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PubMed2006

Computational elucidation of the structural basis of ligand binding to the dopamine 3 receptor through docking and homology modeling.

The dopamine subtype 3 receptor (D3) is a promising therapeutic target for the treatment of cocaine addiction, schizophrenia, Parkinson's disease, and other disorders, but little is known about the binding of ligands to D3 at the atomic level. In the present study, binding of 29 known ligands to the D3 receptor was modeled computationally using four D3 receptor models which were obtained from homology modeling. The predicted binding models were validated with experimental data from site-directed mutagenesis, structure-activity relationship studies, and affinity labeling studies. Docking scores calculated for these 29 ligands correlate reasonably well with the experimentally determined binding affinities. A pharmacophore model is proposed that describes the binding of ligands at a single D3 receptor binding site and offers insights into the binding of structurally diverse D3 ligands to this receptor.

Binding Sites↗
PubMed2003

Configurational entropy and cooperativity between ligand binding and dimerization in glycopeptide antibiotics.

Oligomerization and ligand binding are thermodynamically cooperative processes in many biochemical systems, and the mechanisms giving rise to cooperative behavior are generally attributed to changes in structure. In glycopeptide antibiotics, however, these cooperative processes are not accompanied by significant structural changes. To investigate the mechanism by which cooperativity arises in these compounds, fully solvated molecular dynamics simulations and quasiharmonic normal-mode analysis were performed on chloroeremomycin, vancomycin, and dechlorovancomycin. Configurational entropies were derived from the vibrational modes recovered from ligand-free and ligand-bound forms of the monomeric and dimeric species. Results indicate that both ligand binding and dimerization incur an entropic cost as vibrational activity in the central core of the antibiotic is shifted to higher frequencies with lower amplitudes. Nevertheless, ligand binding and dimerization are cooperative because the entropic cost of both processes occurring together is less than the cost of these processes occurring separately. These reductions in configurational entropy are more than sufficient in magnitude to account for the experimentally observed cooperativity between dimerization and ligand binding. We conclude that biochemical cooperativity can be mediated through changes in vibrational activity, irrespective of the presence or absence of concomitant structural change. This may represent a general mechanism of allostery underlying cooperative phenomena in diverse macromolecular systems.

Anti-Bacterial Agents↗
PubMed1997

Circular dichroism studies in conformation of cellobiohydrolase and endoglucanase from Trichoderma pseudokiningii S-38: effects of pH and ligand binding.

Effects of pH and ligand binding upon the conformation of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from Trichoderma pseudokiningii S-38 have been studied by circular dichroism measurements. In the high-pH range (6-9), increasing pH resulted in a similar conformational change occurring in free CBHI and EGI, while such treatment gave different changes of the two enzyme conformations in the presence of cellobiose. On the other hand, in the low-pH region, with both CBHI an EGI in the active form, decreasing pH resulted in a large conformational change of free EGI compared to that of free CBHI, whereas ligand binding resulted in a similar change of both CBHI and EGI, independent of pH change.

Cellobiose↗
PubMed2003

Characterization of two forms of mouse salivary androgen-binding protein (ABP): implications for evolutionary relationships and ligand-binding function.

Mouse salivary androgen-binding protein (ABP) is a member of the secretoglobin family produced in the submaxillary glands of house mice (Mus musculus). We report the cDNA sequences and amino acid sequences of the beta and gamma subunits of ABP from a mouse cDNA library, identifying the two subunits by their pIs and molecular weights. An anomalously high molecular weight of the alpha subunit is likely due to glycosylation at a single site. A phylogenetic comparison of the three subunits of ABP with the chains of other mammalian secretoglobins shows that ABP is most closely related to mouse lachrymal protein and to the major cat allergen Fel dI. An evaluation of the most conserved residues in ABP and the other secretoglobins, in light of structural data reported by others [Callebaut, I., Poupon, A., Bally, R., Demaret, J.-P., Housset, D., Delettre, J., Hossenlopp, P., and Mornon, J.-P. (2000) Ann. N.Y. Acad. Sci. 923, 90-112; Pattabiraman, N., Matthews, J., Ward, K., Mantile-Selvaggi, G., Miele, L., and Mukherjee, A. (2000) Ann. N.Y. Acad. Sci. 923, 113-127], allows us to draw conclusions about the critical residues important in ligand binding by the two different ABP dimers and to assess the importance of ligand binding in the function of the molecule. In addition to the cDNAs, which represent those of the musculus subspecies of Mus musculus, we also report the coding regions of the beta and gamma subunit cDNAs from two other mouse inbred strains which represent the other two subspecies: M. musculus domesticus and M. musculus castaneus. The high nonsynonymous/synonymous substitution rate ratios (K(a)/K(s)) for both the beta and gamma subunits suggest that these two proteins are evolving under strong directional selection, as has been reported for the alpha subunit [Hwang, J., Hofstetter, J., Bonhomme, F., and Karn, R. (1997) J. Hered. 88, 93-97; Karn, R., and Clements, M. (1999) Biochem. Genet. 37, 187-199].

Amino Acid Sequence↗
PubMed2001

Different effects of lectins on the ligand binding of the NMDA receptors and sigma sites in rat brain hippocampus synaptic membranes.

The effects of the lectins concanavalin A, WGA, ricin, abrin, and the mistletoe lectins from Viscum album MLI, MLII, and MLIII on the binding of ligands of the NMDA and sigma receptors in rat hippocampus synaptic plasma membranes were investigated. Binding of [3H]MK-801, [3H]glutamate, [3H]5,7-DCKA, and [3H]glycine to the membranes was decreased by 40-60% after addition of galactose-specific lectins (mistletoe lectins MLI, MLII, ricin, abrin) at concentrations of 0.01 mg/ml, but was not affected by the glucose- and mannose-specific lectin Con A, an acetylglucosamine-specific lectin WGA, or an acetylgalactosamine-specific lectin MLIII. The binding of [3H]SKF 10047 was decreased only in the presence of MLIII and did not change after addition of the other lectins. It is suggested that lectin-sensitive ligand binding sites of sigma- and NMDA receptors are located separately, and that the carbohydrate side chains of the sigma receptor do not participate in the modulation of the NMDA-receptor.

Animals↗
PubMed1991

Studies of ligand binding to Escherichia coli adenylosuccinate synthetase.

Dissociation constants of Escherichia coli adenylosuccinate synthetase with IMP, GTP, adenylosuccinate, and AMP (a competitive inhibitor for IMP) were determined by measuring the extent of quenching of the intrinsic tryptophan fluorescence of the enzyme. The enzyme has one binding site for each of these ligands. Aspartate and GDP did not quench the fluorescence to any great extent, and their dissociation constants could not be determined. These ligand binding studies were generally supportive of the kinetic mechanism proposed earlier for the enzyme. Cys291 was modified with the fluorescent chromophores N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate and tetramethylrhodamine maleimide in order to measure enzyme conformational changes attending ligand binding. The excitation and emission spectra of these fluorophores are not altered by the addition of active site binding ligands. TbGTP and TbGDP were used as native reporter groups, and changes in their fluorescence on complexing with the enzyme and various ligands made it possible to detect conformational changes occurring at the active site. Evidence is presented for abortive complexes of the type: enzyme-TbGTP-adenylosuccinate and enzyme-TbGTP-adenylosuccinate-aspartate. These results suggest that the IMP and aspartate binding sites are spatially separated.

Adenosine Monophosphate↗
PubMed1994

A carboxyl-terminally truncated mutant and nonglycosylated A2a adenosine receptors retain ligand binding.

The amino acids that comprise the ligand binding sites of adenosine receptors have not been identified. Adenosine and its agonist analogues differ from ligands for the well studied biogenic amine receptors and rhodopsin in that the adenosine receptor agonists are larger, contain a ribose moiety, and are uncharged at physiological pH. Thus, the locations of the ligand binding pockets of the adenosine receptors could differ significantly from those of the biogenic amine receptors. This report describes the characterization of a purification-amenable truncated mutant of the canine A2a adenosine receptor and demonstrates that neither the long carboxyl-terminal tail nor the glycosidic moiety appears to be required for ligand binding. The dog thyroid A2a adenosine receptor cDNA (RDC8) was subcloned into the mammalian expression vector pCMV4. A mutant A2a construct, in which six histidines replaced residues 310-412 as the carboxyl terminus of the protein, also was prepared. When overexpressed transiently in COS M6 cells, the wild-type and mutant A2a receptors exhibited similar 2-[p-(2-[3H]carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine saturation binding and competition curve profiles. The following biochemical techniques confirmed that the COS M6 cells were transcribing and translating A2a receptors of the expected molecular masses: (a) immunoblotting with an antipeptide antibody directed against the putative carboxyl-terminal side of the second extracellular loop (Tyr155-Val172) of the canine A2a adenosine receptor, (b) photoaffinity labeling with the A2a-selective agonist 125I-2-[4-[2-[2-[(4-azidophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl]ethylamino-5'-N-ethylcarboxamidoad enosine (125I-azido-PAPA-APEC), and (c) partial purification of the hexahistidine-tagged receptor on Ni2+.nitrilotriacetic acid resin. A presumed A2a receptor (44 kDa) from rabbit striatal membranes also was detected with the antisera against amino acids Tyr155-Val172 of the RDC8 receptor. Not only could the mutant A2a receptor be photolabeled specifically with 125I-azido-PAPA-APEC but so too could unglycosylated A2a receptors (i.e., from tunicamycin-treated COS M6 cells), either full length or truncated. In all of these cases, photolabeling was attenuated by both agonist and antagonist competitors.

Adenosine↗
PubMed1991

Total versus unspecific binding: standardization and optimization of receptor-ligand binding assays.

Intra- and interassay imprecision was evaluated in 3 in vitro ligand binding assays using different types of filtering devices, glass fiber filters and variations in methodology. In the [3H]-spiroperidol test, the already low unspecific binding seemed to be dependent on the type of filters employed, whereas in the [3H]-ketanserin- and [3H]-GR 65630-binding tests, differences were within the range of the normal variabilities. However, in the latter test, which is problematic owing to the high unspecific binding of [3H]-GR 65630, it was found that although the percentage of specific binding was fairly constant on different sheets, large differences in the absolute amounts of total and unspecific binding were observed on consecutive sheets in the same experiment. Thus, it is critically important to filter samples for total and unspecific binding together on the same filter sheet for the calculation of specific binding. Under these precautions, highly reproducible results for IC50-values in the screening of potential 5HT3-receptor ligands were obtained in spite of using rat entorhinal cortex, a relatively large area with suboptimal 5HT3-receptor density. In contrast, when using rat area postrema, which is optimal with respect to receptor density, more than 10 times the number of rats is necessary for a competition experiment due to the small size of this brain part. Since IC50-values for both areas compare favorably, entorhinal cortex should be used for ethical reasons and to minimize costs.

Animals↗
PubMed1997

Sequences in the ligand-binding domains of the human androgen and progesterone receptors which determine their distinct ligand identities.

The natural ligands of the progesterone (PR) and androgen (AR) receptors, progesterone and testosterone, differ only by their 17 beta-substitution. To identify within the AR and PR ligand-binding domains (LBDs) the sequences responsible for the differential recognition of these ligands, chimeric LBDs assembled from five homologous AR/PR 'cassettes' linked to the GAL4-DNA binding domain were constructed, and their ligand binding and transactivation characteristics were determined. Replacing the central cassette 3 of PR by that of AR generated a progesterone- and testosterone-responsive PR LBD with the AR residues 788-RHLS-791 being specifically involved in testosterone recognition, while the introduction of the C-terminal PR cassette 5 into AR conferred progestin responsiveness onto the AR LBD. These results suggest that residues within AR 788-RHLS-791 interact with the testosterone 17 beta-OH, while PR cassette 5 apparently contains the amino acid(s) specifically involved in the recognition of the progesterone 17 beta-acetyl group. However, ligand binding and transactivation by these chimeras were significantly decreased compared with those of the parental LBDs, indicating that residues located outside of these cassettes contribute to the proper positioning of the steroids in the AR and PR ligand-binding pockets (LBPs). Indeed, certain AR/PR chimeras acquired efficient ligand binding, but were unable to transactivate, indicating that the ligand was improperly bound in the chimeric. LBP and could not induce the conformational changes leading to a transcriptionally competent activation function (AF-2) within the LBD. The properties of the various LBD chimeras are discussed in view of the recently solved three-dimensional structures of the retinoid X receptor alpha apo- and retinoic acid receptor gamma holo-LBDs.

Amino Acid Sequence↗
PubMed2001

Identification and characterization of two distinct ligand binding regions of cubilin.

Using polymerase chain reaction-amplified fragments of cubilin, an endocytic receptor of molecular mass 460 kDa, we have identified two distinct ligand binding regions. Region 1 of molecular mass 71 kDa, which included the 113-residue N terminus along with the eight epidermal growth factor (EGF)-like repeats and CUB domains 1 and 2, and region 2 of molecular mass 37 kDa consisting of CUB domains 6-8 bound both intrinsic factor-cobalamin (vitamin B(12); Cbl) (IF-Cbl) and albumin. Within these two regions, the binding of both ligands was confined to a 110-115-residue stretch that encompassed either the 113-residue N terminus or CUB domain 7 and 8. Ca(2+) dependence of ligand binding or the ability of cubilin antiserum to inhibit ligand binding to the 113-residue N terminus was 60-65%. However, a combination of CUB domains 7 and 8 or 6-8 was needed to demonstrate significant Ca(2+) dependence or inhibition of ligand binding by cubilin antiserum. Antiserum to EGF inhibited albumin but not IF-Cbl binding to the N-terminal cubilin fragment that included the eight EGF-like repeats. While the presence of excess albumin had no effect on binding to IF-Cbl, IF-Cbl in excess was able to inhibit albumin binding to both regions of cubilin. Reductive alkylation of the 113-residue N terminus or CUB 6-8, CUB 7, or CUB 8 domain resulted in the abolishment of ligand binding. These results indicate that (a) cubilin contains two distinct regions that bind both IF-Cbl and albumin and that (b) binding of both IF-Cbl and albumin to each of these regions can be distinguished and is regulated by the nonassisted formation of local disulfide bonds.

Albumins↗
PubMed2001

Raman signatures of ligand binding and allosteric conformation change in hexameric insulin.

Hexameric insulin is an allosteric protein that undergoes transitions between three conformational states (T(6), T(3)R(3), and R(6)). These allosteric states are stabilized by the binding of ligands to the phenolic pockets and by the coordination of anions to the His B10 metal sites. Raman difference (RD) spectroscopy is utilized to examine the binding of phenolic ligands and the binding of thiocyanate, p-aminobenzoic acid (PABA), or 4-hydroxy-3-nitrobenzoic acid (4H3N) to the allosteric sites of T(3)R(3) and R(6). The RD spectroscopic studies show changes in the amide I and III bands for the transition of residues B1-B8 from a meandering coil to an alpha helix in the T-R transitions and identify the Raman signatures of the structural differences among the T(6), T(3)R(3), and R(6) states. Evidence of the altered environment caused by the approximately 30 A displacement of phenylalanine (Phe) B1 is clearly seen from changes in the Raman bands of the Phe ring. Raman signatures arising from the coordination of PABA or 4H3N to the histidine (His) B10 Zn(II) sites show these carboxylates give distorted, asymmetric coordination to Zn(II). The RD spectra also reveal the importance of the position and the type of substituents for designing aromatic carboxylates with high affinity for the His B10 metal site.

Allosteric Site↗
PubMed2006

Methods for the prediction of protein-ligand binding sites for structure-based drug design and virtual ligand screening.

Structure Based Drug Design (SBDD) is a computational approach to lead discovery that uses the three-dimensional structure of a protein to fit drug-like molecules into a ligand binding site to modulate function. Identifying the location of the binding site is therefore a vital first step in this process, restricting the search space for SBDD or virtual screening studies. The detection and characterisation of functional sites on proteins has increasingly become an area of interest. Structural genomics projects are increasingly yielding protein structures with unknown functions and binding sites. Binding site prediction was pioneered by pocket detection, since the binding site is often found in the largest pocket. More recent methods involve phylogenetic analysis, identifying structural similarity with proteins of known function and identifying regions on the protein surface with a potential for high binding affinity. Binding site prediction has been used in several SBDD projects and has been incorporated into several docking tools. We discuss different methods of ligand binding site prediction, their strengths and weaknesses, and how they have been used in SBDD.

Animals↗
PubMed1995

Expression of the rat alpha 1 thyroid hormone receptor ligand binding domain in Escherichia coli and the use of a ligand-induced conformation change as a method for its purification to homogeneity.

The rat alpha 1 thyroid hormone receptor (rTR alpha 1) mediates hormone-dependent gene regulation by utilizing several distinct structural domains, including those containing DNA and ligand binding sites. Binding of the hormone to the ligand binding domain (TR-LBD) induces conformational changes in the receptor that are involved in affecting the receptor's transcriptional regulatory and other functions. A 33-kDa protein fragment (Met122-Val410) of rTR alpha 1, which includes the entire TR-LBD, was expressed in Escherichia coli, yielding typically 1.5 mg of soluble TR-LBD/liter of bacteria. The protein was purified to > 99% homogeneity with a final yield of 24% by hydrophobic interaction, DEAE anionic exchange, and heparin cationic exchange chromatographic steps. The Kd of the purified TR-LBD for 3,3',5-triiodo-L-thyronine (T3) was 0.06 nM, identical to that for full-length rTR alpha 1. T3 analogs had affinities consistent with values obtained for full-length rTR alpha 1. In all three chromatography steps, TR-LBD prebound to [125I]T3 eluted earlier than the unliganded TR-LBD, like the full-length receptor. These studies indicate that the binding affinity and specificity of the TR-LBD are similar to those of the intact rTR alpha 1 and that the ligand-induced conformational changes occur in the LBD itself. These studies also provide methodology for obtaining milligram quantities of protein useful for biochemical and biophysical studies of the thyroid hormone receptor and its ligand-induced changes.

Amino Acid Sequence↗
PubMed2005

The beta subunit determines the ligand binding properties of synaptic glycine receptors.

Inhibitory glycine receptors (GlyRs) regulate motor coordination and sensory signal processing in spinal cord and other brain regions. GlyRs are pentameric proteins composed of membrane-spanning alpha and beta subunits. Here, site-directed mutagenesis combined with homology modeling based on the crystal structure of the acetylcholine binding protein identified key ligand binding residues of recombinant homooligomeric alpha1 and heterooligomeric alpha1beta GlyRs. This disclosed two highly conserved, oppositely charged residues located on adjacent subunit interfaces as being crucial for agonist binding. In addition, the beta subunit was found to determine the ligand binding properties of heterooligomeric GlyRs. Expression of an alpha1beta tandem construct and affinity purification of metabolically labeled GlyRs confirmed a subunit stoichiometry of 2alpha3beta. Because the beta subunit anchors GlyRs at synaptic sites, our results have important implications for the biosynthesis, clustering, and pharmacology of synaptic GlyRs.

Amino Acid Sequence↗
PubMed2001

Mapping the ligand-binding region of Borrelia burgdorferi fibronectin-binding protein BBK32.

The cellular attachment and entry of pathogenic microorganisms can be facilitated by the expression of microbial adhesins that bind fibronectin. We have previously described a Borrelia burgdorferi gene, bbk32, that encodes a 47-kDa fibronectin-binding protein. In this study, the ligand-binding region of BBK32 from B. burgdorferi isolate B31 was localized to 32 amino acids. The bbk32 gene was cloned and sequenced from three additional B. burgdorferi isolates representing different genospecies of B. burgdorferi sensu lato. All four bbk32 genes encoded proteins having fibronectin-binding activity when expressed in Escherichia coli, and the deduced proteins shared 81 to 91% amino acid sequence identity within the ligand-binding domain. In addition, the ligand-binding region of BBK32 was found to share sequence homology with a fibronectin-binding peptide defined for protein F1 of Streptococcus pyogenes. The structural and functional similarity between the ligand-binding region of BBK32 and the UR region of protein F1 suggests a common mechanism of cellular adhesion and entry for B. burgdorferi and S. pyogenes.

Amino Acid Sequence↗
PubMed2003

Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster.

Mutants created by site-directed mutagenesis were used to elucidate the function of amino acids involved in ligand binding to ecdysteroid receptor (EcR) and heterodimer formation with ultraspiracle (USP). The results demonstrate the importance of the C-terminal part of the D-domain and helix 12 of EcR for hormone binding. Some amino acids are involved either in ligand binding to EcR (E476, M504, D572, I617, N626) or ligand-dependent heterodimerization as determined by gel mobility shift assays (A612, L615, T619), while others are involved in both functions (K497, E648). Some amino acids are suboptimal for ligand binding (L615, T619), but mediate ligand-dependent dimerization. We conclude that the enhanced regulatory potential by ligand-dependent modulation of dimerization in the wild type is achieved at the expense of optimal ligand binding. Mutation of amino acids (K497, E648) involved in the salt bridge between helix 4 and 12 impair ligand binding to EcR more severely than hormone binding to the heterodimer, indicating that to some extent heterodimerization compensates for the deleterious effect of certain mutations. Different effects of the same point mutations on ligand binding to EcR and EcR/USP (R511, A612, L615, I617, T619, N626) indicate that the ligand-binding pocket is modified by heterodimerization.

Amino Acid Sequence↗
PubMed2004

Identification of residues in an orthopoxvirus interleukin-18 binding protein involved in ligand binding and species specificity.

Interleukin-18 (IL-18) is a critical cytokine in inflammation and adaptive immune responses. The IL-18 binding proteins (IL-18BP) are a family of proteins that bind to, and inhibit the activity of, IL-18. Using point mutagenesis, we analyzed the ectromelia virus IL-18BP to identify residues involved in binding. Because p13 can bind both human and murine IL-18, and because it is highly homologous to the variola virus IL-18BP, we set out to identify residues that may be involved in species specificity. Several of the mutations resulted in complete abrogation of binding affinity. Three (F49A, E77A, and E69A) significantly affected binding with both species of IL-18, but not to the same extent. Mutant H70A showed reduced affinity for human IL-18 while binding to murine IL-18 was not affected. This study demonstrated that interaction of IL-18 with p13 was similar to other IL-18BPs, however, novel species-specific interactions were identified.

Amino Acid Sequence↗
PubMed1997

Identification of a ligand-binding site on the granulocyte colony-stimulating factor receptor by molecular modeling and mutagenesis.

Granulocyte colony-stimulating factor (G-CSF) initiates its effects on cells of the neutrophil lineage by inducing formation of a homodimeric receptor complex. The structure of the G-CSF receptor has not yet been determined, therefore we used molecular modeling to identify regions of the receptor that were likely to be involved in ligand binding. The G-CSF receptor sequence was aligned with all the available sequences of the gp130 and growth hormone receptor families and a model of the cytokine receptor homologous domain was constructed, based on the growth hormone receptor structure. Alanine substitution mutagenesis was performed on loops and individual residues that were predicted to bind ligand. Mutant receptors were expressed in factor-dependent Ba/F3 cells and assessed for proliferation response and ligand binding. Six residues were identified that significantly reduced receptor function, with Arg288 in the F'-G' loop having the greatest effect. These residues formed a binding face on the receptor model resembling the growth hormone receptor site, which suggests that the model is reasonable. However, electrostatic analysis of the model provided further evidence that the mechanism of receptor dimerization is different from that of the growth hormone receptor.

Amino Acid Sequence↗