Enthalpic cost of water removal from a glucose binding cavity on HK620 TSP

2014 ◽  
Vol 70 (a1) ◽  
pp. C1600-C1600
Author(s):  
Ulrich Gohlke ◽  
Nina Broeker ◽  
Udo Heinemann ◽  
Robert Seckler ◽  
Stefanie Barbirz
Keyword(s):  
Complex Formation ◽  
Binding Site ◽  
Dissociation Constants ◽  
Point Mutations ◽  
Crystal Structure Analysis ◽  
Titration Calorimetry ◽  
High Affinity ◽  
Sugar Binding ◽  
Glucose Binding ◽  
Binding Cavity

Bacteriophage HK620 recognizes and cleaves the O-antigen polysaccharide of Escherichia coli serogroup O18A1 with its tailspike protein (TSP). HK620TSP binds hexasaccharide fragments with low affinity, but single and double amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants. Isothermal titration calorimetry showed that only small amounts of heat were released upon complex formation via a large number of direct and solvent-mediated hydrogen bonds between carbohydrate and protein. At room temperature, association was both enthalpy- and entropy-driven, emphasizing major solvent rearrangements upon complex formation. Crystal structure analysis of a complete set of combinations of wildtype protein and point mutations with and without polysaccharide ligands was carried out. It could be shown that the extended sugar binding site can be dissected into two regions: first, a hydrophobic pocket at the reducing end with minor affinity contributions. Second, a region where the specific exchange of amino acids creates a site for additional water molecules. Sidechain rearrangements upon sugar binding lead to desolvation and additional hydrogen bonding which define this region of the binding site as the high-affinity scaffold.

A HIGH-AFFINITY BINDING SITE FOR THE ANTIOESTROGENS, TAMOXIFEN AND CI 628, IN IMMATURE RAT UTERINE CYTOSOL WHICH IS DISTINCT FROM THE OESTROGEN RECEPTOR

1981 ◽  
Vol 91 (1) ◽  
pp. 155-161 ◽  
Author(s):  
L. C. MURPHY ◽  
R. L. SUTHERLAND
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Oestrogen Receptor ◽  
Dissociation Constants ◽  
Significant Proportion ◽  
Immature Rat ◽  
High Affinity ◽  
Saturable Binding ◽  
Receptor Sites

A high-affinity, saturable antioestrogen binding site, which does not bind oestradiol, has been reported to exist in a number of oestrogen target tissues but not in the immature rat uterus. This study reports the results of a more thorough search for this site in immature rat uterine cytosol. When concentrations of uterine cytoplasmic oestrogen receptor were selectively depleted by translocation of 90–95% of the cytoplasmic oestrogen receptor to the nucleus, saturation analysis studies revealed that the antioestrogens, tamoxifen and CI 628, were bound to high-affinity, saturable binding sites which were present at about 2·5 times the concentration of the residual oestrogen receptor sites. Oestradiol could only partially inhibit the binding of tritiated antioestrogens to their saturable binding sites in this material indicating that a significant proportion of these sites were distinct from the oestrogen receptor sites. This was confirmed in experiments where oestrogen receptor sites were saturated in vitro with oestradiol and high-affinity, saturable sites for CI 628 and tamoxifen were still present. The CI 628 and tamoxifen had high affinity for these sites with dissociation constants of 1·0–1·6 nmol/l. These specific antioestrogen binding sites were present at about 5% of the concentration of oestrogen receptors in normal immature rat uterine cytosol which probably explains their previous lack of detection in this material.

scholarly journals Characterization of bromosulphophthalein binding to human glutathione S-transferase A1-1: thermodynamics and inhibition kinetics

2004 ◽  
Vol 382 (2) ◽  
pp. 703-709 ◽  
Author(s):  
Doris KOLOBE ◽  
Yasien SAYED ◽  
Heini W. DIRR
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Competitive Inhibition ◽  
Hydrophobic Interactions ◽  
Titration Calorimetry ◽  
Inhibition Kinetics ◽  
High Affinity ◽  
Anionic Ligand ◽  
Catalytic Function ◽  
High Affinity Site

In addition to their catalytic functions, GSTs (glutathione S-transferases) bind a wide variety of structurally diverse non-substrate ligands. This ligandin function is known to result in the inhibition of catalytic function. The interaction between hGSTA1-1 (human class Alpha GST with two type 1 subunits) and a non-substrate anionic ligand, BSP (bromosulphophthalein), was studied by isothermal titration calorimetry and inhibition kinetics. The binding isotherm is biphasic, best described by a set of two independent sites: a high-affinity site and a low-affinity site(s). The binding stoichiometries for these sites are 1 and 3 molecules of BSP respectively. BSP binds to the high-affinity site 80 times more tightly (Kd=0.12 μM) than it does to the low-affinity site(s) (Kd=9.1 μM). Binding at these sites is enthalpically and entropically favourable, with no linkage to protonation events. Temperature- and salt-dependent studies indicate the significance of hydrophobic interactions in the binding of BSP, and that the low-affinity site(s) displays low specificity towards the anion. Binding of BSP results in the release of ordered water molecules at these hydrophobic sites, which more than offsets unfavourable entropic changes during binding. BSP inhibition studies show that the binding of BSP to its high-affinity site does not inhibit hGSTA1-1. This site, located near Trp-20, may be related to the buffer-binding site observed in GSTP1-1. The low-affinity-binding site(s) for BSP is most probably located at or near the active site of hGSTA1-1. Binding to this site(s) results in non-competitive inhibition with respect to CDNB (1-chloro-2,4-dinitrobenzene) (KiBSP=16.8±1.9 μM). Given the properties of the H site and the relatively small size of the electrophilic substrate CDNB, it is plausible that the active site of the enzyme can simultaneously accommodate both BSP and CDNB. This would explain the non-competitive behaviour of certain inhibitors that bind the active site (e.g. BSP).

scholarly journals Kinetic and structural characterization of a product complex of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli

2004 ◽  
Vol 380 (3) ◽  
pp. 867-873 ◽  
Author(s):  
Arnaud GARÇON ◽  
Alun BERMINGHAM ◽  
Lu-Yun LIAN ◽  
Jeremy P. DERRICK
Keyword(s):  
Binding Site ◽  
Quantum Coherence ◽  
Chemical Shifts ◽  
Titration Calorimetry ◽  
Second Phase ◽  
Transient Kinetics ◽  
Antimicrobial Drugs ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Equilibrium Dissociation

HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) catalyses the transfer of pyrophosphate from ATP to HMDP (6-hydroxymethyl-7,8-dihydropterin), to form AMP and DHPPP (6-hydroxymethyl-7,8-dihydropterin pyrophosphate). This transformation is a key step in the biosynthesis of folic acid, and HPPK is consequently a target for antimicrobial drugs. The substrates are known to bind to HPPK in an ordered manner, with ATP binding first followed by HMDP. In the present study we show by isothermal titration calorimetry that the product, DHPPP, can bind to the HPPK apoenzyme with high affinity (equilibrium dissociation constant, Kd=0.2 µM), but without the enhancement of pterin fluorescence that occurs on binding of HMDP. The transient kinetics of the enzyme can be monitored by measuring the change in the fluorescence of the pterin ring using stopped-flow methods. The fluorescence exhibits a pronounced biphasic behaviour: it initially rises and then declines back to its original level. This behaviour is in agreement with a two-state kinetic model, with the first phase of fluorescence increase associated with HMDP binding to the enzyme, and the second phase with a slow event that occurs after the reaction has taken place. The HPPK–DHPPP and HPPK–DHPPP–AMP complexes were examined by NMR, and the binding site for DHPPP partially mapped from changes in chemical shifts identified from two dimensional 1H/15N heteronuclear single-quantum coherence spectra. The results demonstrate that DHPPP, in contrast to HMDP, is able to bind to the HPPK apoenzyme and suggest that the pyrophosphate moieties on the ligand play an important role in establishment of a high affinity binding site for the pterin ring.

scholarly journals Zinc- and iron-dependent cytosolic metallo-β-lactamase domain proteins exhibit similar zinc-binding affinities, independent of an atypical glutamate at the metal-binding site

2004 ◽  
Vol 385 (1) ◽  
pp. 145-153 ◽  
Author(s):  
Oliver SCHILLING ◽  
Andreas VOGEL ◽  
Brenda KOSTELECKY ◽  
Hugo NATAL da LUZ ◽  
Daniel SPEMANN ◽  
...  
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
Dissociation Constants ◽  
Zinc Binding ◽  
Titration Calorimetry ◽  
Good Correspondence ◽  
Metal Binding Site ◽  
E Coli ◽  
Metal Ligands ◽  
Metal Selectivity

ZiPD (zinc phosphodiesterase; synonyms are ElaC, ecoZ, RNaseZ and 3′ tRNase) and the iron-dependent redox enzyme FlRd (flavorubredoxin) from Escherichia coli represent prototypical cases of proteins sharing the metallo-β-lactamase fold that require strict metal selectivity for catalytic activity, yet their metal selectivity has only been partially understood. In contrast with hydrolytic metallo-β-lactamase proteins, iron-dependent FlRd-like enzymes have an atypical glutamate ligand, which replaces one otherwise conserved histidine ligand. X-ray absorption spectroscopy revealed that the FlRd metallo-β-lactamase domain is capable of incorporating two zinc ions into the binuclear metal-binding site. Zinc dissociation constants, determined by isothermal titration calorimetry are similar for zinc binding to E. coli ZiPD (Kd1=2.2±0.2 μM and Kd2=23.0±0.6 μM) and to the E. coli FlRd metallo-β-lactamase domain (Kd1=0.7±0.1 μM and Kd2=26.0±0.1 μM). In good correspondence, apo-ZiPD requires incubation with 10 μM zinc for full reconstitution of the phosphodiesterase activity. Accordingly, metal selectivity of ZiPD and FlRd only partially relies on first shell metal ligands. Back mutation of the atypical glutamate in FlRd to a histidine unexpectedly resulted in an increased first zinc dissociation constant (Kd1=30±4 μM and Kd2=23±2 μM). In combination with a recent mutational study on ZiPD [Vogel, Schilling and Meyer-Klaucke (2004) Biochemistry 43, 10379–10386], we conclude that the atypical glutamate does not guide metal selectivity of the FlRd metallo-β-lactamase domain but suppresses possible hydrolytic cross-activity.

Identification of a binding site on human FGF-2 for fibrinogen

Blood ◽  
2004 ◽  
Vol 103 (6) ◽  
pp. 2114-2120 ◽  
Author(s):  
Hu Peng ◽  
Abha Sahni ◽  
Philip Fay ◽  
Stephen Bellum ◽  
Igor Prudovsky ◽  
...  
Keyword(s):  
Binding Site ◽  
Structural Changes ◽  
Dissociation Constants ◽  
Site Directed Mutagenesis ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
The Third ◽  
Fibrinogen Binding ◽  
Adhesive Interactions

Abstract Endothelial cell adhesive interactions are mediated by both fibrinogen and fibrin, and growth is stimulated by fibroblast growth factor 2 (FGF-2). We have shown previously that FGF-2 binds specifically and with high affinity to fibrinogen and fibrin and that fibrinogen potentiates the proliferative capacity of FGF-2 and also protects it from proteolytic degradation. To further characterize this interaction we have performed FGF-2 mutagenesis to identify the interactive site. Because FGF-1 has a similar structure to FGF-2 but does not bind to fibrinogen, we used a strategy of cassette and site-directed mutagenesis, exchanging residues from FGF-1 and FGF-2 and correlating structural changes with fibrinogen binding. Two cassette interchange mutants, 2212 and 2211, contained either the third cassette or both the third and fourth cassettes from FGF-1, and neither exhibited any affinity for fibrinogen. Exchange of 5 residues (Phe95, Ser100, Asn102, Arg107, and Arg109) from FGF-2 into the corresponding sites in the third cassette of FGF-1 imparted high-affinity binding with apparent dissociation constants (Kd) of 5.3 nM and 8.6 nM, respectively, compared with 1.3 nM for wild-type FGF-2. We conclude that these 5 residues define a high-affinity binding site in FGF-2 for fibrinogen.

scholarly journals The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates

2010 ◽  
Vol 10 (1) ◽  
pp. 110-117 ◽  
Author(s):  
Katty V. Y. Goossens ◽  
Catherine Stassen ◽  
Ingeborg Stals ◽  
Dagmara S. Donohue ◽  
Bart Devreese ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dissociation Constants ◽  
Yeast Cells ◽  
Carbohydrate Binding ◽  
Specific Cell ◽  
Affinity Binding ◽  
Content Type ◽  
High Affinity ◽  
Terminal Domain

ABSTRACTSaccharomyces cerevisiaecells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p tod-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level.

Characteristics of renal Na(+)-D-glucose cotransport in the skate (Raja erinacea) and shark (Squalus acanthias)

1997 ◽  
Vol 273 (1) ◽  
pp. R134-R142 ◽  
Author(s):  
H. Kipp ◽  
E. Kinne-Saffran ◽  
C. Bevan ◽  
R. K. Kinne
Keyword(s):  
Binding Site ◽  
Membrane Vesicles ◽  
Squalus Acanthias ◽  
Coupling Ratio ◽  
Raja Erinacea ◽  
High Affinity ◽  
Glucose Binding ◽  
Renal Brush Border Membrane ◽  
Uptake Studies ◽  
Methylene Group

We have investigated the properties of the skate (Raja erinacea) and shark (Squalus acanthias) kidney Na(+)-D-glucose cotransporters (SGLT) in uptake studies of radiolabeled substrates into isolated renal brush-border membrane vesicles (BBMV). Scatchard plot analysis of the substrate dependence revealed that the Na(+)-D-glucose cotransporter population is homogenous within each species. Skate BBMV showed a relatively high affinity for D-glucose [Michaelis constant (K(m)) = 0.12 mM] with an apparent coupling ratio of approximately 2 Na+ to 1 D-glucose, whereas the shark transporter was much lower in affinity (K(m) = 1.90 mM) and had a lower coupling ratio, more like 1 Na+ to 1 D-glucose. These characteristics resemble the properties of SGLT1 and SGLT2, which are known to coexist in the mammalian kidney. Inhibitor studies using sugar analogs and glucosides suggested structural differences of the D-glucose binding site among these transporters, whereas the hydrophobic transporter domains in the vicinity of the D-glucose binding site appeared to be similar. In the high-affinity skate system, D-glucose was recognized by hydrogen bonds to the hydroxy groups at C-2, C-3, and C-4 and by hydrophobic interaction with the C-6 methylene group. In contrast, the low-affinity shark system seemed to lack the hydrophobic recognition motif for the C-6 methylene group of D-glucose.

scholarly journals Identification of Peptide Ligands Specific for the Sugar-Binding Site of Concanavalin A by Screening a Synthetic Peptide Combinatorial Library

1997 ◽  
Vol 2 (4) ◽  
pp. 225-233 ◽  
Author(s):  
David M. Evans ◽  
Lee W. Herman
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Concanavalin A ◽  
Fixed Time ◽  
Peptide Ligands ◽  
Peptide Sequence ◽  
High Affinity ◽  
Sugar Binding ◽  
Natural Amino Acids ◽  
Individual Peptide

We describe a method, using an automated multiple-column chromatographic approach, for identifying a ligand from a peptide library (containing greater than 2.48 x 106 unique peptides) with specificity for the sugar-binding site of the lectin Concanavalin A. The method used an immobilized target to capture moieties from the library as the latter flowed through a chromatographic column. Due to the complexity of the initial library, it was not possible to select for individual peptide sequences with high affinity and specificity for the sugar binding site. However, identification of peptides which specifically bound to the target at this site was possible using subtractive pool sequencing of affinity captured material. The latter technique involved sequencing the peptides retained (after washing the column for a fixed time) in the presence and absence of an excess of the known ligand for the target, methyl a-D-mannopyranoside. Comparisons between the proportion of each amino acid at each sequencing cycle in the absence or presence of an excess of sugar resulted in a peptide sequence of enriched amino acids of the formula HxxSx (where x represents any one of the natural amino acids except cysteine). This sublibrary (containing-6859 individual peptides) was synthesized and rescreened. Two peptide sequences (HHRSY and HVVSV) were identified with relatively high affinity for the sugar-binding site of Concanavalin A. The described technique of solution-phase subtractive pool sequencing (Patent pending) can be employed for rapidly screening highly complex mixtures of peptides and obtaining information about the amino acids within the sequences that are essential for binding to a particular site on the target. This technique could also be applied to other combinatorial mixtures (e.g., PNAs, nucleic acids, or libraries composed of either non-natural or D-amino acids) where a defined number of discrete components are synthesized in a variety of permutations.

scholarly journals The kinetics of dissociation of the inhibitor of nucleoside transport, nitrobenzylthioinosine, from the high-affinity binding sites of cultured hamster cells

1983 ◽  
Vol 216 (2) ◽  
pp. 299-308 ◽  
Author(s):  
R Koren ◽  
C E Cass ◽  
A R Paterson
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Dissociation Constants ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Animal Cells ◽  
Equilibrium Conditions ◽  
Intact Cells ◽  
High Affinity

Nucleoside transport in various types of animal cells is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to a set of high-affinity sites on the plasma membrane. This work examined the binding of [3H]NBMPR to the nucleoside transporters of cultured Nil 8 hamster fibroblasts and of cells of a virus-transformed clone (Nil SV) derived from Nil 8. Experiments conducted with intact Nil 8 and Nil SV cells and with membrane preparations indicated that the two lines differed significantly in the cellular content of binding sites and only slightly in the affinities of these sites for NBMPR. Nil 8 and Nil SV cells possessed (4.2-8.0) X 10(5) and (2.0-4.0) X 10(6) sites per cell respectively, whereas the dissociation constants of site-bound NBMPR obtained with intact cells and with membrane preparations were similar, ranging from 0.29 to 1.5 nM. Dilazep, a potent inhibitor of nucleoside transport that is structurally unrelated to NBMPR, appeared to compete with NBMPR for binding to the high-affinity sites when tested under equilibrium conditions with Ki values for inhibition of NBMPR binding to Nil 8 and Nil SV cells respectively of 15 +/- 4 and 32 +/- 4 nM. The dissociation of NBMPR from the binding site—NBMPR complex of Nil SV membrane preparations was a first-order decay process with a rate constant of 0.68 +/- 0.26 min-1. The rate of dissociation of NBMPR from the binding-site complex of membrane preparations and intact cells was decreased significantly in the presence of dilazep and increased in the presence of the permeant uridine. These results suggest that the apparent competitive-inhibition kinetics obtained for dilazep under equilibrium conditions should not be interpreted as binding of dilazep to the same site as NBMPR but rather as binding of the two inhibitors to closely associated sites on the nucleoside transporter. Similarly, uridine also appears to bind to a site separate from the NBMPR-binding site.

Sign in / Sign up

Export Citation Format

Share Document