Cholinergic Muscarinic Receptors in Human Fetal Brain: Ontogeny of [3H]Quinuclidinyl Benzilate Binding Sites in Corpus Striatum, Brainstem, and Cerebellum

The ontogeny of muscarinic receptors was studied in human fetal striatum, brainstem, and cerebellum to investigate general principles of synaptogenesis as well as the physiological balance between various chemical synapses during development in a given region of the brain. [3H]Quinuclidinyl benzilate ([-'H]QNB) binding was assayed in total particulate fraction (TPF) from various parts of brain. In the corpus striatum, QNB binding sites are present at 16 weeks of gestation (average concentration 180 fmol/mg protein of TPF), slowly increase up to 24 weeks (average concentration 217 fmol/mg protein), and rapidly increase during the third trimester to 480 fmol/mg protein of TPF. In contrast, dopaminergic receptors exist as two subpopulations. one with low affinity and the other with high affinity up to the 24th week of gestation; all of them acquire the highaffinity characteristic during the third trimester. In brainstem, the muscarinic receptors show maximum concentration by 16 weeks of age (360 fmolimg protein of TPF). Subsequently the muscarinic receptor concentration shows a gradual decline in the brainstem. In cerebellum, except for a slight increase at 24 weeks (average concentration 90 fmol/mg protein of TPF), the receptor concentration remained nearly constant at about 60-70 fmolimg protein of TPF throughout fetal life. This study demonstrates that the ontogeny of muscarinic receptors varies among the different regions, and the patterns observed suggest that receptor formation occurs principally in the third trimester. Also noteworthy is the finding that the QNB binding sites decreased in all regions of the human brain during adult life. Key Words: Cholinergic muscarinic receptors-Quinuclidinyl benzilate-Corpus striaturn-Brainstem-Cerebellum. Ravikumar B. V. and Sastry P. S. Cholinergic muscarinic receptors in human fetal brain: Ontogeny of [3H]quinuclidinyl benzilate binding sites in corpus striatum, brainstem, and cerebellum. J. Neurochem. 45, 1948- 1950 (1985).

Synthesis, Binding and Bioactivity of [gamma]-Methylene [gamma]-Lactam Ecdysone Receptor Ligands: Advantages of QSAR Models for Flexible Receptors

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or [`]attractors'. We describe the synthesis, in vitro binding and selected in vivo toxicity data for [gamma]-methylene [gamma]-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognized by a single conformation of the EcR binding pocket.

Fluorescence And Nmr-Studies Of The Binding Of Cholinergic Fluorescent-Probes To Horse Serum-Cholinesterase

The interaction of the cholinergic fluorescent probes, 1-(5-dimethyl-aminoaphthalene-1-sulfonamido) ethane-2-trimethylammonium perchlorate, 1-(5-dimethylaminonaphthalene-1-sulfonamido) pentane-5-trimethylammonium tartarate and 1-(5-dimethylaminonaphthalene-1-sulfonamido) decane-10- trimethylammonium tartarate with horse serum cholinesterase has been examined by fluorescence and n.m.r. methods. Fluorescence titrations show binding of the decane derivative to two sites on the protein whereas the lower homologs bind largely to one site. Active site inhibitors like curbamylcholine and decamethonium abolish binding of the decane derivative to the high affinity site. The inhibitors are largely without effect on the binding of the lower homologs. N.m.r. studies clearly establish immobilization of both ends of the molecule on binding in the case of the decane derivative, whereas in the lower homologs the dimethylamino group on the naphthalene ring is significantly more affected in the presence of enzyme. The probes are effective inhibitors of the enzyme with the decane derivative being two orders of magnitude more effective than its lower homologs. Based on the n.m.r., fluorescence and inhibition studies, a model for probe binding to the enzyme is advanced. It appears that the decane derivative binds with high affinity to the catalytic anionic site while the lower affinity site is assigned to a peripheral anionic site. The lower homologs probe only the peripheral site. A comparison of fluorescence, n.m.r. and inhibition studies with acetylcholinesterases from electric eel and bovine erythrocytes is presented.

Isolation and characterization of riboflavin-binding protein from pregnant-rat serum

A high-affinity riboflavin -binding protein was isolated and characterized for the first time from pregnant-rat sera by affinity chromatography on a lumiflavin-agarose column. The purified protein was homogeneous by the criteria of analytical polyacrylamide-gel disc electrophoresis, gel-filtration chromatography on Sephadex G-100 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It had a molecular weight of 90000+/-5000 and interacted with [14C]riboflavin with a 1:1 molar ratio with a dissociation constant (Kd) of 0.42 micron.

Prediction of heparin binding sites in bone morphogenetic proteins (BMPs)

Heparin is a glycosaminoglycan known to bind bone morphogenetic proteins (BMPs) and the growth and differentiation factors (GDFs) and has strong and variable effects on BMP osteogenic activity. In this paper we report our predictions of the likely heparin binding sites for BMP-2 and 14. The N-terminal sequences upstream of TGF-β-type cysteine-knot domains in BMP-2, 7 and 14 contain the basic residues arginine and lysine, which are key components of the heparin/HS-binding sites, with these residues being highly non-conserved. Importantly, evolutionary conserved surfaces on the beta sheets are required for interactions with receptors and antagonists. Furthermore, BMP-2 has electropositive surfaces on two sides compared to BMP-7 and BMP-14. Molecular docking simulations suggest the presence of high and low affinity binding sites in dimeric BMP-2. Histidines were found to play a role in the interactions of BMP-2 with heparin; however, a pKa analysis suggests that histidines are likely not protonated. This is indicative that interactions of BMP-2 with heparin do not require acidic pH. Taken together, non-conserved amino acid residues in the N-terminus and residues protruding from the beta sheet (not overlapping with the receptor binding sites and the dimeric interface) and not C-terminal are found to be important for heparin–BMP interactions.

Thermodynamics of metal ion binding and denaturation of a calcium binding protein from Entamoeba histolytica

The thermodynamics of tie binding of calcium and magnesium ions to a calcium binding protein from Entamoeba histolytica was investigated by isothermal titration calorimetry (ITC) in 20 mM MOPS buffer (pH 7.0) at 20 degrees C. Enthalpy titration curves of calcium show the presence of four Ca2+ binding sites, There exist two low-affinity sites for Ca2+, both of which are exothermic in nature and with positive cooperative interaction between them. Two other high affinity sites for Ca2+ exist of which one is endothermic and the other exothermic, again with positive cooperative interaction. The binding constants for Ca2+ at the four sites have been verified by a competitive binding assay, where CaBP competes with a chromophoric chelator 5, 5'-Br-2 BAPTA to bind Ca2+ and a Ca2+ titration employing intrinsic tyrosine fluorescence of the protein, The enthalpy of titration of magnesium in the absence of calcium is single site and endothermic in nature. In the case of the titrations performed using protein presaturated with magnesium, the amount of heat produced is altered. Further, the interaction between the high-affinity sites changes to negative cooperativity. No exchange of heat was observed throughout the addition of magnesium in the presence of 1 mM calcium, Titrations performed on a cleaved peptide comprising the N-terminus and the central linker show the existence of two Ca2+ specific sites, These results indicate that this CaBP has one high-affinity Ca-Mg site, one high-affinity Ca-specific site, and two low-affinity Ca-specific sites. The thermodynamic parameters of the binding of these metal ions were used to elucidate the energetics at the individual site(s) and the interactions involved therein at various concentrations of the denaturant, guanidine hydrochloride, ranging from 0.05 to 6.5 M. Unfolding of the protein was also monitored by titration calorimetry as a function of the concentration of the denaturant. These data show that at a GdnHCl concentration of 0.25 M the binding affinity for the Mg2+ ion is lost and there are only two sites which can bind to Ca2+, with substantial loss cooperativity. At concentrations beyond 2.5 M GdnHCl, at which the unfolding of the tertiary structure of this protein is observed by near UV CD spectroscopy, the binding of Ca2+ ions is lost. We thus show that the domain containing the two low-affinity sites is the first to unfold in the presence of GdnHCl. Control experiments with change in ionic strength by addition of KCI in the range 0.25-1 M show the existence of four sites with altered ion binding parameters.

Toward the Discovery of Vaccine Adjuvants: Coupling In Silico Screening and In Vitro Analysis of Antagonist Binding to Human and Mouse CCR4 Receptors

Background: Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. Methodology: Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cellmigration, including that of CCR4(+) Tregs. Significance: Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Characterization of single-stranded DNA-binding proteins from Mycobacteria. The carboxyl-terminal of domain of SSB is essential for stable association with its cognate RecA protein

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.

The GAF Domain of the cGMP-Binding, cGMP-Specific Phosphodiesterase (PDE5) Is a Sensor and a Sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFaF163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET2 assays. BRET2 ratios of the wild type GAFa fusion protein, but not GAFaF163A, increased in the presence of cGMP but not cAMP. Higher basal BRET2 ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFaF163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET2 technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET2 sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.

Vitamin A and thyroxine carrier proteins in chicken plasma Steady-state control of the plasma level of free retinol-binding protein and free thyroxine

1. 1. The binding parameters of prealbumin-2 with retinol-binding protein and thyroxine (T4) revealed the existence of distinct and multiple sites for both retinol-binding protein and T4. 2. 2. From the analysis of binding parameters of retinol-binding protein with prealbumin-2 it is clear that under steady-state conditions about 99% of the holo-retinol-binding protein remains bound to prealbumin-2. 3. 3. Equilibrium dialysis studies on binding properties of thyroid hormones with prealbumin-2 revealed that it has a single high affinity site and three low affinity sites. 4. 4. The occurrence of three carrier proteins for thyroid hormones, thyroxine-binding globulin, prealbumin-2 and albumin has been demonstrated. However, the chicken thyroxine-binding globulin differs from human thyroxine-binding globulin by being relatively less acidic and occuring at a two-fold lower concentration. But the thyroid hormone binding parameters are comparable. 5. 5. Highly sensitive methods were developed for determination of T4 binding capacities of the various proteins and plasma level of total T4 by fractionation of carrier proteins and further quantitatively employing in electrophoresis and equilibrium dialysis. 6. 6. The thyroxine-binding proteins were found to be two types, one (viz., thyroxine-binding globulin) of great affinity but of low binding capacity, which mainly acts as reservoir of T4, and another (viz.,prealbumin-2) of low affinity but of high binding capacity, which can participate predominantly in the control of the free T4 pool.

Purification and characterization of three toxins and two agglutinins from Abrus precatorius seed by using lactamyl-Sepharose affinity chromatography

Three toxins, abrin-I, -II, and -III, and two agglutinins, APA-I and -II, were purified from the seeds of Abrus precatorius by lactamyl-Sepharose affinity chromatography followed by gel filtration and DEAE-Sephacel column chromatography. abrin-I did not bind on DEAE-Sephacel column chromatography and the bound abrin-II, abrin-III, APA-I, and APA-II were eluted with a sodium acetate gradient. The identity of each protein was established by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The relative molecular weights are abrin-I, 64,000; abrin-II and abrin-III, 63,000 each: APA-I, 130,000; and APA-II, 128,000. Isoelectric focusing revealed microheterogeneity due to the presence of isoforms in each protein. Toxicity and binding studies further confirmed the differences among the lectins. The time course of inhibition of protein synthesis in thymocytes by the toxins showed lag times of 78, 61, and 72 min with Ki's of 0.55, 0.99, and 0.74 ms−1 at a 0.63 nImage concentration of each of abrin-I, -II, and -III, respectively. A Scatchard plot obtained from the equilibrium measurement for the lectins binding to lactamyl-Sepharose beads showed nonlinearity, indicating a cooperative mode of binding which was not observed for APA-I binding to Sepharose 4B beads. Further, by the criterion of the isoelectric focusing profile, it was shown that the least toxic abrin-I and the highly toxic abrin-II isolated by lactamyl-Sepharose chromatography were not retained on a low-affinity Sepharose 4B matrix, which signifies the necessity of using a high-affinity matrix for the purification of the lectins.

Dopamine receptors in human foetal brains:Characterization, regulation and ontogeny of [3H]spiperone binding sites in striatum

Eighteen corpora striata from normal human foetal brains ranging in gestational age from 16 to 40 weeks and five from post natal brains ranging from 23 days to 42 years were analysed for the ontogeny of dopamine receptors using [3H]spiperone as the ligand and 10 mM dopamine hydrochloride was used in blanks. Spiperone binding sites were characterized in a 40-week-old foetal brain to be dopamine receptors by the following criteria: (1) It was localized in a crude mitochondrial pellet that included synaptosomes; (2) binding was saturable at 0.8 nM concentration; (3) dopaminergic antagonists spiperone, haloperidol, pimozide, trifluperazine and chlorpromazine competed for the binding with IC50 values in the range of 0.3–14 nM while agonists—apomorphine and dopamine gave IC50 values of 2.5 and 10 μM, respectively suggesting a D2 type receptor.Epinephrine and norepinephrine inhibited the binding much less efficiently while mianserin at 10 μM and serotonin at 1 mM concentration did not inhibit the binding. Bimolecular association and dissociation rate constants for the reversible binding were 5.7 × 108 M−1 min−1 and 5.0 × 10−2 min−1, respectively. Equilibrium dissociation constant was 87 pM and the KD obtained by saturation binding was 73 pM.During the foetal age 16 to 40 weeks, the receptor concentration remained in the range of 38–60 fmol/mg protein or 570–1080 fmol/g striatum but it increased two-fold postnatally reaching a maximum at 5 years Significantly, at lower foetal ages (16–24 weeks) the [3H]spiperone binding sites exhibited a heterogeneity with a high (KD, 13–85 pM) and a low (KD, 1.2–4.6 nM) affinity component, the former accounting for 13–24% of the total binding sites. This heterogeneity persisted even when sulpiride was used as a displacer. The number of high affinity sites increased from 16 weeks to 24 weeks and after 28 weeks of gestation, all the binding sites showed only a single high affinity.GTP decreased the agonist affinity as observed by dopamine competition of [3H]spiperone binding in 20-week-old foetal striata and at all subsequent ages. GTP increased IC50 values of dopamine 2 to 4.5 fold and Hill coefficients were also increased becoming closer to one suggesting that the dopamine receptor was susceptible to regulation from foetal life onwards.

DNA Binding, Coprotease, and Strand Exchange Activities of Mycobacterial RecA Proteins: Implications for Functional Diversity among RecA Nucleoprotein Filaments

One of the fundamental questions concerning homologous recombination is how RecA or its homologues recognize several DNA sequences with high affinity and catalyze all the diverse biological activities. In this study, we show that the extent of single-stranded DNA binding and strand exchange (SE) promoted by mycobacterial RecA proteins with DNA substrates having various degrees of GC content was comparable with that observed for Escherichia coli RecA. However, the rate and extent of SE promoted by these recombinases showed a strong negative correlation with increasing amounts of sequence divergence embedded at random across the length of the donor strand. Conversely, a positive correlation was seen between SE efficiency and the degree of sequence divergence in the recipient duplex DNA. The extent of heteroduplex formation was not significantly affected when both the pairing partners contained various degrees of sequence divergence, although there was a moderate decrease in the case of mycobacterial RecA proteins with substrates containing larger amounts of sequence divergence. Whereas a high GC content had no discernible effect on E. coli RecA coprotease activity, a negative correlation was apparent between mycobacterial RecA proteins and GC content. We further show clear differences in the extent of SE promoted by E. coli and mycobacterial RecA proteins in the presence of a wide range of ATP:ADP ratios. Taken together, our findings disclose the existence of functional diversity among E. coli and mycobacterial RecA nucleoprotein filaments, and the milieu of sequence divergence (i.e., in the donor or recipient) exerts differential effects on heteroduplex formation, which has implications for the emergence of new genetic variants.

Allostery and Conformational Dynamics in cAMP-binding Acyltransferases

Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein.

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

Background: mIHF belongs to a subfamily of proteins, distinct from E. coli IHF. Results: Functionally important amino acids of mIHF and the mechanism(s) underlying DNA binding, DNA bending, and site-specific recombination are distinct from that of E. coli IHF. Conclusion: mIHF functions could contribute beyond nucleoid compaction. Significance: Because mIHF is essential for growth, the molecular mechanisms identified here can be exploited in drug screening efforts. The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ihfA and ihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHF. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.