Net sugar transport is a multistep process. Evidence for cytosolic sugar binding sites in erythrocytes

Biochemistry ◽  
1995 ◽  
Vol 34 (47) ◽  
pp. 15395-15406 ◽  
Author(s):  
Erin K. Cloherty ◽  
Lisa A. Sultzman ◽  
Ralph J. Zottola ◽  
Anthony Carruthers
Keyword(s):  
Binding Sites ◽  
Sugar Transport ◽  
Sugar Binding ◽  
Multistep Process

scholarly journals Structure of uPAR, plasminogen, and sugar-binding sites of the 300 kDa mannose 6-phosphate receptor

2004 ◽  
Vol 23 (10) ◽  
pp. 2019-2028 ◽  
Author(s):  
Linda J Olson ◽  
Rama D Yammani ◽  
Nancy M Dahms ◽  
Jung-Ja P Kim
Keyword(s):  
Binding Sites ◽  
Sugar Binding ◽  
Mannose 6 Phosphate

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 Determinants of target prioritization and regulatory hierarchy for the bacterial small RNA SgrS

2017 ◽  
Author(s):  
Maksym Bobrovskyy ◽  
Jane K. Frandsen ◽  
Jichuan Zhang ◽  
Anustup Poddar ◽  
Muhammad S. Azam ◽  
...  
Keyword(s):  
Small Rna ◽  
Binding Sites ◽  
Sugar Transport ◽  
Enteric Bacteria ◽  
Sugar Uptake ◽  
Rna Chaperone ◽  
Target Mrnas ◽  
Mrna Targets ◽  
Molecular Features ◽  
Response To Stress

ABSTRACTThe mechanisms by which small RNA (sRNA) regulators select and prioritize target mRNAs remain poorly understood, but serve to promote efficient responses to environmental cues and stresses. We sought to uncover mechanisms that establish regulatory hierarchy for a model sRNA, SgrS, found in enteric bacteria and produced under conditions of metabolic stress when sugar transport and metabolism are unbalanced. SgrS post-transcriptionally controls a nine-gene regulon to restore growth and homeostasis under stress conditions. An in vivo reporter system was used to quantify SgrS-dependent regulation of target genes and established that SgrS exhibits a clear preference for certain targets, and regulates those targets efficiently even at low SgrS levels. Higher SgrS concentrations are required to regulate other targets. The position of targets in the regulatory hierarchy is not well-correlated with the predicted thermodynamic stability of SgrS-mRNA interactions or the SgrS-mRNA binding affinity as measured in vitro. Detailed analyses of SgrS interaction with asd mRNA demonstrate that SgrS binds cooperatively to two sites and remodels asd mRNA secondary structure. SgrS binding at both sites increases the efficiency of asd mRNA regulation compared to mutants that have only a single SgrS binding site. Our results suggest that sRNA selection of target mRNAs and regulatory hierarchy are influenced by several molecular features. The sRNA-mRNA interaction, including the number and position of sRNA binding sites on the mRNA and cofactors like the RNA chaperone Hfq, seem to tune the efficiency of regulation of specific mRNA targets.IMPORTANCETo survive, bacteria must respond rapidly to stress and simultaneously maintain metabolic homeostasis. The small RNA (sRNA) SgrS mediates the response to stress arising from imbalanced sugar transport and metabolism. To coordinate the stress response, SgrS regulates genes involved in sugar uptake and metabolism. Intrinsic properties of sRNAs such as SgrS allow them to regulate extensive networks of genes. To date, sRNA regulation of targets has largely been studied in the context of “one sRNA-one target”, and little is known about coordination of multi-gene regulons and sRNA regulatory network structure. Here, we explore the molecular basis for regulatory hierarchy in sRNA regulons. Our results reveal a complex interplay of factors that influence the outcome of sRNA regulation. The number and location of sRNA binding sites on mRNA targets and the participation of an RNA chaperone dictate prioritized regulation of targets to promote an efficient response to stress.

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.

Evidence for a Role of Helix IV in Connecting Cation- and Sugar-Binding Sites ofEscherichia coliMelibiose Permease†

Biochemistry ◽  
2000 ◽  
Vol 39 (15) ◽  
pp. 4493-4499 ◽  
Author(s):  
Emmanuelle Cordat ◽  
Gérard Leblanc ◽  
Isabelle Mus-Veteau
Keyword(s):  
Binding Sites ◽  
Sugar Binding

scholarly journals Analysis of the structural requirements of sugar binding to the liver, brain and insulin-responsive glucose transporters expressed in oocytes

1993 ◽  
Vol 294 (3) ◽  
pp. 753-760 ◽  
Author(s):  
C A Colville ◽  
M J Seatter ◽  
G W Gould
Keyword(s):  
Hydrogen Bond ◽  
Binding Sites ◽  
Glucose Transporters ◽  
Binding Pocket ◽  
Structural Basis ◽  
Sugar Binding ◽  
Glucose Analogues ◽  
Sugar Recognition

We have expressed the liver (GLUT 2), brain (GLUT 3) and insulin-responsive (GLUT 4) glucose transporters in oocytes from Xenopus laevis by microinjection of in vitro-transcribed mRNA. Using a range of halogeno- and deoxy-glucose analogues, and other hexoses, we have studied the structural basis of sugar binding to these different isoforms. We show that a hydrogen bond to the C-3 position is involved in sugar binding for all three isoforms, but that the direction of this hydrogen bond is different in GLUT 2 from either GLUT 1, 3 or 4. Hydrogen-bonding at the C-4 position is also involved in sugar recognition by all three isoforms, but we propose that in GLUT 3 this hydrogen bond plays a less significant role than in GLUT 2 and 4. In all transporters we propose that the C-4 position is directed out of the sugar-binding pocket. The role of the C-6 position is also discussed. In addition, we have analysed the ability of fructopyranose and fructofuranose analogues to inhibit the transport mediated by GLUT2. We show that fructofuranose analogues, but not fructopyranose analogues, are efficient inhibitors of transport mediated by GLUT 2, and therefore suggest that GLUT 2 accommodates D-glucose as a pyranose ring, but D-fructose as a furanose ring. Models for the binding sites of GLUT 2, 3 and 4 are presented.

In situ ultrastructural localization of sugar-binding sites in lizard granulosa cell nuclei

1985 ◽  
Vol 55 (1-2) ◽  
pp. 15-20 ◽  
Author(s):  
J. Hubert ◽  
A. P. Seve ◽  
D. Bouvier ◽  
C. Masson ◽  
M. Bouteille ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Binding Sites ◽  
Ultrastructural Localization ◽  
Cell Nuclei ◽  
Sugar Binding
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