Evaluation of the rate constants of sugar transport through maltoporin (LamB) of Escherichia coli from the sugar-induced current noise

LamB (maltoporin) of Escherichia coli outer membrane was reconstituted into artificial lipid bilayer membranes. The channel contains a binding site for sugars and is blocked for ions when the site is occupied by a sugar. The on and off reactions of sugar binding cause an increase of the noise of the current through the channel. The sugar-induced current noise of maltoporin was used for the evaluation of the sugar-binding kinetics for different sugars of the maltooligosaccharide series and for sucrose. The on rate constant for sugar binding was between 10(6) and 10(7) M-1.s-1 for the maltooligosaccharides and corresponds to the movement of the sugars from the aqueous phase to the central binding site. The off rate (corresponding to the release of the sugars from the channel) decreased with increasing number of glucose residues in the maltooligosaccharides from approximately 2,000 s-1 for maltotriose to 180 s-1 for maltoheptaose. The kinetics for sucrose movement was considerably slower. The activation energies of the stability constant and of the rate constants for sugar binding were evaluated from noise experiments at different temperatures. The role of LamB in the transport of maltooligosaccharides across the outer membrane is discussed.     

Mechanism of sugar transport through the sugar-specific LamB channel ofEscherichia coli outer membrane

Summary Lipid bilayer experiments were performed with the sugar-specific LamB (maltoporin) channel ofEscherichia coli outer membrane. Single-channel analysis of the conductance steps caused by LamB showed that there was a linear relationship between the salt concentration in the aqueous phase and the channel conductance, indicating only small or no binding between the ions and the channel interior. The total or the partial blockage of the ion movement through the LamB channel was not dependent on the ion concentration in the aqueous phase. Both results allowed the investigation of the sugar binding in more detail, and the stability constants of the binding of a large variety of sugars to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the sugars. The channel was highly cation selective, both in the presence and absence of sugars, which may be explained by the existence of carbonyl groups inside the channel. These carbonyl groups may also be involved in the sugar binding via hydrogen bonds. The kinetics of the sugar transport through the LamB channel were estimated relative to maltose by assuming a simple one-site, two-barrier model from the relative rates of permeation taken from M. Luckey and H. Nikaido (Proc. Natl. Acad. Sci. USA 77:165–171 (1980a)) and the stability constants for the sugar binding given in this study.     

Role of a disulfide bond in the thermal stability of the LamB protein trimer in Escherichia coli outer membrane

In order to understand the unusual heat resistance of LamB protein (the outer membrane component of the maltose transport system in Escherichia coli and its receptor for bacteriophage lambda), we investigated the role of its 2 cysteinyl residues. Our studies show that Cys22 and Cys38 form an intrasubunit disulfide bond which contributes to the heat stability of the LamB protein trimer. Physical evidence for the disulfide was obtained by using site-directed mutagenesis to convert Asn36 to Met, which allowed cyanogen bromide cleavage between the 2 cysteines. Upon reduction one of the N36M fragments migrated as two pieces, resolved by two-dimensional polyacrylamide gel electrophoresis. Other mutagenized LamB proteins, in which 1 or both Cys residues were converted to Ser, exhibited a sharp loss of thermal stability. In contrast to wild-type LamB protein trimer, which does not dissociate to monomers even after 60 min at 100 degrees C, only 10-15% of the mutant LamB proteins remain trimeric after boiling 10 min. The disulfide bond in LamB protein is not required for its transport function, since both mutagenized LamB protein and N-ethylmaleimide-labeled LamB protein exhibit normal uptake of sugars in proteoliposomes. Finally, the disulfide bond must not be between subunits of the LamB trimer since reversible dissociation of trimer is achieved by low pH or denaturants in the absence of reducing agent.     

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.     

Structural Basis for Outer Membrane Sugar Uptake in Pseudomonads

Substrate-specific outer membrane channels of Gram-negative bacteria mediate uptake of many small molecules, including carbohydrates. The mechanism of sugar uptake by enterobacterial channels, such as Escherichia coli LamB (maltoporin), has been characterized in great detail. In pseudomonads and related organisms, sugar uptake is not mediated by LamB but by OprB channels. Beyond the notion that OprB channels seem to prefer monosaccharides as substrates, very little is known about OprB-mediated sugar uptake. Here I report the X-ray crystal structure of an OprB channel from Pseudomonas putida F1. The structure shows that OprB forms a monomeric, 16-stranded β-barrel with a constriction formed by extracellular loops L2 and L3. The side chains of two highly conserved arginine residues (Arg⁸³ and Arg¹¹⁰) and a conserved glutamate (Glu¹⁰⁶) line the channel constriction and interact with a bound glucose molecule. Liposome swelling uptake assays show a strong preference for monosaccharide transport over disaccharides. Moreover, substrates with a net negative charge are disfavored by the channel, probably due to the negatively charged character of the constriction. The architecture of the eyelet and the absence of a greasy slide provide an explanation for the observed specificity of OprB for monosaccharides rather than the oligosaccharides preferred by LamB and related enterobacterial channels.     

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.     

The sugar-specific outer membrane channel ScrY contains functional characteristics of general diffusion pores and substrate-specific porins

Escherichia coli K-12 strain PS1-28-37 carries the multicopy plasmid pPSO28-37 containing a DNA fragment coding for two of the proteins that enable bacteria to utilize sucrose as sole carbon source. One of the different gene products of the plasmid is the outer membrane protein, ScrY. This protein was isolated and purified by chromatography across a gel filtration column. Reconstitution experiments with lipid bilayer membrane demonstrated that ScrY formed ion-permeable channels with properties very similar to those of general diffusion pores of enteric bacteria. The presence of sugars in the aqueous phase led to a dose-dependent block of ion transport through the channel, like the situation found with LamB (maltoporin) of Escherichia coli and Salmonella typhimurium. The binding constants of a variety of different sugars were determined. The stability constant for malto-oligosaccharide binding increased with increasing numbers of glucose residues. Disaccharides generally had a larger binding constant than monosaccharides. The binding of different sugars to ScrY and LamB of E. coli is discussed with respect to the kinetics of sugar movement through the channel.     

Site-directed mutagenesis of tyrosine 118 within the central constriction site of the LamB (maltoporin) channel of Escherichia coli. II. Effect on maltose and maltooligosaccharide binding kinetics

The 3-D structure of the maltooligosaccharide-specific LamB channel of Escherichia coli (also called maltoporin) is known from x-ray crystallography. The central constriction of the channel formed by the external loop 3 is controlled by tyrosine 118. Y118 was replaced by site-directed mutagenesis by 10 other amino acids (alanine (A), isoleucine (I), asparagine (N), serine (S), cysteine (C), aspartic acid (D), arginine (R), histidine (H), phenylalanine (F), and tryptophan (W)) including neutral ones, negatively and positively charged amino acids to study the effect of their size, their hydrophobicity index, and their charge on maltose and maltooligosaccharide binding to LamB. The mutants were reconstituted into lipid bilayer membranes and the stability constants for binding of maltose, maltotriose, maltopentaose, and maltoheptaose to the channel were measured using titration experiments. The mutation of Y118 to any other non-aromatic amino acid led to a substantial decrease of the stability constant of binding by factors between about two and six. The highest effect was observed for the mutant Y118A. Replacement of Y118 by the two other aromatic amino acids, phenylalanine (F) and tryptophan (W), resulted in a substantial increase of the stability constant maximally by a factor of almost 400 for the Y118W mutant. The carbohydrate-induced block of the channel function was used for the study of current noise through the different mutant LamB channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k(1) and k(-1)) of sugar binding. The results suggest that both rate constants were affected by the mutations. For most mutants, with the exception of Y118F and Y118W, k(1) decreased and k(-1) increased, whereas the opposite was found for the aromatic amino acid mutants. The results suggest that tyrosine 118 has a crucial effect on carbohydrate transport through LamB.     

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.     

Sugar transport through maltoporin of Escherichia coli: role of the greasy slide

The lining of the maltodextrin-specific maltoporin (LamB) channel exhibits a string of aromatic residues, the greasy slide, part of which has been shown previously by crystallography to be involved in substrate binding. To probe the functional role of the greasy slide, alanine scanning mutagenesis has been performed on the six greasy slide residues and Y118 at the channel constriction. The mutants were characterized by an in vivo uptake assay and sugar-induced-current-noise analysis. Crystallographic analysis of the W74A mutant showed no perturbation of the structure. All mutants showed considerably decreased maltose uptake rates in vivo (<10% of the wild-type value), indicating the functional importance of the investigated residues. Substitutions at the channel center revealed appreciably increased (up to 100-fold) in vitro half-saturation concentrations for maltotriose and maltohexaose binding to the channel. Sugar association rates, however, were significantly affected also by the mutations at either end of the slide (W74A, W358A, and F227A), an effect which became most apparent upon nonsymmetrical sugar addition. The kinetic data are discussed on the basis of an asymmetric one-site two-barrier model, which suggests that, at low substrate concentrations, as are found under physiological conditions, only the heights of the extracellular and periplasmic barriers, which are reduced by the presence of the greasy slide, determine the efficiency of this facilitated diffusion channel.     

Severity of osteogenesis imperfecta and structure of a collagen-like peptide modeling a lethal mutation site

We show that there are correlations between the severities of osteogenesis imperfecta (OI) phenotypes and changes in the residues near the mutation site. Our results show the correlations between the severity of various forms of the inherited disease OI and alteration of residues near the site of OI causing mutations. Among our many observed correlations are particularly striking ones between the presence of nearby proline residues and lethal mutations, and the presence of nearby alanines residues and nonlethal mutations. We investigated the possibility that these correlations have a structural basis using molecular dynamics simulations of collagen-like molecules designed to mimic the site of a lethal OI mutation in collagen type I. Our significant finding is that interchain hydrogen bonding is greatly affected by variations in residue type. We found that the strength of hydrogen bond networks between backbone atoms on different chains depends on the local residue sequence and is weaker in proline-rich regions of the molecule. We also found that an alanine at a site near an OI mutation causes less structural disruption than a proline, and that residue side chains also form interchain hydrogen bonds with frequencies that are dependent on residue type. For example, arginine side chains form strong hydrogen bonds with the backbone of the subsequent peptide chain, while lysine and glutamine less frequently form similar hydrogen bonds. This decrease in the observed hydrogen bond frequency correlates with a decrease in the experimentally determined thermal stability. We contrasted general structural properties of model collagen peptides with and without the mutation to examine the effect of the single-point mutation on the surrounding residues.     

Noise analysis of ion current through the open and the sugar-induced closed state of the LamB channel of Escherichia coli outer membrane: evaluation of the sugar binding kinetics to the channel interior.

LamB, a sugar-specific channel of Escherichia coli outer membrane was reconstituted into lipid bilayer membranes and the current noise was investigated using fast Fourier transformation. The current noise through the open channels had a rather small spectral density, which was a function of the inverse frequency up to about 100 Hz. The spectral density of the noise of the open LamB channels was a quadratic function of the applied voltage. Its magnitude was not correlated to the number of channels in the lipid bilayer membrane. Upon addition of sugars to the aqueous phase the current decreased in a dose-dependent manner. Simultaneously, the spectral density of the current noise increased drastically, which indicated interaction of the sugars with the binding site inside the channel. The frequency dependence of the spectral density was of Lorentzian type, although the power of its frequency dependence was not identical to -2. Analysis of the power density spectra using a previously proposed simple model (Benz, R., A. Schmid, and G. H. Vos-Scheperkeuter. 1987. J. Membr. Biol. 100: 12-29), allowed the evaluation of the on- and the off-rate constants for the maltopentaose binding to the binding site inside the LamB channels. This means also that the maltopentaose flux through the LamB channel could be estimated by assuming a simple one-site, two-barrier model for the sugar transport from the results of the noise analysis.     

Conformation-Determining role for the N-acetyl group in the O-glycosidic linkage, α-GalNAc-Thr

An ¹H-nmr study of 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-D-galactopyranose (AcGalNAc) glycosylated Thr-containing tripeptides in Me₂SO-d₆ solution reveals two mutually exclusive intramolecular hydrogen bonds. In Z-Thr(AcGalNAc)-Ala-Ala-OMe, there is an intramolecular hydrogen bond between the Thr amide proton and the sugar N-acetyl carbonyl oxygen. The strength of this hydrogen bond will be dependent on the amino acid residues on the Thr C terminal side to some undetermined distance. In Ac-Thr(AcGalNAc)-Ala-Ala-OMe, a different intramolecular hydrogen bond between the sugar N-acetyl amide proton and the Thr carbonyl oxygen exists. The choice of hydrogen bonds seems dependent on the bulkiness of the residues on the Thr N terminal side. The consequence of such strong hydrogen bonds is a clearly defined orientation of the sugar moiety with respect to the peptide backbone. In the former, the plane of the sugar pyranose ring is roughly oriented perpendicularly to the peptide backbone. The latter orientation is where the plane of the sugar ring is roughly in line with the peptide backbone. In both orientations, the sugar moiety can increase the shielding of the neighboring amino acid residues from the solvent. The idea that the amino acid residues near the glycosylated Thr influence orientation of the sugar moiety with respect to the peptide backbone and in turn possibly hinder peptide backbone flexibility has interesting implications in the conformational as well as the biological role of O-glycoproteins.     

Extended sugar slide function for the periplasmic coiled coil domain of ScrY

Several bacterial outer membrane proteins have a periplasmic extension whose structure and function remain elusive. Here, the structure/function relationship of the N-terminal periplasmic domain of the sucrose-specific outer membrane channel ScrY was investigated. Circular dichroism and analytical centrifugation demonstrated that the N-terminal domain formed a parallel, three-stranded coiled coil. When this domain was fused to the maltose-specific channel LamB, permeation of maltooligosaccharides in liposomes increased with increasing sugar chain length whereas wild-type LamB showed the opposite effect. Current fluctuation analysis demonstrated increased off-rates for sugar transport through the fusion protein. Moreover, equilibrium dialysis showed an affinity of sucrose for the isolated N-terminal peptide. Together these results demonstrate a novel function for coiled coil domains, operating as an extended sugar slide.     

On the evolutionary conservation of hydrogen bonds made by buried polar amino acids: the hidden joists,braces and trusses of protein architecture

Background  

The hydrogen bond patterns between mainchain atoms in protein structures not only give rise to regular secondary structures but also satisfy mainchain hydrogen bond potential. However, not all mainchain atoms can be satisfied through hydrogen bond interactions that arise in regular secondary structures; in some locations sidechain-to-mainchain hydrogen bonds are required to provide polar group satisfaction. Buried polar residues that are hydrogen-bonded to mainchain amide atoms tend to be highly conserved within protein families, confirming that mainchain architecture is a critical restraint on the evolution of proteins. We have investigated the stabilizing roles of buried polar sidechains on the backbones of protein structures by performing an analysis of solvent inaccessible residues that are entirely conserved within protein families and superfamilies and hydrogen bonded to an equivalent mainchain atom in each family member.     

Statistical and molecular dynamics studies of buried waters in globular proteins

Buried solvent molecules are common in the core of globular proteins and contribute to structural stability. Folding necessitates the burial of polar backbone atoms in the protein core, whose hydrogen-bonding capacities should be satisfied on average. Whereas the residues in alpha-helices and beta-sheets form systematic main-chain hydrogen bonds, the residues in turns, coils and loops often contain polar atoms that fail to form intramolecular hydrogen bonds. The statistical analysis of 842 high resolution protein structures shows that well-resolved, internal water molecules preferentially reside near residues without alpha-helical and beta-sheet secondary structures. These buried waters most often form primary hydrogen bonds to main-chain atoms not involved in intramolecular hydrogen bonds, providing strong evidence that hydrating main-chain atoms is a key structural role of buried water molecules. Additionally, the average B-factor of protein atoms hydrogen-bonded to waters is smaller than that of protein atoms forming intramolecular hydrogen bonds, and the average B-factor of water molecules involved in primary hydrogen bonds with main-chain atoms is smaller than the average B-factor of water molecules involved in secondary hydrogen bonds to protein atoms that form concurrent intramolecular hydrogen bonds. To study the structural coupling between internal waters and buried polar atoms in detail we simulated the dynamics of wild-type FKBP12, in which a buried water, Wat137, forms one side-chain and multiple main-chain hydrogen bonds. We mutated E60, whose side-chain hydrogen bonds with Wat137, to Q, N, S or A, to modulate the multiplicity and geometry of hydrogen bonds to the water. Mutating E60 to a residue that is unable to form a hydrogen bond with Wat137 results in reorientation of the water molecule and leads to a structural readjustment of residues that are both near and distant to the water. We predict that the E60A mutation will result in a significantly reduced affinity of FKBP12 for its ligand FK506. The propensity of internal waters to hydrogen bond to buried polar atoms suggests that ordered water molecules may constitute fundamental structural components of proteins, particularly in regions where alpha-helical or beta-sheet secondary structure is not present.     

A model of maltodextrin transport through the sugar-specific porin, LamB, based on deletion analysis.

LamB facilitates the uptake of maltose and maltodextrins across the bacterial outer membrane and acts as a general porin for small molecules. Using directed deletion mutagenesis we removed several regions of the LamB polypeptide and identified a polypeptide loop that both constricts the maltoporin channel and binds maltodextrins. In conjunction with a second sugar binding site that we identified at the rim of the channel, these data clarify, for the first time, the mechanism of transport through a substrate-specific porin. Furthermore, unlike the transverse loops of general porins, which originate from a central location in their primary structure, the loop that regulates LamB permeability originates from a C-terminal site. Thus LamB represents a second distinct class of porins in the bacterial outer membrane that is differently organized and separately evolved from OmpF-type, general porins.     

Prudent modeling of core polar residues in computational protein design

Hydrogen bond interactions were surveyed in a set of protein structures. Compared to surface positions, polar side-chains at core positions form a greater number of intra-molecular hydrogen bonds. Furthermore, the majority of polar side-chains at core positions form at least one hydrogen bond to main-chain atoms that are not involved in hydrogen bonds to other main-chain atoms. Based on this structural survey, hydrogen bond rules were generated for each polar amino acid for use in protein core design. In the context of protein core design, these prudent polar rules were used to eliminate from consideration polar amino acid rotamers that do not form a minimum number of hydrogen bonds. As an initial test, the core of Escherichia coli thioredoxin was selected as a design target. For this target, the prudent polar strategy resulted in a minor increase in computational complexity compared to a strategy that did not allow polar residues. Dead-end elimination was used to identify global minimum energy conformations for the prudent polar and no polar strategies. The prudent polar strategy identified a protein sequence that was thermodynamically stabilized by 2.5 kcal/mol relative to wild-type thioredoxin and 2.2 kcal/mol relative to a thioredoxin variant whose core was designed without polar residues.     

Sucrose transport through maltoporin mutants of Escherichia coli.

Maltoporin (LamB) and sucrose porin (ScrY) reside in the bacterial outer membrane and facilitate the passive diffusion of maltodextrins and sucrose, respectively. To gain further insight into the determinants of solute specificity, LamB mutants were designed to allow translocation of sucrose, which hardly translocates through wild-type LamB. Three LamB mutants were studied. (a) Based on sequence and structure alignment of LamB with ScrY, two LamB triple mutants were generated (R109D, Y118D,D121F; R109N,Y118D,D121F) to mimic the ScrY constriction. The crystal structure of the first of these mutants was determined to be 3.2 A and showed an increased ScrY-like cross-section except for D109 that protrudes into the channel. (b) Based on this crystal structure a double mutant was generated by truncation of the two residues that obstruct the channel most in LamB (R109A,Y118A). Analysis of liposome swelling and in vivo sugar uptake demonstrated substantial sucrose permeation through all mutants with the double alanine mutant performing best. The triple mutants did not show a well-defined binding site as indicated by sugar-induced ion current noise analysis, which can be explained by remaining steric interference as deduced from the crystal structure. Binding, however, was observed for the double mutant that had the obstructing residues truncated to alanines.     

Interhelical hydrogen bonds in the CFTR membrane domain.

Critical mutations in the membrane-spanning domains of proteins cause many human diseases. We report the expression in Escherichia coli of helix-loop-helix segments of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel domain in milligram quantities. Analysis of gel migration patterns of these constructs, in conjunction with circular dichroism spectroscopy, demonstrate that a neutral-to-charged, CF-phenotypic point mutation of a hydrophobic residue (V232D) in the CFTR transmembrane (TM) helix 4 induces a hydrogen bond with neighboring wild type Gln 207 in TM helix 3. As an electrostatic crosslink within a hydrocarbon phase, such a hydrogen bond could alter the normal assembly and alignment of CFTR TM helices and/or impede their movement in response to substrate transport. Our results imply that membrane proteins may be vulnerable to loss of function through formation of membrane-buried interhelical hydrogen bonds by partnering of proximal polar side chains.