Probing sugar translocation through maltoporin at the single channel level

Sugar permeation through maltoporin of Escherichia coli, a trimer protein that facilitates maltodextrin translocation across outer bacterial membranes, was investigated at the single channel level. For large sugars, such as maltohexaose, elementary events of individual sugar molecule penetration into the channel were readily observed. At small sugar concentrations an elementary event consists of maltoporin channel closure by one third of its initial conductance in sugar-free solution. Statistical analysis of such closures at higher sugar concentrations shows that all three pores of the maltoporin channel transport sugars independently. Interestingly, while channel conductance is only slightly asymmetric showing about 10% higher values at -200 mV than at +200 mV (from the side of protein addition), asymmetry in dependence of the sugar binding constant on the voltage polarity is about 20 times higher. Combining our data with observations made with bacteriophage-lambda we conclude that the sugar residence time is much more sensitive to (and is decreased by) voltages that are negative from the intra-cell side of the bacterial membrane.     

Probing the orientation of reconstituted maltoporin channels at the single-protein level

Recently we have shown that maltoporin channels reconstituted into black lipid membranes have pronounced asymmetric properties in both ion conduction and sugar binding. This asymmetry revealed also that maltoporin insertion is directional. However, the orientation in the lipid bilayer remained an open question. To elucidate the orientation, we performed point mutations at each side of the channel and analyzed the ion current fluctuation caused by an asymmetric maltohexaose addition. In a second series we used a chemically modified maltohexaose sugar molecule with inhibited entry possibility from the periplasmic side. In contrast to the natural outer cell wall of bacteria, we found that the maltoporin inserts in artificial lipid bilayer in such a way that the long extracellular loops are exposed to the same side of the membrane than protein addition. Based on this orientation, the directional properties of sugar binding were correlated to physiological conditions. We found that nature has optimized maltoporin channels by lowering the activation barriers at each extremity of the pore to trap sugar molecules from the external medium and eject them most efficiently to the periplasmic side.     

Substrate specificity of the Escherichia coli maltodextrin transport system and its component proteins

Maltooligosaccharides up to maltoheptaose are transported by the maltodextrin transport system of Escherichia coli. The overall substrate specificity of the transport system was investigated by using 15 maltodextrin analogues with various modifications at the reducing end of the oligosaccharides as competing substrates. The binding interaction of the analogues with maltoporin in the outer membrane and the periplasmic maltose-binding protein, the two protein components of the transport system with known specificity for maltodextrins, was also investigated. All analogues containing several α,1 → 4-glucosyl linkages were bound with high affinity by maltoporin and maltose-binding protein, regardless of O-methyl, O-nitrophenyl, β-glucosyl or β-fructosyl substitutions at the reducing end of the dextrins. Introduction of a negative charge or lack of a ring structure at the reducing end were also ineffective in abolishing binding by these two proteins. These results suggest that the structure of the reducing glucose is not important in the binding specificity of maltoporin or maltose-binding protein. However, the high affinity of these proteins for analogues was not in itself sufficient for recognition by the transport system overall. Maltohexaitol, 4-nitrophenyl α-maltotetraoside and 4-β-d-maltopentaosyl-d-glucopyranose were bound with the same affinity as comparable maltodextrins by both maltoporin and maltose-binding protein but were poorly recognized by the transport system. These results suggest that another, yet uninvestigated component of the transport system has a more restricted specificity towards changes at the reducing end of the maltodextrin molecule.     

On translocation through a membrane channel via an internal binding site: kinetics and voltage dependence

Here we present a model for maltodextrin translocation through maltoporin channels. In a first step, our theoretical analysis does consider the case of a single binding site for a given substrate in a structurally unaffected channel with a possibly different entrance barrier on either side. It is shown how by means of conventional electrical conductance measurements (including current noise analysis) the basic equilibrium and rate constants can be determined as functions of the applied voltage. Then also the net translocation rate of the substrate becomes accessible quantitatively. This most simple model mechanism has been extended to include a voltage-dependent fast conformational change of the channel that prevents the binding process. The so developed approach has been tested with experimental data for a single maltoporin trimer being reconstituted in black lipid membranes when studied in the presence of maltohexaose as the substrate. The experimental results turned out to be clearly incompatible with binding alone. They are, however, very satisfactorily fitted by pertinent theoretical curves if also inhibition of binding by a conformational transition is taken into account. Accordingly, quantitative evaluations of the underlying parameters and eventually of the translocation rate have been carried out successfully. Our analysis reveals a set of parameters necessary for an optimal translocation that nicely corresponds to natural conditions.     

Genetic analysis of sequences in maltoporin that contribute to binding domains and pore structure.

Maltoporin (LamB protein) is a maltodextrin transport protein in the outer membrane of Escherichia coli with binding sites for bacteriophage lambda and maltosaccharides. Binding of starch by bacteria was found to inhibit swarming of Escherichia coli in soft agar plates; the inhibition was dependent on the maltodextrin affinity of maltoporin. On the basis of this observation, chemotactic cell-sorting techniques were developed for the isolation and analysis of mutants with an altered starch-binding phenotype. Fifteen lamB mutations generated by hydroxylamine and linker mutagenesis, as well as spontaneous mutations, were analyzed. The effects of the mutations on starch and lambda-binding, as well as transport specificity, were assayed. Mutations that affect residues near 8 to 18, 74 to 82, and 118 to 121 were found to affect starch binding and maltodextrin-selective functions strongly, confirming and extending previous results with substitutions at these regions. Substitutions and insertions in two previously undefined regions in the protein, in or near residues 194 and 360, also resulted in defects in maltodextrin-specific functions and indicate that C-terminal parts of the protein also contribute to the discontinuous binding and pore domains. There was a detectable transport defect in all binding-affected mutants, and one mutation caused near-total pore blocking towards both maltose and nonmaltoside. The highly discontinuous phage lambda-binding site was affected by mutations near residues 9 and 10 and 194, as well as previously established regions near residues 18, 148 to 165, 245 to 259, and 380 to 400. The significance of these mutations is discussed in the context of a model of the functional topology of maltoporin. The additional role of regions near residues 10 and 120 in maltoporin assembly, as well as starch binding, was suggested by the temperature-sensitive biogenesis of maltoporin in strains with one- or two-codon insertion at these sites.     

Sequence determinants in the lamB gene of Escherichia coli influencing the binding and pore selectivity of maltoporin

Maltoporin (LamB protein) is a malto-oligosaccharide-selective pore protein in the outer membrane of Escherichia coli. The genetic basis of binding and transport specificity was investigated through cloning, mapping and sequencing lamB genes from seven independent mutants with various changes in maltodextrin binding affinities; these mutants were unchanged in binding phage lambda. Single amino acid substitutions specifically resulting in maltodextrin affinity changes were as follows: Arg8----His in two independent mutants resulted in much reduced affinity for all ligands and a smaller pore no longer selective for maltodextrins. A Trp74----Arg substitution resulted in a lower affinity for starch, a slight increase in maltose affinity but no striking pore changes. An Arg82----Ser resulted in lowered maltodextrin affinity, but increased affinity for sucrose in both binding and pore function. A Tyr118----Phe resulted in a higher affinity for both starch and maltose, a slightly larger pore and increased transport of maltohexaose by the pores. Asp121----Gly in two independent isolates resulted in a higher affinity for large dextrins and a marginally larger pore. These results suggest that the maltodextrin-selective functions reside in the N-terminal sequence of maltoporin and are separate from the phage lambda binding domains.     

Exclusion of high-molecular-weight maltosaccharides by lipopolysaccharide O-antigen of Escherichia coli and Salmonella typhimurium.

The barrier properties of lipopolysaccharide were studied by testing the influence of O-antigen on the binding of ligand to maltoporin in the outer membranes of Escherichia coli and Salmonella typhimurium. Maltoporin (LamB protein) of Escherichia coli K-12 was capable of interacting with macromolecular starch polysaccharides, as was previously shown by the binding of intact bacteria to fluorescein-labeled amylopectin or to starch-Sepharose columns. In contrast, strains with complete O-antigenic lipopolysaccharide showed reduced binding to these substrates. A similar result was obtained with Salmonella typhimurium LT2, which did not bind to starch unless rfa mutations removed noncore polysaccharide. The exclusion limit of the lipopolysaccharide permeability barrier to alpha-glucans was tested by measuring the maltoporin-dependent transport of maltose and its inhibition by maltodextrins of various sizes. Only amylopectin (molecular weight, greater than 25,000) was excluded in transport experiments, whereas maltodextrins with molecular weights of up to 2,000 were not excluded by the presence of an O-polysaccharide layer.     

Transposon Tn10-dependent expression of the lamB gene in Escherichia coli.

Among Tn10 insertions isolated in or near the malB region of Escherichia coli, one (zjb-729::Tn10) mapped between malK and lamB or late in malK and allowed MalT-independent expression of lamB. Tn10-dependent expression of a lamB-lacZ protein fusion was 25% of the expression of the fusion from the malK-lamB operon promoter in malTc constitutive strains. The maltoporin content of a strain carrying this Tn10 was about 20% that of a malTc malB+ strain. Transport of maltose at concentrations of below 10(-6) M was reduced about threefold. When maltoporin was present at about 50% of the level of malTc malB+ strains, maltose transport was largely restored. We conclude that maltoporin is not rate limiting for maltose transport in wild-type cells but becomes rate limiting when the ratio of maltoporin to other maltose transport components is reduced more than twofold.     

Maltose-binding protein does not modulate the activity of maltoporin as a general porin in Escherichia coli.

Maltoporin (lambda receptor) is part of the maltose transport system in Escherichia coli and is necessary for the facilitated diffusion of maltose and maltodextrins across the outer membrane. Maltoporin also allows the diffusion of nonmaltodextrin substrates, albeit with less efficiency. The preference of maltoporin for maltodextrins in vivo is thought to be the result of an interaction of maltoporin with the maltose-binding protein, the malE gene product. In a recent report Heuzenroeder and Reeves (J. Bacteriol. 144:431-435, 1980) suggested that this interaction establishes a gating mechanism which inhibits the diffusion of nonmaltodextrin substrates, such as lactose. To reinvestigate this important conclusion, we constructed ompR malTc strains carrying either the malE+ gene, the nonpolar malE444 deletion, or the malE254 allele, which specifies an interaction-deficient maltose-binding protein. Lactose uptake was measured at different concentrations below the Km of this transport system and under conditions where transport was limited by the diffusion through maltoporin. We found no difference in the kinetics of lactose uptake irrespective of the malE allele. We conclude that the maltose-binding protein does not modulate the activity of maltoporin as a general outer membrane porin.     

Docking of a single phage lambda to its membrane receptor maltoporin as a time-resolved event

We have been able to observe the first step in bacteriophage infection, the docking of phage lambda to its membrane receptor maltoporin, at the single-particle level. High-resolution conductance recording from a single trimeric maltoporin channel reconstituted into a planar lipid bilayer has allowed detection of the simultaneous and irreversible interaction of the phage tail with all three monomers of the receptor. The formation of a phage-maltoporin complex affects the channel transport properties. Our analysis demonstrates that phage attaches symmetrically to all three receptor monomers. The statistics of sugar binding to the phage-receptor complex on the side opposite to phage docking show that the monomers of maltoporin still bind sugar independently, with the kinetic constants expected from those of the phage-free receptor. This finding suggests that phage docking does not distort the structure of the receptor, and that the phage-binding regions are close to, but do not overlap with, the sugar-binding domains of the maltoporin monomers. However, ion fluxes through the pores of maltoporin in the phage-receptor complex share a new common pathway. We expect that the present study contributes to the current needs for structural information on the functional complexes involved in intercellular recognition.     

Oriented channels reveal asymmetric energy barriers for sugar translocation through maltoporin of Escherichia coli.

Sugar transport through maltoporin of Escherichia coli was investigated. This protein facilitates maltooligosaccharide translocation via a binding site in the channel. Because incorporation of the protein into the bilayer results in randomly orientated channels, we re-examined the postulated symmetric translocation model by reconstitution of maltoporin under an externally applied field. Upon binding of bacteriophage lambda, which exploit surface-exposed loops of maltoporin as the receptor, sugar permeation, but not the ion current, was blocked. Thus using the phage-to-probe orientation we were able to show that the channels were approximately 80% directionally inserted into the bilayer. Moreover, asymmetry of the channel was revealed because sugar entrance through the 'open' periplasmic side of maltoporin was similarly reduced. Here a new asymmetrical two-barrier model is presented. Based on liposome-swelling assays and current-fluctuation analysis we conclude that the periplasmic side of the porin shows a two- to threefold higher energy barrier than the extracellular loop-side of the channels.     

Formation and Excretion of Acetylmaltose After Accumulation of Maltose in Escherichia coli

malB⁺malQ strains accumulate maltose via the maltose-binding-protein-dependent transport system but are unable to metabolize it. Nevertheless, some of the maltose is modified after entering the cell. This newly formed compound exhibited a higher R_f value than did maltose upon thin-layer and paper chromatography with the usual sugar-separating solvents. Treatment of this compound with acid and alkali reformed maltose. The identity of this compound with acetylmaltose was derived from mass spectrometry. Nuclear magnetic resonance spectra of the compound confirmed the presence of the acetyl group but did not allow its precise location on the maltose moiety. However, linkage to the 1-position of maltose could be excluded. Analysis of the mass spectra indicated that the nonreducing end of maltose was acetylated. Other substrates of the maltose transport system, such as maltotetraose, maltopentaose, and maltohexaose, were also modified after accumulation into the cell. Several products were formed; the heterogeneity of these products was probably caused by different degrees of acetylation. The enzymatic activity responsible for maltose and maltodextrin acetylation is unknown. However, it is clear that the lacA-dependent thiogalactoside transacetylase was not necessary for the acetylation of maltose. Strains that accumulate maltose via a bypass of the normal malB-dependent transport system also acetylated maltose even in the absence of any malB gene products. Thus, the acetylating activity was not connected to the malB system. Acetylmaltose as well as acetylated maltodextrins was excreted into the medium. Acetylmaltose is not a substrate of the maltose transport system. Thus, maltose acetylation may be an effective detoxification mechanism.     

Translocation mechanism of long sugar chains across the maltoporin membrane channel

Maltoporin allows permeation of long maltodextrin chains. It tightly binds the amphiphilic sugar, offering both hydrophobic interactions with a helical lane of aromatic residues and H bonds with ionic side chains. The minimum-energy path of maltohexaose translocation is obtained by the conjugate peak refinement method, which optimizes a continuous string of conformers without applying constraints. This reveals that the protein is passive while the sugar glides screw-like along the aromatic lane. Near instant switching of sugar hydroxyl H bond partners results in two small energy barriers (of approximately 4 kcal/mol each) during register shift by one glucosyl unit, in agreement with a kinetic analysis of experimental dissociation rates for varying sugar chain lengths. Thus, maltoporin functions like an efficient translocation "enzyme," and the slow rate of the register shift (approximately 1/ms) is due to high collisional friction.     

Channel architecture in maltoporin: dominance studies with lamB mutations influencing maltodextrin binding provide evidence for independent selectivity filters in each subunit.

Maltoporin trimers constitute maltodextrin-selective channels in the outer membrane of Escherichia coli. To study the organization of the maltodextrin-binding site within trimers, dominance studies were undertaken with maltoporin variants of altered binding affinity. It has been established that amino acid substitutions at three dispersed regions of the maltoporin sequence (at residues 8, 82, and 360) resulted specifically in maltodextrin-binding defects and loss of maltodextrin channel selectivity; a substitution at residue 118 increased both binding affinity and maltodextrin transport. Strains heterodiploid for lamB were constructed in which these substitutions were encoded by chromosomal and plasmid-borne genes, and the relative level of maltoporin expression from these genes was estimated. Binding assays with bacteria forming maltoporin heterotrimers were performed in order to test for complementation between binding-negative alleles, negative dominance of negative over wild-type alleles, and possible dominance of negatives over the high-affinity allele. Double mutants with mutations affecting residues 8 and 118, 82 and 118, and 118 and 360 were constructed in vitro, and the dominance properties of the mutations in cis were also tested. There was no complementation between negatives and no negative dominance in heterotrimers. The high-affinity mutation was dominant over negatives in trans but not in cis. The affinity of binding sites in heterotrimer populations was characteristic of the high-affinity allele present and uninfluenced by the negative allele. These results are consistent with the presence of three discrete binding sites in a maltoporin trimer and suggest that the selectivity filter for maltodextrins is not at the interface between the three subunits.     

Bacterial chemotaxis to saccharides is governed by a trade-off between sensing and uptake

To swim up gradients of nutrients, E. coli senses nutrient concentrations within its periplasm. For small nutrient molecules, periplasmic concentrations typically match extracellular concentrations. However, this is not necessarily the case for saccharides, such as maltose, which are transported into the periplasm via a specific porin. Previous observations have shown that, under various conditions, E. coli limits maltoporin abundance so that, for extracellular micromolar concentrations of maltose, there are predicted to be only nanomolar concentrations of free maltose in the periplasm. Thus, in the micromolar regime, the total uptake of maltose from the external environment into the cytoplasm is limited not by the abundance of cytoplasmic transport proteins but by the abundance of maltoporins. Here, we present results from experiments and modeling suggesting that this porin-limited transport enables E. coli to sense micromolar gradients of maltose despite having a high-affinity ABC transport system that is saturated at these micromolar levels. We used microfluidic assays to study chemotaxis of E. coli in various gradients of maltose and methyl-aspartate and leveraged our experimental observations to develop a mechanistic transport-and-sensing chemotaxis model. Incorporating this model into agent-based simulations, we discover a trade-off between uptake and sensing: although high-affinity transport enables higher uptake rates at low nutrient concentrations, it severely limits the range of dynamic sensing. We thus propose that E. coli may limit periplasmic uptake to increase its chemotactic sensitivity, enabling it to use maltose as an environmental cue.     

Maltose transport and starch binding in phage-resistant point mutants of maltoporin. Functional and topological implications

The relationships between the bacteriophage lambda binding site, the starch binding site and the pore formed by maltoporin (LamB protein, lambda receptor protein) were investigated. Bacteria with single amino acid substitutions in the maltoporin sequence, which were previously shown to be strongly reduced in phage lambda sensitivity, were assayed for maltose- (and maltodextrin) selective pore functions. Maltose transport assays was performed at low substrate concentrations, under conditions where LamB is limiting for transport. It revealed three classes of mutants. Class A is composed of mutants with no effect on transport (substitutions at amino acid residues 154, 155, 259, 382 and 401); class B corresponds to mutants with a significant but variable reduction in transport (sites 148, 151, 152, 163, 164, 245, 247 and 250); class C is represented by a single mutant for which transport is almost completely abolished (site 18). Starch binding was assayed by two different methods that gave compatible results. In class A mutants, binding was normal, while no binding was observed in the class C mutant. Binding was impaired to various extents in category B mutants. There was a correlation between the level of impairment of starch binding and impairment of maltose transport, consistent with the notion that the residues influencing starch binding are inside, or in close proximity to, the pore. These results, together with previous data on starch-binding mutants that were not affected in phage binding (substitutions at residues 8, 74, 82, 118 and 121), suggest that the binding sites for starch and phage lambda overlap but are distinct. Mutations affecting transport and starch binding are located in the first third of the protein and in the region of residues 245 to 250. Mutations affecting phage adsorption are located mainly in the last two-thirds of the protein. The topological constraints suggested by the results with the available mutants altered in the lamB gene were used to propose a revised model of maltoporin folding across the outer membrane as well as to define the outlines of footprints of macromolecular binding sites (phage, starch and monoclonal antibodies) on the surface of the protein.     

The Periplasmic Cyclodextrin Binding Protein CymE from Klebsiella oxytoca and Its Role in Maltodextrin and Cyclodextrin Transport

Klebsiella oxytoca M5a1 has the capacity to transport and to metabolize α-, β- and γ-cyclodextrins. Cyclodextrin transport is mediated by the products of the cymE, cymF, cymG, cymD, and cymA genes, which are functionally homologous to the malE, malF, malG, malK, and lamB gene products of Escherichia coli. CymE, which is the periplasmic binding protein, has been overproduced and purified. By substrate-induced fluorescence quenching, the binding of ligands was analyzed. CymE bound α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, with dissociation constants (K_d) of 0.02, 0.14 and 0.30 μM, respectively, and linear maltoheptaose, with a K_d of 70 μM. In transport experiments, α-cyclodextrin was taken up by the cym system of K. oxytoca three to five times less efficiently than maltohexaose by the E. coli maltose system. Besides α-cyclodextrin, maltohexaose was also taken up by the K. oxytoca cym system, but because of the inability of maltodextrins to induce the cym system, growth of E. coli mal mutants on linear maltodextrin was not observed when the cells harbored only the cym uptake system. Strains which gained this capacity by mutation could easily be selected, however.     

Selectivity for maltose and maltodextrins of maltoporin, a pore-forming protein of E. coli outer membrane

Homogenous maltoporin (lamB protein), an Escherichia coli outer membrane spanning protein, was incorporated in phospholipid planar bilayers. It generates aqueous channels distinct from those formed by the non-specific porin (OmpF) or by phosphoporin (phoE protein). The single conductance, 150 pS in 1 M NaCl, is much smaller than that of the porins. The channels, which are poorly selective for cations and voltage independent, are specifically inhibited by maltose and maltodextrins. This inhibition, observed in the absence of maltose binding protein, demonstrates that the selectivity of maltoporin for maltose and maltodextrins is an intrinsic property of the protein.     

Affinity engineering of maltoporin: variants with enhanced affinity for particular ligands

Affinity-chromatographic selection on immobilized starch was used to selectively enhance the affinity of the maltodextrin-specific pore protein ( maltoporin , LamB protein, or lambda receptor protein) in the outer membrane of E. coli. Selection strategies were established for rare bacteria in large populations producing maltoporin variants with enhanced affinities for both starch and maltose, for starch but not maltose and for maltose but not starch. Three classes of lamB mutants with up to eight-fold increase in affinity for particular ligands were isolated. These mutants provide a unique range of modifications in the specificity of a transport protein.     

Three-dimensional reconstruction of maltoporin from electron microscopy and image processing.

Two dimensional crystals of maltoporin (or phage lambda receptor) were obtained by reconstitution of purified maltoporin trimers and Escherichia coli phospholipids by detergent dialysis. Two different trimer packing forms were observed. One was hexagonal (a = 7.8 nm) and one rectangular (a = 7.8 nm, b = 13.6 nm). In this paper we describe the three-dimensional structure of maltoporin, deduced from the study of the rectangular form by electron microscopy and image processing. At a resolution of approximately 2.5 nm, maltoporin trimers form aqueous channel triplets which appear to merge into a single outlet at the periplasmic surface of the outer membrane. The pore defined by maltoporin has a similar structure to that outlined by the matrix protein. From the results of functional studies by conductance measurement, it is concluded that the three channels defined by maltoporin act, contrary to those formed by the porin (OmpF protein), as a single conducting unit. A tentative outline of the maltoporin promoter is given. Maltoporin appears to be constituted by three different domains: a major rod-like domain spanning the membrane, a minor domain located near the periplasmic surface of the membrane and finally a central domain responsible for the splitting of the channel.