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

1995 ◽  
Vol 105 (3) ◽  
pp. 385-401 ◽  
Author(s):  
C Andersen ◽  
M Jordy ◽  
R Benz
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Outer Membrane ◽  
Rate Constants ◽  
Sugar Transport ◽  
Current Noise ◽  
Induced Current ◽  
Lipid Bilayer Membranes ◽  
Sugar Binding ◽  
Different Temperatures

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.

scholarly journals Pore-forming activity of the Tsx protein from the outer membrane of Escherichia coli. Demonstration of a nucleoside-specific binding site.

1988 ◽  
Vol 263 (5) ◽  
pp. 2493-2499
Author(s):  
C Maier ◽  
E Bremer ◽  
A Schmid ◽  
R Benz
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Outer Membrane ◽  
Specific Binding

scholarly journals Bridging the gap between structure and kinetics of human SGLT1

2012 ◽  
Vol 302 (9) ◽  
pp. C1293-C1305 ◽  
Author(s):  
Monica Sala-Rabanal ◽  
Bruce A. Hirayama ◽  
Donald D. F. Loo ◽  
Vincent Chaptal ◽  
Jeff Abramson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Sugar Transport ◽  
Gene Families ◽  
Structural Studies ◽  
Sugar Binding ◽  
Substituted Cysteine Accessibility ◽  
Biochemical Assays ◽  
Functional Consequences ◽  
Binding Residues

The Na+-glucose cotransporter hSGLT1 is a member of a class of membrane proteins that harness Na+ electrochemical gradients to drive uphill solute transport. Although hSGLT1 belongs to one gene family (SLC5), recent structural studies of bacterial Na+ cotransporters have shown that Na+ transporters in different gene families have the same structural fold. We have constructed homology models of hSGLT1 in two conformations, the inward-facing occluded (based on vSGLT) and the outward open conformations (based on Mhp1), mutated in turn each of the conserved gates and ligand binding residues, expressed the SGLT1 mutants in Xenopus oocytes, and determined the functional consequences using biophysical and biochemical assays. The results establish that mutating the ligand binding residues produces profound changes in the ligand affinity (the half-saturation concentration, K0.5); e.g., mutating sugar binding residues increases the glucose K0.5 by up to three orders of magnitude. Mutation of the external gate residues increases the Na+ to sugar transport stoichiometry, demonstrating that these residues are critical for efficient cotransport. The changes in phlorizin inhibition constant ( Ki) are proportional to the changes in sugar K0.5, except in the case of F101C, where phlorizin Ki increases by orders of magnitude without a change in glucose K0.5. We conclude that glucose and phlorizin occupy the same binding site and that F101 is involved in binding to the phloretin group of the inhibitor. Substituted-cysteine accessibility methods show that the cysteine residues at the position of the gates and sugar binding site are largely accessible only to external hydrophilic methanethiosulfonate reagents in the presence of external Na+, demonstrating that the external sugar (and phlorizin) binding vestibule is opened by the presence of external Na+ and closes after the binding of sugar and phlorizin. Overall, the present results provide a bridge between kinetics and structural studies of cotransporters.

scholarly journals Maltose/Maltodextrin System of Escherichia coli: Transport, Metabolism, and Regulation

1998 ◽  
Vol 62 (1) ◽  
pp. 204-229 ◽  
Author(s):  
Winfried Boos ◽  
Howard Shuman
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Transcriptional Control ◽  
Sugar Transport ◽  
Receptor Protein ◽  
Dimensional Structure ◽  
Atp Binding ◽  
Functional Sites ◽  
Maltose Transporter ◽  
Atp Binding Cassette

SUMMARY The maltose system of Escherichia coli offers an unusually rich set of enzymes, transporters, and regulators as objects of study. This system is responsible for the uptake and metabolism of glucose polymers (maltodextrins), which must be a preferred class of nutrients for E. coli in both mammalian hosts and in the environment. Because the metabolism of glucose polymers must be coordinated with both the anabolic and catabolic uses of glucose and glycogen, an intricate set of regulatory mechanisms controls the expression of mal genes, the activity of the maltose transporter, and the activities of the maltose/maltodextrin catabolic enzymes. The ease of isolating many of the mal gene products has contributed greatly to the understanding of the structures and functions of several classes of proteins. Not only was the outer membrane maltoporin, LamB, or the phage lambda receptor, the first virus receptor to be isolated, but also its three-dimensional structure, together with extensive knowledge of functional sites for ligand binding as well as for phage λ binding, has led to a relatively complete description of this sugar-specific aqueous channel. The periplasmic maltose binding protein (MBP) has been studied with respect to its role in both maltose transport and maltose taxis. Again, the combination of structural and functional information has led to a significant understanding of how this soluble receptor participates in signaling the presence of sugar to the chemosensory apparatus as well as how it participates in sugar transport. The maltose transporter belongs to the ATP binding cassette family, and although its structure is not yet known at atomic resolution, there is some insight into the structures of several functional sites, including those that are involved in interactions with MBP and recognition of substrates and ATP. A particularly astonishing discovery is the direct participation of the transporter in transcriptional control of the mal regulon. The MalT protein activates transcription at all mal promoters. A subset also requires the cyclic AMP receptor protein for transcription. The MalT protein requires maltotriose and ATP as ligands for binding to a dodecanucleotide MalT box that appears in multiple copies upstream of all mal promoters. Recent data indicate that the ATP binding cassette transporter subunit MalK can directly inhibit MalT when the transporter is inactive due to the absence of substrate. Despite this wealth of knowledge, there are still basic issues that require clarification concerning the mechanism of MalT-mediated activation, repression by the transporter, biosynthesis and assembly of the outer membrane and inner membrane transporter proteins, and interrelationships between the mal enzymes and those of glucose and glycogen metabolism.

scholarly journals Defined Inactive FecA Derivatives Mutated in Functional Domains of the Outer Membrane Transport and Signaling Protein of Escherichia coli K-12

2004 ◽  
Vol 186 (16) ◽  
pp. 5303-5310 ◽  
Author(s):  
Annette Sauter ◽  
Volkmar Braun
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Outer Membrane ◽  
Crystal Structures ◽  
Transcription Initiation ◽  
Ferric Citrate ◽  
Globular Domain ◽  
Salt Bridges ◽  
The Individual ◽  
K 12

ABSTRACT The FecA outer membrane protein of Escherichia coli functions as a transporter of ferric citrate and as a signal receiver and signal transmitter for transcription initiation of the fec transport genes. Three FecA regions for which functional roles have been predicted from the crystal structures were mutagenized: (i) loops 7 and 8, which move upon binding of ferric citrate and close the entrance to the ferric citrate binding site; (ii) the dinuclear ferric citrate binding site; and (iii) the interface between the globular domain and the β-barrel. Deletion of loops 7 and 8 abolished FecA transport and induction activities. Deletion of loops 3 and 11 also inactivated FecA, whereas deletion of loops 9 and 10 largely retained FecA activities. The replacement of arginine residue R365 or R380 and glutamine Q570, which are predicted to serve as binding sites for the negatively charged dinuclear ferric citrate, with alanine resulted in inactive FecA, whereas the binding site mutant R438A retained approximately 50% of the FecA induction and transport activities. Residues R150, E541, and E587, conserved among energy-coupled outer membrane transporters, are predicted to form salt bridges between the globular domain and the β-barrel and to contribute to the fixation of the globular domain inside the β-barrel. Mutations E541A and E541R affected FecA induction and transport activity slightly, whereas mutations E587A and E587R more strongly reduced FecA activity. The double mutations R150A E541R and R150A E587R nearly abolished FecA activity. Apparently, the salt bridges are less important than the individual functions these residues seem to have for FecA activity. Comparison of the properties of the FecA, FhuA, FepA, and BtuB transporters indicates that although they have very similar crystal structures, the details of their functional mechanisms differ.

Outer-membrane protein PhoE from Escherichia coli forms anion-selective pores in lipid-bilayer membranes

1984 ◽  
Vol 140 (2) ◽  
pp. 319-324 ◽  
Author(s):  
Roland BENZ ◽  
Richard P. DARVEAU ◽  
Robert E. W. HANCOCK
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Lipid Bilayer ◽  
Outer Membrane Protein ◽  
Lipid Bilayer Membranes ◽  
Bilayer Membranes

scholarly journals The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Karen S. Jakes
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Receptor Protein ◽  
Lipid Bilayer Membranes ◽  
Content Type ◽  
Essential Component ◽  
Colicin E1 ◽  
Protein Toxin ◽  
Subsequent Challenge ◽  
Translocation Domain

ABSTRACT Colicins are protein toxins made by Escherichia coli to kill related bacteria that compete for scarce resources. All colicins must cross the target cell outer membrane in order to reach their intracellular targets. Normally, the first step in the intoxication process is the tight binding of the colicin to an outer membrane receptor protein via its central receptor-binding domain. It is shown here that for one colicin, E1, that step, although it greatly increases the efficiency of killing, is not absolutely necessary. For colicin E1, the second step, translocation, relies on the outer membrane/transperiplasmic protein TolC. The normal role of TolC in bacteria is as an essential component of a family of tripartite drug and toxin exporters, but for colicin E1, it is essential for its import. Colicin E1 and some N-terminal translocation domain peptides had been shown previously to bind in vitro to TolC and occlude channels made by TolC in planar lipid bilayer membranes. Here, a set of increasingly shorter colicin E1 translocation domain peptides was shown to bind to Escherichia coli in vivo and protect them from subsequent challenge by colicin E1. A segment of only 21 residues, the “TolC box,” was thereby defined; that segment is essential for colicin E1 cytotoxicity and for binding of translocation domain peptides to TolC. IMPORTANCE The Escherichia coli outer membrane/transperiplasmic protein TolC is normally an essential component of the bacterium's tripartite drug and toxin export machinery. The protein toxin colicin E1 instead uses TolC for its import into the cells that it kills, thereby subverting its normal role. Increasingly shorter constructs of the colicin's N-terminal translocation domain were used to define an essential 21-residue segment that is required for both colicin cytotoxicity and for binding of the colicin's translocation domain to bacteria, in order to protect them from subsequent challenge by active colicin E1. Thus, an essential TolC binding sequence of colicin E1 was identified and may ultimately lead to the development of drugs to block the bacterial drug export pathway.

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