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.     

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.     

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.     

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.     

Cysteine-22 and cysteine-38 are not essential for the functions of maltoporin (LamB protein)

Maltoporin in the outer membrane of Escherichia coli contains two cysteine residues, at positions 22 and 38 in the primary sequence. The role of these residues in determining structural stability, and their contributions to the maltoporin binding sites for maltodextrins and bacteriophage lambda, was investigated. Site-directed mutagenesis was used to alter each of these residues to a serine. A double mutant lacking both cysteines was also isolated. None of the substitutions affected maltodextrin binding or the binding of phage lambda, suggesting the variant proteins retain a native binding-site conformation. The mutants were assembled at wild-type levels into the outer membrane as maltoporin trimers but the temperature-stability of the trimer greater than monomer dissociation was slightly reduced in the presence of the Cys 38 substitution. However, it is unlikely that the stability of trimers was due to disulfide linkages between subunits since the native trimers are stable under highly reducing conditions in the presence of SDS; more likely the Cys greater than Ser substitutions slightly perturb intra- or inter-subunit hydrophobic interactions in regions predicted to span across the outer membrane.     

Bacteriophage K20 requires both the OmpF porin and lipopolysaccharide for receptor function.

Mutations which prevent absorption of the bacteriophage K20 to Escherichia coli K-12 were selected by using an altered OmpF protein which confers the ability to grow on maltodextrin in the absence of the LamB maltoporin. The mutations map in the rfa gene cluster and alter the structure of the lipopolysaccharide core.     

Effect of point mutations on the in-vitro pore properties of maltoporin, a protein of Escherichia coli outer membrane

Maltoporin (LamB protein), a protein of Escherichia coli outer membrane forms ionic channels with a selectivity for maltose and maltodextrins (Dargent et al., 1987). The effect of different point mutations on maltoporin pore properties was investigated in vitro with planar bilayers. The mutations belong to three classes in terms of selective maltose transport in vivo: class A (substitution at positions 259 and 382) does not affect maltose transport, class B (position 163 and 245) decreases maltose transport down to 20 to 30%, and class C (position 18) almost completely abolishes selective maltose transport. This in-vitro study reveals that class A does not affect the pore properties in contrast to class B substitutions. The class B maltoporins are still able to form channels but display some specific features and altered specificity for maltose and maltodextrins. The substitution (Gly18----Val) alters trimer stability and impedes pore function (class C mutant). Thus, there is a good correlation between the specific transport properties of the mutated maltoporins in vivo and their behavior in vitro. These data, in combination with the asymmetric orientation of the protein within the bilayer and topological considerations, indicate that residues 245 and 163 do not belong to the selectivity filter. Mutations at these sites cause hindrance at the mouth of the pore on the outer domain of maltoporin.     

Residues in the alpha helix 7 of the bacterial maltose binding protein which are important in interactions with the Mal FGK2 complex.

The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even thought the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of alpha helix 7, therefore seem to play some important role in maltose transport, most probably in a productive interaction between the MalE protein and the membrane bound MalFGK2 complex.     

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.     

A cluster of charged and aromatic residues in the C-terminal portion of maltoporin participates in sugar binding and uptake

The maltoporin LamB of Escherichia coli K12 is a trimeric protein which facilitates the diffusion of maltose and maltodextrins through the bacterial outer membrane, and also acts as a non-specific porin for small hydrophilic molecules as well as a receptor for phages. Loop L9 (residues 375 to 405) is the most distal and largest surface-exposed loop of LamB. It comprises a central portion, which varies in size and sequence in the maltoporins of known sequence, flanked by two conserved regions containing charged and aromatic residues. In order to identify the residues within the proximal region that are specifically involved in sugar utilization, we used site-directed mutagenesis to change, individually, each of the charged (five) and aromatic (three) residues in the region 371 to 379 into alanine. None of the eight single amino acid substitutions affected the phage receptor activity of LamB. In contrast, they all affected, to variable extents, maltoporin functions. For all the mutants, very good correlations were observed between the effects on sugar binding and on in vivo uptake. In no case were maltoporin functions completely abolished. Mutants E374 A and W376 A were the most impaired (with over 60% reduction in dextrin binding and in vivo uptake of maltose and maltopentaose). These two mutations also led to an increased bacterial sensitivity to bacitracin and vancomycin. The functional and structural implications are discussed. Received: 29 April 1998 / Accepted: 23 July 1998     

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.     

Mutations affecting antigenic determinants of an outer membrane protein of Escherichia coli.

The Escherichia coli LamB protein is located in the outer membrane. It is both a component of the maltose and maltodextrin transport system, and the receptor for phages lambda and K10. It is a trimer composed of three identical polypeptide chains, each containing 421 residues. Six independent mutants have been isolated, in which the LamB protein is altered in its interaction with one or more monoclonal antibodies specific for regions of the protein that are exposed at the cell surface. Some of the mutations also altered the binding site for phage lambda. All of the mutations were clustered in the same region of the lamB gene, corresponding to residues 333-394 in the polypeptide. This and previous results strongly suggest that a rather large segment of the LamB polypeptide, extending from residue 315 to 401, is exposed at the outer face of the outer membrane. This segment would bear the epitopes for the four available anti-LamB monoclonal antibodies that react with the cell surface, and part of the binding site for phage lambda.     

Mechanisms of solute transport through outer membrane porins: burning down the house

Porins mediate the uptake of nutrients across the outer membrane of Gram-negative bacteria. For general porins like OmpF, electrophysicoloigcal experiments now establish that the charged residues within their channels primarily modulate pore selectivity, rather than voltage-gated switching between open and closed states. Recent studies on the maltoporin, LamB, solidify the importance of its 'greasy slide' aromatic residues during sugar transport, and suggest the involvement of L9, in the exterior vestibule, as the initial maltodextrin binding site. The application of biophysical methodologies to the TonB-dependent porin, FepA, ostensibly reveal the opening and closing of its channel during ligand uptake, a phenomenon that was predicted but not previously demonstrated.     

In vivo and in vitro studies of transmembrane beta-strand deletion, insertion or substitution mutants of the Escherichia coli K-12 maltoporin

LamB of Escherichia coli K12, also called maltoporin, is an outer membrane protein, which specifically facilitates the diffusion of maltose and maltodextrin through the bacterial outer membrane. Each monomer is composed of an 18-stranded antiparallel beta-barrel. In the present work, on the basis of the known X-ray structure of LamB, the effects of modifications of the beta-barrel domain of maltoporin were studied in vivo and in vitro. We show that: (i) the substitution of the pair of strands beta13-beta14 of the E. coli maltoporin with the corresponding pair of strands from the functionally related maltoporin of Salmonella typhimurium yielded a protein active in vivo and in vitro; and (ii) the removal of one pair of beta-strands (deletion beta13-beta14) from the E. coli maltoporin, or its replacement by a pair of strands from the general porin OmpF of E. coli, leads to recombinant proteins that lost in vivo maltoporin activities but still kept channel formation and carbohydrate binding in vitro. We also inserted into deletion beta13-beta14 the portion of the E. coli LamB protein comprising strands beta13 to beta16. This resulted in a protein expected to have 20 beta-strands and which completely lost all LamB-specific activities in vivo and in vitro.     

Two-dimensional crystals of Escherichia coli maltoporin and their interaction with the maltose-binding protein.

We have reconstituted Escherichia coli maltoporin into phospholipid membranes at low lipid-to-protein ratios to produce two-dimensional crystals of this membrane protein. Electron microscopy of negatively stained membranes showed three different types of arrays, two of them hexagonal and the third rectangular, all diffracting to approximately (2 nm)-1. Furthermore, we have core-constituted maltoporin with the maltose-binding protein from E. coli, a soluble periplasmic protein that has been proposed to interact with maltoporin. One of the hexagonal arrays was found to bind maltose-binding protein molecules in a regular way, while the maltose-binding protein binding sites were not accessible in the other crystal forms. Difference maps from averaged decorated arrays and undecorated controls showed three symmetry-related maltose-binding protein binding sites per maltoporin trimer, of which not more than one is likely to be occupied at a given time. Using multivariate statistical analysis to select similar unit cells of the decorated maltoporin array, we have obtained a map showing the rough outline of a maltose-binding protein molecule interacting with the pore formed by a maltoporin trimer.     

Lambda Receptor in the Outer Membrane of Escherichia coli as a Binding Protein for Maltodextrins and Starch Polysaccharides

The starch polysaccharides amylose and amylopectin are not utilized by Escherichia coli, but are bound by the bacteria. The following evidence supports the view that the outer membrane lambda receptor protein, a component of the maltose/ maltodextrin transport system is responsible for the binding. (i) Amylose and amylopectin both inhibit the transport of maltose into E. coli. (ii) Both polysaccharides prevent binding of non-utilizable maltodextrins by the intact bacterium, a process previously shown to be dependent on components of the maltose transport system (T. Ferenci, Eur. J. Biochem., in press). (iii) A fluorescent amylopectin derivative, O-(fluoresceinyl thiocarbamoyl)-amylopectin, has been synthesized and shown to bind to E. coli in a reversible, saturable manner. Binding of O-(fluoresceinyl thiocarbamoyl)-amylopectin is absent in mutants lacking the lambda receptor, but mutations in any of the other components of the maltose transport system do not affect binding as long as lambda receptor is present. (iv) Using the inhibition of lambda receptor-dependent O-(fluoresceinyl thiocarbamoyl)-amylopectin binding as an assay, the affinities of the lambda receptor for maltodextrins and other sugars have been estimated. The affinity for dextrins increases with increasing degree of polymerization (K(d) for maltose, 14 mM; for maltotetraose, 0.3 mM; for maltodecaose, 0.075 mM). Maltose and some other di- and trisaccharides are inhibitory to amylopectin binding, but only at concentrations above 1 mM.     

Site-directed mutagenesis of the greasy slide aromatic residues within the LamB (maltoporin) channel of Escherichia coli: effect on ion and maltopentaose transport

The 3D-structure of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) is known from X-ray crystallography. The 3D structure suggests that a number of aromatic residues (Y6, Y41, W74, F229, W358 and W420) within the channel lumen are involved in carbohydrate and ion transport. All aromatic residues were replaced by alanine-scanning mutagenesis. Furthermore, LamB mutants were created in which two, three, four, five and all six aromatic residues were replaced to study their effects on ion and maltopentaose transport through LamB. The purified mutant proteins were reconstituted into lipid bilayer membranes and the single-channel conductance of the mutants was studied in conductance experiments. The results suggest that all aromatic residues provide some steric hindrance for ion transport through LamB. Highest impact is provided by Y6 and Y41 that are localized opposite Y118, which form the central constriction of the LamB channel. Stability constants for binding of maltopentaose to the mutant channels were measured using titration experiments with the carbohydrate. The mutation of one or several aromatic residue(s) led to a substantial decrease of the stability constant of binding. The highest effect was observed when all aromatic residues were replaced by alanine because no binding of maltopentaose could be detected in such a case. However, binding was again possible when Y118 was replaced by tryptophan. The carbohydrate-induced block of the channel function could be used also for the study of current noise through the different mutant LamB-channels. The analysis of the power density spectra of some of the mutants allowed the evaluation of the on-rate and off-rate constants (k1 and k(-1)) of carbohydrate binding to the binding site inside the channels. The results suggest that both on-rate and off-rate constants were affected by the mutations. For most mutants, k1 decreased and k(-1) increased. The possible influence of the aromatic residues of the greasy slide on carbohydrate and ion transport through LamB is discussed.     

Genetic mapping of starch- and Lambda-receptor sites in maltoporin: identification of substitutions causing direct and indirect effects on binding sites by cysteine mutagenesis

Cysteine mutagenesis was used to test the proximity of 16 residues to protein-ligand interaction sites in maltoporin (LamB protein). LamB protein with additional cysteines was incorporated into the outer membrane of Escherichia coli except with a Ser-30----Cys substitution. Phage Lambda and starch binding was assayed before and after incubation of mutants with six thiol-specific reagents. Four categories of mutation were recognized on the basis of phenotype and modification for each of the Lambda- and starch binding sites. The thiol modification experiments helped to clarify whether the phenotype of a mutation was due to a substitution at the binding site or an indirect perturbation of the structure. This study suggests that the cysteine mutagenesis/thiol modification approach may be usefully applied to the operational mapping of surface-accessible binding sites or epitopes.     

Investigation of the selectivity of maltoporin channels using mutant LamB proteins: mutations changing the maltodextrin binding site.

Wild-type and seven mutant maltoporins were purified and their channel-forming activities studied after reconstitution into black lipid membranes. The proteins were assayed for alterations at the maltodextrin binding site by measuring the sugar-dependent blockage of ion flux through these channels. Some substitutions (R8H, W74R) caused reduced channel affinity for all maltodextrins without changing single channel conductivities. The channel with a GlySer insertion after residue 9 was also poorly blocked by sugars but unique to this protein, the channel showed a striking, almost exponential increase of affinity with increasing maltodextrin chain length. In mutants with AspPro insertions after residues 79 and 183, there was an increase in affinity for glucose and maltose but not longer maltodextrins. The additional negative charge in the AspPro insertion mutants increased the cation selectivity of maltoporin channels, as did the decrease in positive charge resulting from the R8H substitution. A mutant with a W120C substitution also showed an increased affinity for glucose and maltose but reduced affinity for longer maltosaccharides. In contrast, a Y118F substitution resulted in an 8-fold increase in maltotriose affinity, but lesser improvements for other sugars. These results are interpreted to reflect changes in subsites contributing to an extended binding site within the channel, which in turn determines the overall sugar affinity of maltoporin.     

Sequence–function relationships in MalG, an inner membrane protein from the maltose transport system in Escherichia coli

The maIG gene encodes a hydrophobic cytoplasmic membrane protein which is required for the energy-dependent transport of maltose and maltodextrins in Escherichia coli. The MalG protein, together with MalF and MalK proteins, forms a multimeric complex in the membrane consisting of two MalK subunits for each MalF and MalG subunit. Fifteen mutations have been isolated in malG by random linker insertion mutagenesis. Two regions essential for maltose transport have been identified. In particular, a hydro philic region containing the peptidic motif EAA—G———I-LP, highly conserved among inner membrane proteins from binding protein-dependent transport systems, is essential for maltose transport. The results also show that several regions of MalG are not essential for function. A region (residues 30–50) encompassing the first predicted transmembrane segment and the first periplasmic loop in MalG may be modified extensively with little effect on maltose transport and no effect on the stability and the localization of the protein. A region located at the middle of the protein (residues 153–157) is not essential for the function of the protein. A region, essential for maltodextrin utilization but not for maltose transport, has been identified near the C-terminus of the protein.