Aspartate and maltose-binding protein interact with adjacent sites in the Tar chemotactic signal transducer of Escherichia coli.

The Tar protein of Escherichia coli is a chemotactic signal transducer that spans the cytoplasmic membrane and mediates responses to the attractants aspartate and maltose. Aspartate binds directly to Tar, whereas maltose binds to the periplasmic maltose-binding protein, which then interacts with Tar. The Arg-64, Arg-69, and Arg-73 residues of Tar have previously been shown to be involved in aspartate sensing. When lysine residues are introduced at these positions by site-directed mutagenesis, aspartate taxis is disrupted most by substitution at position 64, and maltose taxis is disrupted most by substitution at position 73. To explore the spatial distribution of ligand recognition sites on Tar further, we performed doped-primer mutagenesis in selected regions of the tar gene. A number of mutations that interfere specifically with aspartate taxis (Asp-), maltose taxis (Mal-), or both were identified. Mutations affecting residues 64 to 73 or 149 to 154 in the periplasmic domain of Tar are associated with an Asp- phenotype, whereas mutations affecting residues 73 to 83 or 141 to 150 are associated with a Mal- phenotype. We conclude that aspartate and maltose-binding protein interact with adjacent and partially overlapping regions in the periplasmic domain of Tar to initiate attractant signalling.     

Maltose chemoreceptor of Escherichia coli: interaction of maltose-binding protein and the tar signal transducer.

The maltose chemoreceptor in Escherichia coli consists of the periplasmic maltose-binding protein (MBP) and the Tar signal transducer, which is localized in the cytoplasmic membrane. We previously isolated strains containing malE mutations that cause specific defects in the chemotactic function of MBP. Four of these mutations have now been characterized by DNA sequence analysis. Two of them replace threonine at residue 53 of MBP with isoleucine (MBP-TI53), one replaces an aspartate at residue 55 with asparagine (MBP-DN55), and the fourth replaces threonine at residue 345 with isoleucine (MBP-TI345). The chemotactic defects of MBP-TI53 and MBP-DN55, but not of MBP-TI345, are suppressed by mutations in the tar gene. Of the tar mutations, the most effective suppressor (isolated independently three times) replaces Arg-73 of Tar with tryptophan. Two other tar mutations that disrupt the aspartate chemoreceptor function of Tar also suppress the maltose taxis defects associated with MBP-TI53 and MBP-DN55. One of these mutations introduces glutamine at residue 73 of Tar, the other replaces arginine at residue 69 of Tar with cysteine. These results suggest that regions of MBP that include residues 53 to 55 and residue 345 are important for the interaction with Tar. In turn, arginines at residues 69 and 73 of Tar must be involved in the recognition of maltose-bound MBP and/or in the production of the attractant signal generated by Tar in response to maltose-bound MBP.     

Mutations in tar suppress defects in maltose chemotaxis caused by specific malE mutations.

Maltose-binding protein (MBP), which is encoded by the malE gene, is the maltose chemoreceptor of Escherichia coli, as well as an essential component of the maltose uptake system. Maltose-loaded MBP is thought to initiate a chemotactic response by binding to the tar gene product, the signal transducer Tar, which is also the aspartate chemoreceptor. To study the interaction of MBP with Tar, we selected 14 malE mutants which had specific defects in maltose taxis. Three of these mutants were fully active in maltose transport and produced MBP in normal amounts. The isoelectric points of the MBPs from these three mutants were identical to (malE461 and malE469) or only 0.1 pH unit more basic than (malE454) the isoelectric point of the wild-type protein (pH 5.0). Six of the mutations, including malE454, malE461, and malE469, were mapped in detail; they were located in two regions within malE. We also isolated second-site suppressor mutations in the tar gene that restored maltose taxis in combination with the closely linked malE454 and malE461 mutations but not with the malE469 mutation, which maps in a different part of the gene. This allele-specific suppression confirmed that MBP and Tar interact directly.     

Evolution of chemotactic-signal transducers in enteric bacteria.

The methyl-accepting chemotactic-signal transducers of the enteric bacteria are transmembrane proteins that consist of a periplasmic receptor domain and a cytoplasmic signaling domain. To study their evolution, transducer genes from Enterobacter aerogenes and Klebsiella pneumoniae were compared with transducer genes from Escherichia coli and Salmonella typhimurium. There are at least two functional transducer genes in the nonmotile species K. pneumoniae, one of which complements the defect in serine taxis of an E. coli tsr mutant. The tse (taxis to serine) gene of E. aerogenes also complements an E. coli tsr mutant; the tas (taxis to aspartate) gene of E. aerogenes complements the defect in aspartate taxis, but not the defect in maltose taxis, of an E. coli tar mutant. The sequence was determined for 5 kilobases of E. aerogenes DNA containing a 3' fragment of the cheA gene, cheW, tse, tas, and a 5' fragment of the cheR gene. The tse and tas genes are in one operon, unlike tsr and tar. The cytoplasmic domains of Tse and Tas are very similar to those of E. coli and S. typhimurium transducers. The periplasmic domain of Tse is homologous to that of Tsr, but Tas and Tar are much less similar in this region. However, several short sequences are conserved in the periplasmic domains of Tsr, Tar, Tse, and Tas but not of Tap and Trg, transducers that do not bind amino acids. These conserved regions include residues implicated in amino-acid binding.     

Pleiotropic aspartate taxis and serine taxis mutants of Escherichia coli.

Mutants that at one time were thought to be specifically defective in taxis toward aspartate and related amino acids (tar mutants) or specifically defective in taxis toward serine and related amino acids (tar mutants) are now shown to be pleiotropic in their defects. The tar mutants also lack taxis toward maltose and away from Co2+ and Ni2+. The tsr mutants are altered in their response to a variety of repellents. Double mutants (tar tsr) fail in nearly all chemotactic responses. The tar and tsr mutants provide evidence for two complementary, converging pathways of information flow: certain chemoreceptors feed information into the tar pathway and others into the tsr pathway. The tar and tsr products have been shown to be two different sets of methylated proteins.     

Dependence of maltose transport and chemotaxis on the amount of maltose-binding protein

Maltose-binding protein (MBP) is essential for maltose transport and chemotaxis in Escherichia coli. To perform these functions it must interact with two sets of cytoplasmic membrane proteins, the MalFGK transport complex and the chemotactic signal transducer Tar. MBP is present at high concentrations, on the order of 1 mM, in the periplasm of maltose-induced or malTc constitutive cells. To determine how the amount of MBP affects transport and taxis, we utilized a series of malE signal-sequence mutations that interfere with export of MBP. The MBP content in shock fluid from cells carrying the various mutations ranged from 4 to 23% of the malE+ level. The apparent Km for maltose transport varied by less than a factor of 2 among malE+ and mutant strains. At a saturating maltose concentration 9% (approximately 90 microM) of the malE+ amount of MBP was required for half-maximal uptake rates. Transport exhibited a sigmoidal dependence on the amount of periplasmic MBP, indicating that MBP may be involved in a cooperative interaction at some stage of the transport process. The chemotactic response to a saturating maltose stimulus exhibited a first-order dependence on the amount of periplasmic MBP. Thus, interaction of a single substrate-bound MBP with Tar appears sufficient to initiate a chemotactic signal from the transducer. A half-maximal chemotactic response occurred at 25% of the malE+ MBP level, suggesting that in vivo the KD for binding of maltose-loaded MBP to Tar is quite high (approximately 250 microM).     

A mechanism for simultaneous sensing of aspartate and maltose by the Tar chemoreceptor of Escherichia coli

The Tar chemoreceptor of Escherichia coli exhibits partial sensory additivity. Tar can mediate simultaneous responses to two disparate ligands, aspartate and substrate-loaded maltose-binding protein (MBP). To investigate how one receptor generates concurrent signals to two stimuli, ligand-binding asymmetry was imposed on the rotationally symmetric Tar homodimer. Mutations causing specific defects in aspartate or maltose chemotaxis were introduced pairwise into plasmid-borne tar genes. The doubly mutated tar genes did not restore aspartate or maltose chemotaxis in a strain containing a chromosomal deletion of tar (Δ tar ). However, when Tar proteins with complementing sets of mutations were co-expressed from compatible plasmids, the resulting heterodimeric receptors enabled Δ tar cells to respond to aspartate or maltose. The effect of one attractant on the response to the other depended on the relative orientations of the functional binding sites for aspartate and MBP. When the sites were in the 'same' orientation, saturating levels of one attractant strongly inhibited chemotaxis to the other. In the 'opposite' orientation, such inhibitory effects were negligible. These data demonstrate that opposing subunits of Tar can transmit signals to aspartate and maltose independently if the ligands are restricted to the 'opposite' binding orientation. When aspartate and MBP bind in the 'same' orientation, they compete for signalling through one subunit. In the wild-type Tar dimer, aspartate and MBP can bind in either the 'same' or the 'opposite' orientation, a freedom that can explain the partial additivity of the aspartate and maltose responses that is seen with tar ⁺ cells.     

Genetics of methyl-accepting chemotaxis proteins in Escherichia coli: null phenotypes of the tar and tap genes.

The tar and tap genes are located adjacent to one another in an operon of chemotaxis-related functions. They encode methyl-accepting chemotaxis proteins implicated in tactic responses to aspartate and maltose stimuli. The functional roles of these two gene products were investigated by isolating and characterizing nonpolar, single-gene deletion mutants at each locus. Deletions were obtained by selecting for loss or a defective Mu d1 prophage inserted in either the tar or tap gene. The extent of the tar deletions was determined by genetic mapping with Southern hybridization. Representative deletion mutants were surveyed for chemotactic responses on semisolid agar and by temporal stimulation in a tethered cell assay to assess flagellar rotational responses to chemoeffector compounds. The tar deletion strains exhibited complete loss of aspartate and maltose responses, whereas the tap deletion strains displayed a wild-type phenotype under all conditions tested. These findings indicate that the tap function is unable to promote chemotactic responses to aspartate and maltose, and its role in chemotaxis remains unclear.     

Hybrid Escherichia coli sensory transducers with altered stimulus detection and signaling properties.

The tar and tap loci of Escherichia coli encode methyl-accepting inner membrane proteins that mediate chemotactic responses to aspartate and maltose or to dipeptides. These genes lie adjacent to each other in the same orientation on the chromosome and have extensive sequence homology throughout the C-terminal portions of their coding regions. Many spontaneous deletions in the tar-tap region appear to be generated by recombination between these regions of homology, leading to gene fusions that produce hybrid transducer molecules in which the N terminus of Tar is joined to the C terminus of Tap. The properties of two such hybrids are described in this report. Although Tar and Tap molecules have homologous domain structures, these Tar-Tap hybrids exhibited defects in stimulus detection and flagellar signaling. Both hybrid transducers retained Tar receptor specificity, but had reduced detection sensitivity. This defect was correlated with the presence of the C-terminal methyl-accepting segment of Tap, which may have more methylation sites than its Tar counterpart, leading to elevated steady-state methylation levels in the hybrid molecules. One of the hybrids, which carried a more extensive segment from Tap, appeared to generate constitutive signals that locked the flagellar motors in a counterclockwise rotational mode. Changes in the methylation state of this transducer were ineffective in cancelling this aberrant signal. These findings implicate the conserved C-terminal domain of bacterial transducers in the generation or regulation of flagellar signals.     

Interspecific reconstitution of maltose transport and chemotaxis in Escherichia coli with maltose-binding protein from various enteric bacteria.

In Escherichia coli, the periplasmic maltose-binding protein (MBP), the product of the malE gene, is the primary recognition component of the transport system for maltose and maltodextrins. It is also the maltose chemoreceptor, in which capacity it interacts with the signal transducer Tar (taxis to aspartate and some repellents). In studies of the maltose system in other members of the family Enterobacteriaceae, we found that MBP is produced by Salmonella typhimurium, Klebsiella pneumoniae, Enterobacter aerogenes, and Serratia marcescens. MBP from all of these species cross-reacted with antibody against the E. coli protein and had a similar molecular weight (about 40,000). The Shigella flexneri and Proteus mirabilis strains we examined did not synthesize MBP. The isoelectric points of MBP from different species varied from the acid extreme of E. coli (4.8) to the basic extreme of E. aerogenes (8.9). All species with MBP transported maltose with high affinity, although the Vmax for K. pneumoniae was severalfold lower than that for the other species. Maltose chemotaxis was observed only in E. coli and E. aerogenes. In S. typhimurium LT2, Tar was completely inactive in maltose taxis, although it signaled normally in response to aspartate. MBP isolated from all five species could be used to reconstitute maltose transport and taxis in a delta malE strain of E. coli after permeabilization of the outer membrane with calcium.     

Mutations in the aspartate receptor of Escherichia coli which affect aspartate binding

The effects of five mutations at arginines 64, 69, and 73 of the Tar protein were analyzed using swarm assays, aspartate binding in purified membranes, and methylation both in vitro and in vivo. The defects in the responses of these mutant receptors to aspartate were shown to be directly attributable to reduced binding of aspartate to the receptor rather than to defects in their signaling characteristics. Mutations at residues 64, 69, and 73 reduced aspartate binding by factors of greater than 10(-4), 10(-3), and 10(-2), respectively. Once aspartate was bound, the mutants exhibited normal signaling properties. No cooperativity was observed in the coupling of aspartate binding to methylation, indicating that the monomers of the receptor dimer act independently. The in vitro methylation system was thus shown to be an effective way of measuring aspartate binding constants and examining the functional integrity of the proteins. The maltose responses of the receptor proteins were affected slightly, or not at all, in an in vivo methylation assay. Two models for the roles of these arginine residues in receptor function are discussed.     

Role of the receptor for bacteriophage lambda in the functioning of the maltose chemoreceptor of Escherichia coli.

Chemotaxis towards maltose is specifically defective in many strains of Escherichia coli carrying mutations affecting lamB, the gene coding for the outer membrane receptor for bacteriophage lambda. However, with one exception, the most extreme effect of lamB mutants on the maltose response as determined in the capillary assay is a shift to higher sugar concentrations and a reduction in the number of bacteria accumulated to about 25% of the wild-type level. The severity of the taxis defect is strongly correlated with reduced ability of the cells to take up the maltose present at 1 and 10 muM. Evidence presented here and in the accompanying paper indicates that the lambda receptor is involved in the transport of maltose at these concentrations. The effects of lamB mutations on maltose taxis can be explained by postulating that the high-affinity maltose transport system in which the lambda receptor participates transfers maltose from the surrounding medium across the outer membrane and into the periplasmic space. If the maltose chemoreceptor detects sugar present in the periplasmic space, and not molecules external to the outer membrane, then defective transport of low concentrations of maltose into the periplasm would result in the observed apparent reduction in the sensitivity of the maltose receptor. Thus, the lambda receptor protein would participate in maltose chemorecepton only indirectly through its role in maltose transport.     

Maltose chemotaxis involves residues in the N-terminal and C-terminal domains on the same face of maltose-binding protein.

The periplasmic maltose-binding protein (MBP) of Escherichia coli is the recognition component of the maltose chemoreceptor and of the active transport system for maltose. It interacts with the Tar chemotactic signal transducer and the integral cytoplasmic-membrane components (the MalF and MalG proteins) of the maltose transport system. Maltose binds in a cleft between the globular N-terminal and C-terminal domains of MBP, which are connected by a moveable hinge. The two domains undergo a large motion relative to one another as the protein moves from the open, unbound state to the closed, ligand-bound state. We generated, by doped-primer mutagenesis, amino acid substitutions that specifically disrupt the chemotactic function of MBP. These substitutions cluster in two well-defined regions that are nearly contiguous on the surface of MBP in its closed conformation. One region is in the N-terminal domain and one is in the C-terminal domain. The distance between the two regions is expected to change substantially as the protein goes from the open to the closed form. These results support a model in which ligand binding brings two recognition sites on MBP into the proper spatial relationship to interact with complementary sites on Tar. Mutations in MBP that appear to cause defects in interaction with MalF and MalG are distributed differently from mutations that primarily affect maltose taxis. We conclude that the regions of MBP that contact Tar and those that contact MalF and MalG are adjacent on the face of the protein opposite the hinge connecting the two domains and that those regions are largely, although perhaps not entirely, distinct.     

Maltose-binding protein interacts simultaneously and asymmetrically with both subunits of the Tar chemoreceptor

The Tar chemotactic signal transducer of Escherichia coli mediates attractant responses to L-aspartate and to maltose. Aspartate binds across the subunit interface of the periplasmic receptor domain of a Tar homodimer. Maltose, in contrast, first binds to the periplasmic maltose-binding protein (MBP), which in its ligand-stabilized closed form then interacts with Tar. Intragenic complementation was used to determine the MBP-binding site on the Tar dimer. Mutations causing certain substitutions at residues Tyr-143, Asn-145, Gly-147, Tyr-149, and Phe-150 of Tar lead to severe defects in maltose chemotaxis, as do certain mutations affecting residues Arg-73, Met-76, Asp-77, and Ser-83. These two sets of mutations defined two complementation groups when the defective proteins were co-expressed at equal levels from compatible plasmids. We conclude that MBP contacts both subunits of the Tar dimer simultaneously and asymmetrically. Mutations affecting Met-75 could not be complemented, suggesting that this residue is important for association of MBP with each subunit of the Tar dimer. When the residues involved in interaction with MBP were mapped onto the crystal structure of the Tar periplasmic domain, they localized to a groove at the membrane-distal apex of the domain and also extended onto one shoulder of the apical region.     

Effect of outer membrane permeability on chemotaxis in Escherichia coli.

The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC. Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC. A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays. AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II. Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate. Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability. These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E. coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane.     

The residue composition of the aromatic anchor of the second transmembrane helix determines the signaling properties of the aspartate/maltose chemoreceptor Tar of Escherichia coli

Repositioning of the tandem aromatic residues (Trp-209 and Tyr-210) at the cytoplasmic end of the second transmembrane helix (TM2) modulates the signal output of the aspartate/maltose chemoreceptor of Escherichia coli (Tar(Ec)). Here, we directly assessed the effect of the residue composition of the aromatic anchor by studying the function of a library of Tar(Ec) variants that possess all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. We identified three important properties of the aromatic anchor. First, a Trp residue at position 209 was required to maintain clockwise (CW) signal output in the absence of adaptive methylation, but adaptive methylation restored the ability of all of the mutant receptors to generate CW rotation. Second, when the aromatic anchor was replaced with tandem Ala residues, signaling was less compromised than when an Ala residue occupied position 209 and an aromatic residue occupied position 210. Finally, when Trp was present at position 209, the identity of the residue at position 210 had little effect on baseline signal output or aspartate chemotaxis, although maltose taxis was significantly affected by some substitutions at position 210. All of the mutant receptors we constructed supported some level of aspartate and maltose taxis in semisolid agar swim plates, but those without Trp at position 209 were overmethylated in their baseline signaling state. These results show the importance of the cytoplasmic aromatic anchor of TM2 in maintaining the baseline Tar(Ec) signal output and responsiveness to attractant signaling.     

Chimeric Chemoreceptors in Escherichia coli: Signaling Properties of Tar-Tap and Tap-Tar Hybrids

The Tap (taxis toward peptides) receptor and the periplasmic dipeptide-binding protein (DBP) of Escherichia coli together mediate chemotactic responses to dipeptides. Tap is a low-abundance receptor. It is present in 5- to 10-fold-fewer copies than high-abundance receptors like Tar and Tsr. Cells expressing Tap as the sole receptor, even from a multicopy plasmid at 5- to 10-fold-overexpressed levels, do not generate sufficient clockwise (CW) signal to tumble and thus swim exclusively smoothly (run). To study the signaling properties of Tap in detail, we constructed reciprocal hybrids between Tap and Tar fused in the linker region between the periplasmic and cytoplasmic domains. The Tapr hybrid senses dipeptides and is a good CW-signal generator, whereas the Tarp hybrid senses aspartate but is a poor CW-signal generator. Thus, the poor CW signaling of Tap is a property of its cytoplasmic domain. Eighteen residues at the carboxyl terminus of high-abundance receptors, including the NWETF sequence that binds the CheR methylesterase, are missing in Tap. The Tart protein, created by removing these 18 residues from Tar, has diminished CW-signaling ability. The Tapl protein, made by adding the last 18 residues of Tar to the carboxyl terminus of Tap, also does not support CW flagellar rotation. However, Tart and Tapl cross-react well with antibody directed against the conserved cytoplasmic region of Tsr, whereas Tap does not cross-react with this antibody. Tap does cross-react, however, with antibody directed against the low-abundance chemoreceptor Trg. The hybrid, truncated, and extended receptors exhibit various levels of methylation. However, Tar and Tapl, which contain a consensus CheR-binding motif (NWETF) at their carboxyl termini, exhibit the highest basal levels of methylation, as expected. We conclude that no simple correlation exists between the abundance of a receptor, its methylation level, and its CW-signaling ability.     

Chemotactic responses of Escherichia coli to small jumps of photoreleased L-aspartate

Computer-assisted motion analysis coupled to flash photolysis of caged chemoeffectors provides a means for time-resolved analysis of bacterial chemotaxis. Escherichia coli taxis toward the amino acid attractant L-aspartate is mediated by the Tar receptor. The physiology of this response, as well as Tar structure and biochemistry, has been studied extensively. The beta-2, 6-dinitrobenzyl ester of L-aspartic acid and the 1-(2-nitrophenyl)ethyl ether of 8-hydroxypyrene-1,3,6-tris-sulfonic acid were synthesized. These compounds liberated L-aspartate and the fluorophore 8-hydroxypyrene 1,3,6-tris-sulfonic acid (pyranine) upon irradiation with near-UV light. Photorelease of the fluorophore was used to define the amplitude and temporal stability of the aspartate jumps employed in chemotaxis experiments. The dependence of chemotactic adaptation times on aspartate concentration, determined in mixing experiments, was best fit by two Tar aspartate-binding sites. Signal processing (excitation) times, amplitudes, and adaptive recovery of responses elicited by aspartate jumps producing less than 20% change in receptor occupancy were characterized in photorelease assays. Aspartate concentration jumps in the nanomolar range elicited measurable responses. The response threshold and sensitivity of swimming bacteria matched those of bacteria tethered to glass by a single flagellum. Stimuli of similar magnitude, delivered either by rapid mixing or photorelease, evoked responses of similar strength, as assessed by recovery time measurements. These times remained proportional to change in receptor occupancy close to threshold, irrespective of prior occupancy. Motor excitation responses decayed exponentially with time. Rates of excitation responses near threshold ranged from 2 to 7 s-1. These values are consistent with control of excitation signaling by decay of phosphorylated pools of the response regulator protein, CheY. Excitation response rates increased slightly with stimulus size up to values limited by the instrumentation; the most rapid was measured to be 16 +/- 3 (SE) s-1. This increase may reflect simultaneous activation of CheY dephosphorylation, together with inhibition of its phosphorylation.     

Maltose chemoreceptor of Escherichia coli.

Strains carrying mutations in the maltose system of Escherichia coli were assayed for maltose taxis, maltose uptake at 1 and 10 muM maltose, and maltose-binding activity released by osmotic shock. An earlier conclusion that the metabolism of maltose is not necessary for chemoreception is extended to include the functioning of maltodextrin phosphorylase, the product of malP, and the genetic control of the maltose receptor by the product of malT is confirmed. Mutants in malF and malK are defective in maltose transport at low concentrations as well as high concentrations, as previously shown, but are essentially normal in maltose taxis. The product of malE has been previously shown to be the maltose-binding protein and was implicated in maltose transport. Most malE mutants are defective in maltose taxis, and all those tested are defective in maltose transport at low concentrations. Thus, as previously suggested, the maltose-binding protein probably serves as the recognition component of the maltose receptor, as well as a component of the transport system. tsome malE mutants release maltose-binding activity and are tactic toward maltose, although defective in maltose transport, implying that the binding protein has separate sites for interaction with the chemotaxis and transport systems. Some mutations in lamB, whose product is the receptor for the bacteriophage lamba, cause defects in maltose taxis, indicating some involvement of that product in maltose reception.     

Progress in the identification of interaction sites on the periplasmic maltose binding protein from E coli

The periplasmic maltose binding protein (MBP) is required for the high affinity transport of maltose and maltodextrins and for chemotaxis towards these sugars. In these functions, MBP interacts with proteins of the cytoplasmic membrane: MalF and MalG for transport, Tar for chemotaxis. A large number of MBP mutations have been isolated by us and other laboratories. We grouped these mutations into classes depending on the interactions affected and we represented the corresponding residues on the 3-D model for MBP so as to further identify the sites of MBP interacting with the MalF-MalG complex and with the Tar protein. MBP (like the other binding proteins) is composed of 2 lobes enclosing a cleft where the substrate binds. The face of the protein opposite the cleft seems to interact neither with MalF-MalG nor with Tar. The other face, corresponding to the cleft, contains sites for interactions with MalF-MalG and Tar. These sites appear to cover both sides of the cleft and may overlap in part. The present definition of the interaction sites suggests further that MBP has different in vivo orientations when it interacts with MalF-MalG or with Tar. This work constitutes an additional step in combining the use of genetic and structural analysis to define the interaction sites on MBP. Because of the structural similarities between periplasmic binding proteins, the regions of interaction defined could be relevant for other members of this family.