Rate Constants of Sugar Transport Through Two LamB Mutants ofEscherichia coli: Comparison with Wild-type Maltoporin and LamB ofSalmonella typhimurium

Jordy, Malte; Andersen, Christian; Schülein, K.; Ferenci, Thomas; Benz, Roland

doi:10.1006/jmbi.1996.0348

Cited by 44 publications

(62 citation statements)

References 26 publications

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“…Estimates of half-saturation by L-malate are clearly below 10 mM, which is in the physiological concentration range and could also be expected regarding the affinity of maltose binding in maltoporin (51,52). It is critical to determine the binding constant accurately by means of conductance measurements in a straightforward manner.…”

Section: Resultsmentioning

confidence: 96%

High Resolution Crystal Structures and Molecular Dynamics Studies Reveal Substrate Binding in the Porin Omp32

Zachariae

Klühspies

et al. 2006

Journal of Biological Chemistry

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The porin Omp32 is the major outer membrane protein of the bacterium Delftia acidovorans. The crystal structures of the strongly anion-selective porin alone and in complex with the substrate malate were solved at 1.5 and 1.45 Å resolution, respectively, and revealed a malate-binding motif adjacent to the channel constriction zone. Binding is mediated by interaction with a cluster of two arginine residues and two threonines. This binding site is specific for Omp32 and reflects the physiological adaptation of the organism to organic acids. Structural studies are combined with a 7-ns unbiased molecular dynamics simulation of the trimeric channel in a model membrane. Molecular dynamics trajectories show how malate ions are efficiently captured from the surrounding bulk solution by the electrostatic potential of the channel, translocated to the binding site region, and immobilized in the constriction zone. In accordance with these results, conductance measurements with Omp32 inserted in planar lipid membranes revealed binding of malate. The anion-selective channel Omp32 is the first reported example of a porin with a 16-stranded ␤-barrel and proven substrate specificity. This finding suggests a new view on the correlation of porin structure with substrate binding in specific channels.Porins are channel proteins that facilitate the exchange of ions and small hydrophilic substrate molecules across the outer membrane present in Gram-negative bacteria, mitochondria, and chloroplasts (1, 2). A number of bacterial porins have been studied structurally. They all possess ␤-barrels as the common structural principle (3). The trimeric bacterial porins are usually classified according to their number of barrelforming ␤-strands (3, 4); 18-stranded porins function as substratespecific channels and provide periplasmic binding proteins and uptake systems of the inner membrane with their specific substrates (5, 6), and 16-stranded porins are reported to merely act as unspecific ("general") diffusion pores, however, typically with a distinct ion selectivity (7-11).The current classification is mainly based on the crystal structures of 3 different 18-stranded and seven 16-stranded porins from Proteobacteria. Structural studies of substrate binding to trimeric porins were, thus, restricted to the sugar-specific maltoporins and to ScrY (5, 6). The nucleoside-specific pore protein Tsx and the fatty acid-binding transporter FadL are monomeric 12-and 14-stranded ␤-barrel proteins, respectively (17, 18). They are not regarded as porins proper; however, they also belong to the class of passive outer membrane channel proteins. Thus, in a more general view, substrate binding is not a special feature of 18-stranded outer membrane proteins. Several theories were developed to describe channel-substrate interactions which apply to diffusion pores in general (19). A decisive step of substrate translocation was modeled for maltoporin by conjugate peak refinement (20). Nonequilibrium molecular dynamics (MD) 2 and docking methods were applied to inves...

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Section: Resultsmentioning

confidence: 96%

High Resolution Crystal Structures and Molecular Dynamics Studies Reveal Substrate Binding in the Porin Omp32

Zachariae

Klühspies

et al. 2006

Journal of Biological Chemistry

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“…Since this residue contributes to the shape complementary of the channel with respect to maltodextrins, it is probably important for proper alignment of the sugar with the binding site. The inverse effect (stabilization of the complex) was reported for the Y118F maltoporin mutant (12), probably due to the increased hydrophobicity of the channel constriction and con- The main goal of the current-induced-noise analysis was to get a more detailed insight into the kinetics of sugar binding and into the role of the residues at the two ends of the slide that appear not to be part of the sugar binding site. A simple one-side, two-barrier kinetic model has been proposed previously (5,12) to describe the maltodextrin-maltoporin complex formation (S extrac , sugar at the extracellular side; P, protein [i.e., the maltoporin channel]; S perip , sugar at the periplasmic side) k1 k2…”

Section: Discussionmentioning

confidence: 99%

Sugar Transport through Maltoporin of Escherichia coli : Role of the Greasy Slide

Gelder¹,

Dumas²,

Bartoldus

et al. 2002

J Bacteriol

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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.Solute uptake into cells of gram-negative bacteria requires the crossing of two membranes, the outer and the inner membranes, which are separated by the periplasmic space (16). The outer membrane can be considered a defense wall protecting the cell against harmful compounds such as toxins and bile salts. Since a complete seal would impede the import of essential nutrients into the cell, several pore-forming proteins (porins) are located in the outer membrane (19). Transport of solutes through these water-filled channels most commonly proceeds by simple diffusion. For specific porins, diffusional transport is aided by the presence of a binding site.Maltoporin (LamB) specifically facilitates the translocation of malto-oligosaccharides across the outer membrane barrier. Its crystal structure (20) is trimeric, with each monomer consisting of an 18-stranded ␤-barrel enclosing a channel. Long loops protrude into the extracellular space, and short turns face the periplasm. Three loops fold into the ␤-barrel, with loop L3 constricting the channel about halfway through. In the channel lining, there are six contiguous aromatic residues, which form an elongated apolar stripe, the greasy slide, extending from the vestibule through the channel constriction to the periplasmic outlet (Fig. 1). The first of these residues, W74, is provided by L2 of a neighboring subunit. The next three residues, Y41, Y6, and W420, are situated in the middle of the channel. They are followed by W358 and F227, which reside in the wide periplasmic exit hall. A comparable ...

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“…Known maltoporin sizes range from M r 42,500 in Vibrio cholerae (Lang & Palva, 1993) to 48,000 in Salmonella typhimurium (Schneider et al, 1992). Numerous mutational experiments (Klebba et al, 1994;Chan & Ferenci, 1993) and biophysical studies (Jordy et al, 1996;Andersen et al, 1995) have been carried out to elucidate the permeation mechanism.…”

Section: Introductionmentioning

confidence: 98%

Structure of maltoporin from Salmonella typhimurium ligated with a nitrophenyl-maltotrioside

Meyer

Hofnung

Schulz

1997

Journal of Molecular Biology

118

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Rate Constants of Sugar Transport Through Two LamB Mutants ofEscherichia coli: Comparison with Wild-type Maltoporin and LamB ofSalmonella typhimurium

Cited by 44 publications

References 26 publications

High Resolution Crystal Structures and Molecular Dynamics Studies Reveal Substrate Binding in the Porin Omp32

High Resolution Crystal Structures and Molecular Dynamics Studies Reveal Substrate Binding in the Porin Omp32

Sugar Transport through Maltoporin of Escherichia coli : Role of the Greasy Slide

Structure of maltoporin from Salmonella typhimurium ligated with a nitrophenyl-maltotrioside

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