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.     

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.     

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.     

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.     

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.     

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.     

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     

Interaction of bacteriophage lambda with its cell surface receptor: an in vitro study of binding of the viral tail protein gpJ to LamB (Maltoporin)

The cell surface receptor for bacteriophage Lambda is LamB (maltoporin). Responsible for phage binding to LamB is the C-terminal part, gpJ, of phage tail protein J. To study the interaction between LamB and gpJ, a chimera protein composed of maltose binding protein (MBP or MalE) connected to the C-terminal part of J (gpJ, amino acids 684-1131) of phage tail protein J of bacteriophage Lambda was expressed in Escherichia coli and purified to homogeneity. The interaction of the MBP-gpJ chimera protein with reconstituted LamB and its mutants LamB Y118G and the loop deletion mutant LamB Delta4+Delta6+Delta9v was studied using planar lipid bilayer membranes on a single-channel and multichannel level. Titration with the MBP-gpJ chimera blocked completely the ion current through reconstituted LamB when it was added to the cis side, the extracellular side of LamB with a half-saturation constant of approximately 6 nM in 1 M KCl. Control experiments with LamB Delta4+Delta6+Delta9v from which all major external loops had been removed showed similar blocking, whereas MBP alone caused no visible effect. Direct conductance measurement with His(6)-gpJ that contained a hexahistidyl tag (His(6) tag) at the N-terminal end of the protein for easy purification revealed no blocking of the ion current, requiring other measurements for the binding constant. However, when maltoporin was preincubated with His-gpJ, MBP-gpJ could not block the channel, which indicated that also His(6)-gpJ bound to the channel. High-molecular mass bands on SDS-PAGE and Western blots, confirming the planar lipid bilayer experiment results, also demonstrated stable complex formation between His(6)-gpJ and LamB or LamB mutants. The results revealed that phage Lambda binding includes not only the extracellular loops.     

Bacteriophage lambda receptor site on the Escherichia coli K-12 LamB protein.

We have analyzed eight new phage-resistant missense mutations in lamB. These mutations identify five new amino acid residues essential for phage lambda adsorption. Two mutations at positions 245 and 382 affect residues which were previously identified, but lead to different amino acid changes. Three mutations at residues 163, 164, and 250 enlarge and confirm previously proposed phage receptor sites. Two different mutations at residue 259 and one at 18 alter residues previously suggested as facing the periplasmic face. The mutation at residue 18 implicates for the first time the amino-terminal region of the LamB protein in phage adsorption. The results are discussed in terms of the topology of the LamB protein.     

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.     

LamB (maltoporin) of Salmonella typhimurium: isolation, purification and comparison of sugar binding with LamB of Escherichia coli

LamB (maltoporin) of Salmonella typhimurium was found to be more strongly associated with the murein than OmpF. It was purified in one step using a hydroxyapatite (HTP) column. Reconstitution of the pure protein with lipid bilayer membrane showed that LamB of S. typhimurium formed small ion-permeable channels with a single channel conductance of about 90 pS in 1 M KCl and some preference for cations over anions. The conductance concentration curve was linear, which suggested that LamB of S. typhimurium does not contain any binding site for ions. Pore conductance was completely inhibited by the addition of 20 mM maltotriose. Titration of the LamB-induced membrane conductance with different sugars, including all members of the maltooligosaccharide series up to seven glucose residues, suggested that the channel contains, like LamB (maltoporin) of Escherichia coli, a binding site for sugars. The binding constant of sugars of the maltooligosaccharide series increased with increasing number of glucose residues up to five (saturated). Small sugars had a higher stability constant for sugar binding relative to LamB of E. coli. The advantage of a binding site inside a specific porin for the permeation of solutes is discussed with respect to the properties of a general diffusion porin.     

Epitope mapping by cysteine mutagenesis: Identification of residues involved in recognition by three monoclonal antibodies directed against LamB glycoporin in the outer membrane of Escherichia coli

Abstract Site-directed mutagenesis of the lamB gene was used to introduce individual cysteine substitutions at 20 sites in two regions (surface loops L7 and L8) of LamB protein significant in antibody recognition. Characterisation of cysteine mutants involved immunoblotting with three surface-specific monoclonal antibodies (mAb72, mAb302, mAb347) before and after incubation with thiol-specific reagents. In contrast to an earlier study that showed no amino acid changes affecting recognition by all three antibodies, changes at six amino acids were found to influence a common core epitope. These core sites included one residue (T336) in the predicted loop L7 containing amino acids 329–342 and four (Y379, N387, N389, K392, F398) in the large surface loop involving residues 370–412. Individual antibodies made additional but distinct contacts within the two studied regions, with mAb347 binding the most different and affected by seven substitutions in the 328–338 regions. The lamB mutants were also tested for phage λ receptor activity and starch binding before and after thiol modification and were useful in extending previous maps of these ligand binding sites.     

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.     

Combinatorial mutagenesis of the lamB gene: residues 41 through 43, which are conserved in Escherichia coli outer membrane proteins, are informationally important in maltoporin structure and function.

A new strategy for combinatorial mutagenesis was developed and applied to residues 40 through 60 of LamB protein (maltoporin), with the aim of identifying amino acids important for LamB structure and function. The strategy involved a template containing a stop codon in the target sequence and a pool of random degenerate oligonucleotides covering the region. In vitro mutagenesis followed by selection for function (Dex+, ability to utilize dextrins) corrected the nonsense mutation and simultaneously forced incorporation of a random mutation(s) within the region. The relative importance of each residue within the target was indicated by the frequency and nature of neutral and deleterious mutations recovered at each position. Residues 41 through 43 in LamB accepted few neutral substitutions, whereas residues 55 through 57 were highly flexible in this regard. Consistent with this finding was that the majority of defective mutants were altered at residues 41 to 43. Characterization of these mutants indicated that the nature of residues 41 to 43 influenced the amount of stable protein in the outer membrane. These results, as well as the conserved nature of this stretch of residues among outer membrane proteins, suggest that residues 41 to 43 of LamB play an important role in the process of outer membrane localization.     

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.     

Further sequence analysis of the phage lambda receptor site. Possible implications for the organization of the lamB protein in Escherichia coli K12

We present the DNA sequence alterations due to seven lamB missense mutations yielding resistance to phages lambda and K10. They reveal five different amino acid positions in the LamB protein. Three positions (245, 247 and 249) define a new region required for phage adsorption. The two other positions (148 and 152) belong to a region where mutations to phage resistance has already been detected. These two regions are hydrophilic and could belong to turns of the protein located at the surface of the cell. All the missense mutational alterations to phage resistance sequenced in the LamB protein correspond to 10 sites located in four different segments of the polypeptide chain. We discuss their location in terms of the notion of phage receptor site and of a working model for the organization of this protein in the outer membrane of Escherichia coli.     

Signal sequence processing is required for the assembly of LamB trimers in the outer membrane of Escherichia coli.

Proteins destined for either the periplasm or the outer membrane of Escherichia coli are translocated from the cytoplasm by a common mechanism. It is generally assumed that outer membrane proteins, such as LamB (maltoporin or lambda receptor), which are rich in beta-structure, contain additional targeting information that directs proper membrane insertion. During transit to the outer membrane, these proteins may pass, in soluble form, through the periplasm or remain membrane associated and reach their final destination via sites of inner membrane-outer membrane contact (zones of adhesion). We report lamB mutations that slow signal sequence cleavage, delay release of the protein from the inner membrane, and interfere with maltoporin biogenesis. This result is most easily explained by proposing a soluble, periplasmic LamB assembly intermediate. Additionally, we found that such lamB mutations confer several novel phenotypes consistent with an abortive attempt by the cell to target these tethered LamB molecules. These phenotypes may allow isolation of mutants in which the process of outer membrane protein targeting is altered.     

The gene bglH present in the bgl operon of Escherichia coli, responsible for uptake and fermentation of beta-glucosides encodes for a carbohydrate-specific outer membrane porin

The cryptic gene bglH from the Escherichia coli chromosome was cloned into a tacOP-driven expression vector. The resulting plasmid was transferred into the porin-deficient E. coli strain KS26 and the protein was expressed by addition of IPTG. The BglH protein was localized in the outer membrane. It was purified to homogeneity using standard methods. Reconstitution experiments with lipid bilayer membranes defined BglH as a channel-forming component, i.e. it is an outer membrane porin. The single-channel conductance of BglH (560 pS in 1 M KCl) was only one-third of that of the general diffusion porins of E. coli outer membrane. The presence of carbohydrates in the aqueous phase led to a dose-dependent block of ion transport through the channel, similar to that found for LamB (maltoporin) of E. coli and Salmonella typhimurium, which means that BglH is a porin specific for the uptake of carbohydrates. The binding constants of a variety of different carbohydrates were calculated from titration experiments of the BglH-induced membrane conductance. The tightest binding was observed with the aromatic beta-D-glucosides arbutin and salicin, and with gentibiose and cellobiose. Binding of maltooligosaccharides to BglH was in contrast to their binding to LamB in that it was much weaker, indicating that the binding site of BglH for carbohydrates is different from that of LamB (maltoporin). The kinetics of cellopentaose binding to BglH was investigated using the carbohydrate-induced current noise and was compared with that of cellopentaose binding to LamB (maltoporin) and ScrY (sucroseporin).     

High-conductance channel induced by the interaction of phage lambda with its receptor maltoporin

Bacteriophage lambda that binds to liposomes bears its receptor maltoporin (LamB) and is able to inject its DNA into the internal space. During this process, the liposomes are permeabilized, suggesting that a transmembrane channel has formed (Roessner and Ihler (1986) J. Biol. Chem. 261, 386-390). This pore possibly constitutes the pathway used by lambda DNA to cross the membrane. We reconstituted purified LamB from Shigella in liposomes that were incubated with lambda phages. Addition of this mixture to a bilayer chamber resulted in the incorporation in planar bilayers of high-conductance channels whose conductance, kinetics and voltage dependence were totally different from those of maltoporin channels.