Entropy drives integrin alphaIIbbeta3:echistatin binding--evidence from surface plasmon resonance spectroscopy.

This investigation examined the molecular mechanisms that enable the alphaIIbbeta3 integrin to bind efficiently, tightly, and selectively to echistatin, an RGD disintegrin. We used surface plasmon resonance spectroscopy to measure the rate, extent, and stability of complexes formed between micellar alphaIIbbeta3 and recombinant echistatin (rEch) mutants, immobilized on the surface of a biosensor chip. alphaIIbbeta3 bound readily and tightly to wild-type RGD-rEch and RGDF-rEch but not to RGA-rEch or AGD-rEch, demonstrating that both of those charged moieties contribute to integrin recognition. van't Hoff analysis of the temperature dependence of the RGD-rEch K d data yielded an unfavorable enthalpy change, Delta H degrees = 14 +/- 3 kcal/mol, offset by a favorable entropy term, TDelta S degrees = 23 +/- 3 kcal/mol. Eyring analysis of the temperature dependence of the kinetic parameters yielded Delta H a degrees (++) = 9 +/- 2 kcal/mol and TDelta S a degrees (++) = -4 +/- 2 kcal/mol, indicating that a substantial activation enthalpy barrier and a smaller activation entropy hinder assembly of the encounter complex. Thus, equilibrium thermodynamic data demonstrate that entropy is the dominant factor stabilizing integrin:echistatin binding, while transition-state thermodynamic parameters indicate that the rate of complex formation is enthalpy-limited. When electrostatic contacts are the major source of receptor:ligand stability, theory and experiment indicate that entropy-favorable ion-pair desolvation often provides the driving force for molecular recognition.     

A new allosteric site in glycogen phosphorylase b as a target for drug interactions

BACKGROUND: In muscle and liver, glycogen concentrations are regulated by the coordinated activities of glycogen phosphorylase (GP) and glycogen synthase. GP exists in two forms: the dephosphorylated low-activity form GPb and the phosphorylated high-activity form GPa. In both forms, allosteric effectors can promote equilibrium between a less active T state and a more active R state. GP is a possible target for drugs that aim to prevent unwanted glycogen breakdown and to stimulate glycogen synthesis in non-insulin-dependent diabetes. As a result of a data bank search, 5-chloro-1H-indole-2-carboxylic acid (1-(4-fluorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2-oxoethy l)amide, CP320626, was identified as a potent inhibitor of human liver GP. Structural studies have been carried out in order to establish the mechanism of this unusual inhibitor. RESULTS: The structure of the cocrystallised GPb-CP320626 complex has been determined to 2.3 A resolution. CP320626 binds at a site located at the subunit interface in the region of the central cavity of the dimeric structure. The site has not previously been observed to bind ligands and is some 15 A from the AMP allosteric site and 33 A from the catalytic site. The contacts between GPb and CP320626 comprise six hydrogen bonds and extensive van der Waals interactions that create a tight binding site in the T-state conformation of GPb. In the R-state conformation of GPa these interactions are significantly diminished. CONCLUSIONS: CP320626 inhibits GPb by binding at a new allosteric site. Although over 30 A from the catalytic site, the inhibitor exerts its effects by stabilising the T state at the expense of the R state and thereby shifting the allosteric equilibrium between the two states. The new allosteric binding site offers a further recognition site in the search for improved GP inhibitors.     

Interaction of soluble and surface-bound heparin binding growth-associated molecule with heparin.

The interaction of heparin with heparin binding growth-associated molecule (HB-GAM) was studied using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). ITC studies showed that, in solution, heparin bound HB-GAM with a deltaH of -30 kcal/mole corresponding to a dissociation constant (Kd) of 460 nM. The stoichiometry of interaction was 3 moles of HB-GAM per mole of heparin, corresponding to a minimum heparin binding site for HB-GAM of 12-16 saccharide residues. Kinetic measurements of heparin interaction with HB-GAM made by SPR afforded a Kd of 4 nM, suggesting considerably tighter binding when HB-GAM was immobilized on a surface. Affinity chromatography of a sized mixture of heparin oligosaccharides, having a degree of polymerization (dp) of > 14 saccharide units, on HB-GAM-Sepharose demonstrated that oligosaccharides having more than 18 saccharide residues showed the tightest interaction.     

1-(3-Deoxy-3-fluoro-β-d-glucopyranosyl) pyrimidine derivatives as inhibitors of glycogen phosphorylase b: Kinetic,crystallographic and modelling studies

Design of inhibitors of glycogen phosphorylase (GP) with pharmaceutical applications in improving glycaemic control in type 2 diabetes is a promising therapeutic strategy. The catalytic site of muscle glycogen phosphorylase b (GPb) has been probed with five deoxy-fluro-glucose derivatives. These inhibitors had fluorine instead of hydroxyl at the 3′ position of the glucose moiety and a variety of pyrimidine derivatives at the 1′ position. The best of this carbohydrate-based family of five inhibitors displays a K_i value of 46 μM. To elucidate the mechanism of inhibition for these compounds, the crystal structures of GPb in complex with each ligand were determined and refined to high resolution. The structures demonstrated that the inhibitors bind preferentially at the catalytic site and promote the less active T state conformation of the enzyme by making several favorable contacts with residues of the 280s loop. Fluorine is engaged in hydrogen bond interactions but does not improve glucose potency. The pyrimidine groups are located between residues 284–286 of the 280s loop, Ala383 of the 380s loop, and His341 of the β-pocket. These interactions appear important in stabilizing the inactive quaternary T state of the enzyme. As a follow up to recent computations performed on β-d-glucose pyrimidine derivatives, tautomeric forms of ligands 1–5 were considered as potential binding states. Using Glide-XP docking and QM/MM calculations, the ligands 2 and 5 are predicted to bind in different tautomeric states in their respective GPb complexes. Also, using α-d-glucose as a benchmark model, a series of substitutions for glucose –OH at the 3′ (equatorial) position were investigated for their potential to improve the binding affinity of glucose-based GPb catalytic site inhibitors. Glide-XP and quantum mechanics polarized ligand (QPLD-SP/XP) docking calculations revealed favorable binding at this position to be dominated by hydrogen bond contributions; none of the substitutions (including fluorine) out-performed the native –OH substituent which can act both as hydrogen bond donor and acceptor. The structural analyses of these compounds can be exploited towards the development of better inhibitors.     

Binding of the potential antitumour agent indirubin-5-sulphonate at the inhibitor site of rabbit muscle glycogen phosphorylase b. Comparison with ligand binding to pCDK2-cyclin A complex.

The binding of indirubin-5-sulphonate (E226), a potential anti-tumour agent and a potent inhibitor (IC(50) = 35 nm) of cyclin-dependent kinase 2 (CDK2) and glycogen phosphorylase (GP) has been studied by kinetic and crystallographic methods. Kinetic analysis revealed that E226 is a moderate inhibitor of GPb (K(i) = 13.8 +/- 0.2 micro m) and GPa (K(i) = 57.8 +/- 7.1 micro m) and acts synergistically with glucose. To explore the molecular basis of E226 binding we have determined the crystal structure of the GPb/E226 complex at 2.3 A resolution. Structure analysis shows clearly that E226 binds at the purine inhibitor site, where caffeine and flavopiridol also bind [Oikonomakos, N.G., Schnier, J.B., Zographos, S.E., Skamnaki, V.T., Tsitsanou, K.E. & Johnson, L.N. (2000) J. Biol. Chem.275, 34566-34573], by intercalating between the two aromatic rings of Phe285 and Tyr613. The mode of binding of E226 to GPb is similar, but not identical, to that of caffeine and flavopiridol. Comparative structural analyses of the GPb-E226, GPb-caffeine and GPb-flavopiridol complex structures reveal the structural basis of the differences in the potencies of the three inhibitors and indicate binding residues in the inhibitor site that can be exploited to obtain more potent inhibitors. Structural comparison of the GPb-E226 complex structure with the active pCDK2-cyclin A-E226 complex structure clearly shows the different binding modes of the ligand to GPb and CDK2; the more extensive interactions of E226 with the active site of CDK2 may explain its higher affinity towards the latter enzyme.     

The binding of beta- and gamma-cyclodextrins to glycogen phosphorylase b: kinetic and crystallographic studies

A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in human clinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, alpha-, beta-, and gamma-cyclodextrins were identified as moderate mixed-type competitive inhibitors of GPb (with respect to glycogen) with K(i) values of 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined the structure of GPb complexed with beta- and gamma-cyclodextrins at 1.94 A and 2.3 A resolution, respectively. The structures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage site of T-state GPb with very little change of the tertiary structure and provide a basis for understanding their potency and subsite specificity. Structural comparisons of the two complexes with GPb in complex with either maltopentaose (G5) or maltoheptaose (G7) show that beta- and gamma-cyclodextrins bind in a mode analogous to the G5 and G7 binding with only some differences imposed by their cyclic conformations. It appears that the binding energy for stabilization of enzyme complexes derives from hydrogen bonding and van der Waals contacts to protein residues. The binding of alpha-cyclodextrin and octakis (2,3,6-tri-O-methyl)-gamma-cyclodextrin was also investigated, but none of them was bound in the crystal; moreover, the latter did not inhibit the phosphorylase reaction.     

Cytochrome c peroxidase-cytochrome c complex: locating the second binding domain on cytochrome c peroxidase with site-directed mutagenesis

Cytochrome c peroxidase (CcP) can bind as many as two cytochrome c (Cc) molecules in an electrostatic complex. The location of the two binding domains on CcP has been probed by photoinduced interprotein electron transfer (ET) between zinc-substituted horse cytochrome c (ZnCc) and CcP with surface charge-reversal mutations and by isothermal titration calorimetry (ITC). These results, which are the first experimental evidence for the location of domain 2, indicate that the weak-binding domain includes residues 146-150 on CcP. CcP(E290K) has a charge-reversal mutation in the tight-binding domain, which should weaken binding, and it weakens the 1:1 complex; K(1) decreases 20-fold at 18 mM ionic strength. We have employed two mutations to probe the proposed location for the weak-binding domain on the CcP surface: (i) D148K, a "detrimental" mutation with a net (+2) change in the charge of CcP, and (ii) K149E, a "beneficial" mutation with a net (-2) change in the charge. The interactions between FeCc and CcP(WT and K149E) also have been studied with ITC. The CcP(D148K) mutation causes no substantial change in the 2:1 binding but an increase in the reactivity of the 2:1 complex. The latter can be interpreted as a long-range influence on the heme environment or, more likely, the enhancement of a minority subset of binding conformations with favorable pathways for ET. CcP(K149E) has a charge-reversal mutation in the weak-binding domain that produces a substantial increase in the 2:1 binding constant as measured by both quenching and ITC. For the 1:1 complex of CcP(WT), DeltaG(1) = -8.2 kcal/mol (K(1) = 1.3 x 10(6) M(-)(1)), DeltaH(1) = +2.7 kcal/mol, and DeltaS(1) = +37 cal/K.mol at 293 K; for the second binding stage, K(2) < 5 x 10(3) M(-)(1), but accurate thermodynamic parameters were not obtained. For the 1:1 complex of CcP(K149E), DeltaG(1) = -8.5 kcal/mol (K(1) = 2 x 10(6) M(-)(1)), DeltaH(1) = +2. 0 kcal/mol, and DeltaS(1) = +36 cal/K.mol; for the second stage, DeltaG(2) = -5.5 kcal/mol (K(1) = 1.3 x 10(4) M(-)(1)), DeltaH(2) = +2.9 kcal/mol, and DeltaS(2) = +29 cal/K.mol.     

Amino-terminal leucine-rich repeats in gonadotropin receptors determine hormone selectivity.

Recombinant expression of truncated receptors for luteinizing hormone/chorionic gonadotropin (LH/CG) revealed that the amino-terminal leucine-rich repeats 1-8 of the extracellular receptor domain bind human chorionic gonadotropin (hCG) with an affinity (Kd = 0.72 +/- 0.2 nM) similar to that of the native LH/CG receptor (Kd = 0.48 +/- 0.05 nM). LH/CG receptor leucine-rich repeats 1-8 were used to replace homologous sequences in the closely related receptor for follicle stimulating hormone (FSH). Cells expressing such chimeric LH/CG-FSH receptors bind hCG and show elevated cylic AMP levels when stimulated by hCG but not by recombinant human FSH (rhFSH). Similarly, a chimeric LH/CG receptor in which leucine-rich repeats 1-11 originated from the FSH receptor is activated by rhFSH but not by hCG. For this chimera, no residual [125I] hCG binding was observed in a range of 2 pM to 10 nM. Our results demonstrate that specificity of gonadotropin receptors is determined by a high affinity hormone binding site formed by the amino-terminal leucine-rich receptor repeats.     

Thermodynamics and structural analysis of positive allosteric modulation of the ionotropic glutamate receptor GluA2

Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer's disease. These modulators bind within the dimer interface of the LBD (ligand-binding domain) and stabilize the agonist-bound conformation slowing receptor desensitization and/or deactivation. In the present study, we employ isothermal titration calorimetry to determine binding affinities and thermodynamic details of binding of modulators of GluA2. A mutant of the LBD of GluA2 (LBD-L483Y-N754S) that forms a stable dimer in solution was used. The potent GluA2 modulator BPAM-97 was used as a reference compound. Evidence that BPAM-97 binds in the same pocket as the well-known GluA2 modulator cyclothiazide was obtained from X-ray structures. The LBD-L483Y-N754S:BPAM-97 complex has a Kd of 5.6?μM (ΔH=-4.9 kcal/mol, -TΔS=-2.3 kcal/mol; where 1?kcal≈4.187?kJ). BPAM-97 was used in a displacement assay to determine a Kd of 0.46?mM (ΔH=-1.2 kcal/mol, -TΔS=-3.3 kcal/mol) for the LBD-L483Y-N754S:IDRA-21 complex. The major structural factors increasing the potency of BPAM-97 over IDRA-21 are the increased van der Waals contacts to, primarily, Met496 in GluA2 imposed by the ethyl substituent of BPAM-97. These results add important information on binding affinities and thermodynamic details, and provide a new tool in the development of drugs against cognitive disorders.     

Thermodynamic dissection of the Ezrin FERM/CERMAD interface

ERM (Ezrin-Radixin-Moesin) proteins are key cross-linkers of the plasma membrane and the actin cytoskeleton. They are regulated by the intramolecular association of the N-terminal FERM (band-four point one, Ezrin, Radixin, Moesin) and C-terminal CERMAD (ERM association domain) domains (N/C interaction), which masks the binding surfaces of the domains for other molecules. The N/C interface is characterized by the highly distributed binding of CERMAD through a beta-strand and four alpha-helices to a globular FERM. Though it is a target for multiple regulatory signals, little is known about the dynamics/thermodynamics governing this interface. Recent implications of Ezrin in cancer metastasis have increased the necessity to understand this regulatory switch. In this study, we report residue-specific stabilities of Ezrin CERMAD at the Ezrin N/C interface obtained using hydrogen-deuterium exchange NMR. These stabilities vary across secondary structural elements and identify F583 and L586 as key anchor residues for the most stable element, alphaD. Macroscopic N/C binding energetics, obtained using isothermal titration calorimetry (ITC) reveals a high affinity (Kd =176 nM) enthalpy-driven binding (DeltaH = -26 kcal/mol, TDeltaS = -17 kcal/mol) at 25 degrees C at pH 7 in MES and phosphate buffers. A 10-fold increase in affinity was observed for measurements in acetate buffer, suggesting that an acetate-like molecule might promote the repressed form of the complex, possibly through interaction with the F2 subdomain of FERM, which resembles the acyl-CoA binding protein. In summary, our results have illustrated the dynamic nature of this regulatory interface and provide a foundation for investigating the role of regulatory signals on the stability of this interface.     

Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism

Yeast AMP deaminase is allosterically activated by ATP and MgATP and inhibited by GTP and PO4. The tetrameric enzyme binds 2 mol each of ATP, GTP, and PO4/subunit with Kd values of 8.4 +/- 4.0, 4.1 +/- 0.6, and 169 +/- 12 microM, respectively. At 0.7 M KCl, ATP binds to the enzyme, but no longer activates. Titration with coformycin 5'-monophosphate, a slow, tight-binding inhibitor, indicates a single catalytic site/subunit. ATP and GTP bind at regulatory sites distinct from the catalytic site and their binding is mutually exclusive. Inorganic phosphate competes poorly with ATP for the ATP sites (Kd = 20.1 +/- 4.1 mM). However, near-saturating ATP reduces the moles of phosphate bound per subunit to 1 PO4, which binds with a Kd = 275 +/- 22 microM. In the presence of ATP, PO4 cannot effectively compete with ATP for the nucleotide triphosphate sites. The PO4 which binds in the presence of ATP is competitive with AMP at the catalytic site since the Kd equals the kinetic inhibition constant for PO4. Initial reaction rate curves are a cooperative function of AMP concentration and activation by ATP is also cooperative. However, no cooperativity is observed in the binding of any of the regulator ligands and ATP binding and kinetic activation by ATP is independent of substrate analog concentration. Cooperativity in initial rate curves results, therefore, from altered rate constants for product formation from each (enzyme.substrate)n species and not from cooperative substrate binding. The traditional cooperative binding models of allosteric regulation do not apply to yeast AMP deaminase, which regulates catalytic activity by kinetic control of product formation. The data are used to estimate the rates of AMP hydrolysis under reported metabolite concentrations in yeast.     

A hybrid ligand method for androgen receptor measurement

Existing techniques for androgen receptor (AR) assay are complicated by cross-reactivity of ligand binding affinities that can lead to incorrect estimation of receptor concentration. Two most frequently used ligands are [3H]dihydrotestosterone [( 3H]DHT) and [3H]methyltrienolone [( 3H]R1881), which in addition to binding to AR also bind to sex hormone binding globulin (SHBG; Kd = 1.5 nM) and progesterone receptors (PgR; Human Kd = 1 nM, rat Kd = 6 nM) respectively. Triamcinolone acetonide (TMA) is commonly used to block binding of [3H]R1881 to PgR, however at high concentrations TMA itself will bind AR (Kd = 7 microM). We have developed a hybrid ligand method for the measurement of AR in the presence of SHBG and PgR. This method used [3H]R1881 as the high specific activity labelled tracer and DHT as the unlabelled competitor of specific AR binding. Using this assay, 20% of human colorectal carcinomas were found to contain AR.     

Allosteric inhibition of glycogen phosphorylase a by the potential antidiabetic drug 3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbo xylate.

The effect of the potential antidiabetic drug (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarbox ylate (W1807) on the catalytic and structural properties of glycogen phosphorylase a has been studied. Glycogen phosphorylase (GP) is an allosteric enzyme whose activity is primarily controlled by reversible phosphorylation of Ser14 of the dephosphorylated enzyme (GPb, less active, predominantly T-state) to form the phosphorylated enzyme (GPa, more active, predominantly R-state). Upon conversion of GPb to GPa, the N-terminal tail (residues 5-22), which carries the Ser14(P), changes its conformation into a distorted 3(10) helix and its contacts from intrasubunit to intersubunit. This alteration causes a series of tertiary and quaternary conformational changes that lead to activation of the enzyme through opening access to the catalytic site. As part of a screening process to identify compounds that might contribute to the regulation of glycogen metabolism in the noninsulin dependent diabetes diseased state, W1807 has been found as the most potent inhibitor of GPb (Ki = 1.6 nM) that binds at the allosteric site of T-state GPb and produces further conformational changes, characteristic of a T'-like state. Kinetics show W1807 is a potent competitive inhibitor of GPa (-AMP) (Ki = 10.8 nM) and of GPa (+1 mM AMP) (Ki = 19.4 microM) with respect to glucose 1-phosphate and acts in synergism with glucose. To elucidate the structural features that contribute to the binding, the structures of GPa in the T-state conformation in complex with glucose and in complex with both glucose and W1807 have been determined at 100 K to 2.0 A and 2.1 A resolution, and refined to crystallographic R-values of 0.179 (R(free) = 0.230) and 0.189 (R(free) = 0.263), respectively. W1807 binds tightly at the allosteric site and induces substantial conformational changes both in the vicinity of the allosteric site and the subunit interface. A disordering of the N-terminal tail occurs, while the loop of chain containing residues 192-196 and residues 43'-49' shift to accommodate the ligand. Structural comparisons show that the T-state GPa-glucose-W1807 structure is overall more similar to the T-state GPb-W1807 complex structure than to the GPa-glucose complex structure, indicating that W1807 is able to transform GPa to the T'-like state already observed with GPb. The structures provide a rational for the potency of the inhibitor and explain GPa allosteric inhibition of activity upon W1807 binding.     

High-affinity binding of cytochalasin B to the B-end of F-actin loses its inhibitory effect on subunit exchange when the bound nucleotide is ADP.

We investigated the mode of binding of cytochalasin B (CB) to F-actin in an ADP-solution with and without inorganic phosphate (Pi). In the presence of Pi (20 mM), a filament of F-actin had a single high-affinity CB binding site (Kd = 1.4 nM), just like in the case of an ATP-solution [Kd = 5.0 nM: Suzuki, N. & Mihashi, K. (1991) J. Biochem. 109, 19-23]. But in the absence of Pi, there were two low-affinity (Kd = 200 nM) CB binding sites as well as one high-affinity site (Kd = 1.6 nM). We determined the concentration of CB necessary for half-maximal inhibition of growth or shortening of F-actin (Ki) using of pyrene-labeled actin. We obtained Ki = 80 nM for growth and Ki = 800 nM for shortening in the presence of ATP. The addition of Pi to the ATP-solution reduced Ki for growth to 9 nM. We propose a model explaining these results. In the model, high-affinity CB binding to the terminal subunit dimer can inhibit subunit exchange at the B-end only when the terminal subunits bind ATP or ADP.Pi. When the terminal subunits bind ADP, additional low-affinity CB bindings to the terminal subunits are needed to inhibit the subunit exchange.     

Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.     

The interaction of vasoactive intestinal peptide (VIP) with isolated bovine thyroid plasma membranes

The binding of vasoactive intestinal peptide (VIP) and stimulation of adenylate cyclase were studied in bovine thyroid plasma membranes. The binding depended on time, temperature and was saturable and specific. Binding studies suggested the presence of two classes of binding sites: a class with high affinity (Kd = 13 nM) and low capacity (6411 sites/pg), and a class with low affinity (Kd = 480 nm) and high capacity (105,300 sites/pg) at 15 degrees C. Secretin, glucagon, insulin and somatostatin did not displace the tracer from the membranes. VIP stimulated cyclic AMP production. Maximal cyclic AMP production (2-fold above basal values) was observed with 100 nM VIP and half-maximal response was obtained at 5 nM VIP at 15 degrees C.     

Thermodynamic analysis of allosamidin binding to a family 18 chitinase

Inhibition of family 18 chitinases is emerging as a target for pest and fungal control as well as asthma and inflammatory therapy. One of the best known inhibitors for these enzymes is allosamidin, a natural product. While interactions of this compound with family 18 chitinases have been studied in much detail by X-ray crystallography and standard enzymology, details of the driving forces behind its tight binding remain unknown. We have studied the thermodynamics of allosamidin binding to chitinase B (ChiB), a family 18 chitinase from Serratia marcescens, using isothermal titration calorimetry. At pH 6.0, Kd is 0.16 +/- 0.04 microM, and the binding reaction is entropically driven (DeltaSr = 44 cal/K mol) with an enthalpic penalty (DeltaHr = 3.8 +/- 0.2 kcal/mol). Dissection of the entropic term shows that a favorable conformational change in the allosamidin-ChiB complex (DeltaSconf = 37 cal/K mol) is the main contributor to the reaction. At pH 8.5, Kd decreases to 0.03 muM and the binding reaction is less entropically favorable (DeltaSr = 30 cal/K mol). While the solvation entropy change (DeltaSsolv) increases from 15 cal/K mol at pH 6.0 to 46 cal/K mol at pH 8.5, DeltaSconf becomes small and negative (-8 cal/K mol) because of an enthalpy-entropy compensation. Analyses of proton transfer showed that at pH 6.0 binding of allosamidin requires deprotonation of the Asp142-Glu144 catalytic diad. At pH 8.5, the 142-144 diad is ionized in the native enzyme, relieving the deprotonation penalty of binding and explaining why binding becomes enthalpically favorable (DeltaHr = -1.2 +/- 0.2 kcal/mol).     

Oxalate binding to rat intestinal brush-border membrane in pyridoxine deficiency: a kinetic study

Oxalate bound specifically to the intestinal brush-border membrane (BBM) of pyridoxine-deficient rats, but not to BBM of control rats. The binding of oxalate to intestinal BBM of pyridoxine-deficient rats was rapid, reversible, dependent on concentration of oxalate, temperature sensitive and competitively inhibited by oxalate analogues. Kinetic analysis of the oxalate binding data revealed induction of two distinct classes of receptor site for oxalate. The high-affinity oxalate binding sites, reached saturation at 60-70 nM oxalate, had a Kd of 24.29 nM and the number of binding sites were 30 pmoles (i.e., 1.8.10(13) molecules). The low-affinity oxalate binding sites, could not be saturated under experimental conditions upto 1 microM oxalate. It had a Kd of 487.5 nM and the number of binding sites were 156 pmoles (i.e., 9.4.10(13) molecules). The apparent energy of activation was 19 kcal/mol. The half-saturation concentration of inhibitor (IC50) of oxalate was 0.4.10(-5) M, while all other structural analogues of oxalate had higher IC50 values. Among the competitive inhibitors tested IC50 was in the following order, pyruvate greater than maleate greater than oxaloacetate greater than glyoxylate greater than parabonate greater than oxalate. These kinetic characteristics indicate involvement of a membrane protein in oxalate binding and transport in rat intestinal brush-border membrane in pyridoxine deficiency.     

Activation of glycogen phosphorylase with 5-aminoimidazole-4-carboxamide riboside (AICAR). Assessment of glycogen as a precursor of mannosyl residues in glycoconjugates

The experimental evaluation of the contribution of glycogen phosphorylase (GP) to biochemical pathways is limited to methods that raise cAMP, activating the cAMP-dependent protein kinase/phosphorylase kinase/GP cascade. Such methods convert the unphosphorylated form, "GPb," which catalyzes glycogenolysis only in the presence of appropriate allosteric activators such as AMP, to the phosphorylated, constitutively activated form, "GPa." However, activation of GP in this way is indirect, requires a functional cAMP kinase cascade, and is complicated by other actions of cAMP. Here, we demonstrate a strategy for the experimental manipulation of GP in intact dermal fibroblasts, involving activation by the membrane-permeable adenosine analog 5-aminoimidazole-4-carboxamide riboside (AICAR) and inhibition by caffeine and Pfizer compound CP-91149, which bind to GP at distinct sites. Potential complications because of activation of AMP-activated protein kinase by AICAR were assessed with metformin, which activates this kinase but does not activate GP. Using this strategy, we show that glycogen can be a significant and regulatable precursor of mannosyl units in lipid-linked oligosaccharides and glycoproteins.