be able to usethis unactivated subunit to trigger a response evenwhen one molecule of aspartate is present. Minordifferences in the structures of Salmonella and E.coli Tar would be sufficient to dictate that, when thedimers have bound to a single molecule of aspartate,the former protein would be competent to bind asecond aspartate, while the latter would prefer tointeract with MBP.

MODULATION OF CHEA BY MCPS

Clearly, motions of a single a4 within an MCPdimer can affect both intrasubunit and intersubunitstructure in the cytoplasmic regions, with correspond-ing consequences for receptor interactions with CheAand CheW. That the intrasubunit effects dominatewithin any individual MCP dimer is suggested bystudies showing kinase modulation (Gardina andManson, 1996; Tatsuno et al., 1996) and methylation(Milligan and Koshland, 1991) with receptor dimerswhere one subunit has been truncated (but stillincludes the linker). Since the activation of CheA isbelieved to involve its dimeric form (Surette et al.,1996; Levit et al., 1996), these results are difficult toexplain within the context of a one receptor dimer:one CheA dimer:two CheW monomer complex thathas been demonstrated in vitro (Gegner et al., 1992).They are, however, more easily rationalized for thetype of multi-MCP aggregates that seem to be neces-sary for maximal stimulation of CheA (Liu et al.,1997). (The truncated receptors have not, however,been demonstrated to form clusters.) In reality, theamounts of CheA and CheW present in the cell areinsufficient to form 2:2:2 complexes with all avail-able MCP (data on quantities summarized in Falkeet al., 1997, and Stock et al., 1991), suggesting that,in vivo, a mixture of larger and smaller complexes islikely to exist. Indeed, the spectrum of activationlevels obtainable from the various species may be animportant aspect of receptor function.

In any case, conformational signals associatedwith ligand binding must be converted into changesin CheA activity, presumably via changes in MCPstructure near a7 and a8 (the conserved signalingdomain). The ‘‘crude’’ nature of the signal required atthe periplasmic side is suggested by the success ofmany functional chimeric constructs. Indeed, a recep-tor with both transmembrane segments removed isstill capable of sending a signal (Ottemann andKoshland, 1997).

Observations that CheA by itself is in equilibriumbetween an inactive monomeric form and an activedimer (Surette et al., 1996) suggested that receptor-induced superactivation of CheA (Borkovich et al.,1989; Ninfa et al., 1991; Borkovich and Simon, 1990)


involves some variation on the dimer theme. Incontrast, the inhibition of CheA by attractant-boundreceptors may involve a return to a monomer-likestate of CheA. The mechanisms by which eitheractivation or inhibition of CheA occur are far fromclear. Control does not appear to rely on association/dissociation of MCP/CheA/CheW complexes, as their

lifetime is too long (Gegner et al., 1992; Schuster etal., 1993). Dimers of Tar with appropriate cross-linksbetween their a1/TM1s can affect the kinase eitherpositively or negatively (Chervitz and Falke, 1995),further suggesting that changes in function areaccomplished allosterically rather than through asso-ciation/dissociation of the cytoplasmic regions. Two

FIG. 6. Model for the MBP/Tar complex. A Tar dimer (light blues) and MBP (gold) are shown with ribbon representations. The view ofTar differs slightly from that used in Fig. 4; the first domain of MBP is at left. Red highlights residues of MBP (41, 45, 46, 49, 50, 53, 55, 342,345, 354, 359, and 367) and Tar (69, 73, 75–77, 83, 143, 145, 147, 149, and 150) that have been implicated in maltose chemotaxis (Kossmanet al., 1988; Zhang et al., 1992; Gardina et al., 1997). The model coordinates used to prepare this figure were kindly provided by Y. Zhang,P. J. Gardina, J. Christopher, and M. D. Manson of Texas A&M.


distinctly different types of ‘‘cytoplasmic’’ dimershave indeed been reported. Dimers in which a6/a68contacts are directed toward a specific coiled-coilpattern are associated with CheA activation (Suretteand Stock, 1996; Cochran and Kim, 1996). Thepresence of a9 and a98 somehow reduces this stimu-lation (Ames and Parkinson, 1994; Surette andStock, 1996), perhaps by changing the relationshipof a6 and a68. Another type of dimer is observed(Long and Weis, 1992a,b) for cytoplasmic constructsthat represent smooth-swimming (CheA-inhibiting)mutants of Tar (Mutoh et al., 1986; Oosawa et al.,1988). This second type of dimer has been shown inat least one case to be dominant when expressedtogether with intact receptor, indicating effectivebinding of CheA and/or CheW (Oosawa et al., 1988).It could, therefore, represent an important alternatestate of the MCP, much like the open forms of thebinding proteins. Fundamentally different interac-tions between CheA and the MCPs in the two stateshave been suggested by other studies, with thekinase-on interaction being more dependent on CheW(Ames and Parkinson, 1994). Such a two-state modelis illustrated schematically in Fig. 7. Although itseems likely that there are a limited number of waysof turning the kinase on, there is almost certainlymore than one way of turning it off. The piston-likemotion seen in the structures of the periplasmicfragments illustrates one way of inactivating thekinase and rotational movements of a6/a68 withrespect to each other represents another possibility(Cochran and Kim, 1996). Kinase inactivation result-ing from small-molecule or binding protein associa-tions may reflect different combinations of these two

components, although rotational inactivation hasnot been demonstrated in intact receptors.

Increased methylation of an MCP acts in theopposite manner to attractant binding, i.e., morehighly methylated MCP stimulates the kinase moreefficiently (Ninfa et al., 1991; Borkovich et al., 1992).If aspartate binding works to weaken the bundling ofa6s and a9s in the dimer, then glutamate modifica-tion may well favor the interaction by reducingelectrostatic repulsion between the helices (Stock etal., 1991). In this situation, it is easy to imaginemultiple methylation events having qualitativelysimilar results.

Dramatic changes in secondary and tertiary struc-ture seem to be rather common for proteins involvingcoiled coils (Lupas, 1996). It has been proposed (Brayet al., 1998) that such changes within a dimer couldbe passed on to ‘‘infect’’ neighbors, making clusteredMCPs supersensitive to the effects of aspartatebinding. A mixture of different, and varying, levels ofaggregation seems most consistent with the charac-teristics of sensing in vivo. Further, it is predictedthat clustering will be extensive at low concentra-tions of aspartate and much less at high concentra-tions of aspartate. Other workers have suggestedthat slight fluctuations in the levels of CheW provideanother means of controlling the formation, and thuseffects, of clusters (Liu et al., 1997).

The behavior of any cell is thus the result of abalance of stresses that originate in the periplasmwith compensating cytoplasmic interactions. A re-sponse, positive or negative, is due to a temporarydisturbance in that balance. The probability of the

FIG. 7. A two-state model. Ligand binding and covalent modification appear to act by altering the probability of CheA kinaseactivation. The interconversion between the two states in shown here (from the ‘‘top’’ of the dimer) as a dramatic (30°) rotation, fordemonstration purposes. The activation of CheA could, of course, be ‘‘interdimeric,’’ but the basic ideas remain the same. Experimentalevidence suggests that alterations in the regions of the a6/a9 contacts, and the associated a6/a68 contacts, are involved. Both kinase-on andkinase-off states of the receptor appear to involve a5/a58 (Butler and Falke, 1998) and a6/a68 contacts (Danielson et al., 1997; Cochran andKim, 1996), but the relationship to a9 and a98 to the rest of the structure is not really established for either case.


kinase being activated under given conditions is thekey issue.

INTERACTIONS WITH METHYLTRANSFERASEAND METHYLESTERASE

Great progress has been made recently in under-standing the interactions of MCPs with the enzymesthat covalently modify them. Realization that meth-yltransferase actually binds most tightly to a five-residue sequence at the C-terminal end of somereceptors (Wu et al., 1996) was quickly followed by anx-ray structure of a methyltransferase/Tar–peptidecomplex that demonstrated that such binding oc-curred at a site distant from the methyltransferaseactive site (Djordjevic and Stock, 1998). Whenmounted on this flexible tether, a methyltransferasecan act on the various methylation sites in its own,as well as other nearby, dimers (Li et al., 1997; LeMoual et al., 1997). Continuously exchanging tetra-mers may offer a convenient mechanism for inter-dimer methylation. In solubilized systems such as(Bogonez and Koshland, 1985), the intradimer modi-fication will almost certainly dominate. Both localiz-ing effects, and the ability to methylate multiplesites from the same vantage point, seem to offer clearadvantages. Cellulases, which have similar needs intheir processing of solid cellulose substrates, have astrikingly similar design: a cellulose-binding domainis attached to the catalytic domain via a flexiblelinker (see, e.g., Rouvinen et al., 1990). Not all MCPshave such a methyltransferase-binding tail (Le Moualand Koshland, 1996), but interdimer action allowsthem to be methylated in a fashion reflecting thecellular level of CheA (and thus CheB) activation.The presence of CheA and CheW (i.e., the potentialfor more extensive clustering) did not appear toinfluence the rates of methylation (Le Moual et al.,1997), although it was not proved that clusteringactually occurred under the assay conditions. Theprediction of Bray et al. (1998) that clustering will bevirtually absent at high aspartate concentrationsmay be pertinent.

Recognition of MCP at the methylation sites mayinvolve up to five turns of the helix in which theyreside (Shapiro and Koshland, 1994; Le Moual andKoshland, 1996; Djordjevic and Stock, 1997), al-though the exact mode of binding here is not yetknown. The catalytic site of the methyltransferase iscertainly wide enough to accept a single helix, orperhaps a coiled pair, but may discriminate againstlarger assemblies and thus be affected by the kinase-modulation state of the MCP. That attractant-boundMCPs are inherently better substrates for the meth-yltransferase has been documented in numerousstudies.

Structures of the methylesterase have also been

solved and illustrate how MCP demethylation iscontrolled. A catalytic domain by itself is as active asone in the intact CheR where the associated regula-tory domain has been phosphorylated (Simms et al.,1985; Lupas and Stock, 1989). In the x-ray structureof the intact, unphosphorylated methylesterase(Djordjevic et al., 1998), the active site is physicallyblocked by the regulatory domain, giving rise to thesuggestion that phosphorylation causes the regula-tory domain to physically move out of the way. Anopen methylesterase active site appears broaderthan that of the methyltransferase, which may makeit more accommodating to the ‘‘larger’’ bundle assem-bly of the kinase-activating MCP. That this should bethe only form of the MCP recognized by the methyles-terase has been suggested by the kinetic modelingstudies of Barkai and Leibler (1997).

WORK FOR THE FUTURE

It is obvious from the above discussion that not allquestions have been answered. This is an exciting,but often confusing, time in the field, as manyprevious results need to be reevaluated with newerconcepts in mind. Structures are urgently needed,particularly for the cytoplasmic elements of thesystem and for the partners together in complexes.This is a highly dynamic and complex system, so asingle snapshot of any protein by itself will not tell usall we need to know about its function. It seemsprobable that lower resolution studies such as elec-tron microscopy will produce much of value in thenear future, helping to establish when, and howmany, of the different components are in particularplaces under different conditions. Modeling of thekinetics of the system as a whole is also likely toprove invaluable. The promising starts made in thatdirection (e.g., Bray and Bourret, 1995; Hauri andRoss, 1995; Barkai and Leibler, 1997; Spiro et al.,1997; Levin et al., 1998) have certainly helped intesting whether current assumptions fit the factsand in suggesting new avenues for experimentalwork. Such efforts must go hand in hand, however,with frequent and careful reevaluations of the under-lying assumptions and parameters used.

The authors thank those who assisted us in gathering therecent data, including Ann Stock, Joe Falke, LynnMarie Thomp-son, Gerry Hazelbauer, and Bob Weis. Ida-Maria Sintorn providedinvaluable editorial assistance. Special gratitude is owed to Y.Zhang, P. J. Gardina, J. Christopher, and M. D. Manson of TexasA&M for the model coordinates used to prepare Figure 6.

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Chemotaxis Receptors: A Progress Report on Structure and Function

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