Science.gov

Sample records for allosteric ligand binding

  1. Ligand Binding to Macromolecules: Allosteric and Sequential Models of Cooperativity.

    ERIC Educational Resources Information Center

    Hess, V. L.; Szabo, Attila

    1979-01-01

    A simple model is described for the binding of ligands to macromolecules. The model is applied to the cooperative binding by hemoglobin and aspartate transcarbamylase. The sequential and allosteric models of cooperative binding are considered. (BB)

  2. Anisotropic energy flow and allosteric ligand binding in albumin

    PubMed Central

    Li, Guifeng; Magana, Donny; Dyer, R. Brian

    2014-01-01

    Allosteric interactions in proteins generally involve propagation of local structural changes through the protein to a remote site. Anisotropic energy transport is thought to couple the remote sites, but the nature of this process is poorly understood. Here, we report the relationship between energy flow through the structure of bovine serum albumin and allosteric interactions between remote ligand binding sites of the protein. Ultrafast infrared spectroscopy is used to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic and anisotropic energy flow through the protein structure following input of thermal energy into the flexible ligand binding sites, without local heating of the rigid helix bundles that connect these sites. This efficient energy transport mechanism enables the allosteric propagation of binding energy through the connecting helix structures. PMID:24445265

  3. Anisotropic energy flow and allosteric ligand binding in albumin

    NASA Astrophysics Data System (ADS)

    Li, Guifeng; Magana, Donny; Dyer, R. Brian

    2014-01-01

    Allosteric interactions in proteins generally involve propagation of local structural changes through the protein to a remote site. Anisotropic energy transport is thought to couple the remote sites, but the nature of this process is poorly understood. Here, we report the relationship between energy flow through the structure of bovine serum albumin and allosteric interactions between remote ligand binding sites of the protein. Ultrafast infrared spectroscopy is used to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic and anisotropic energy flow through the protein structure following input of thermal energy into the flexible ligand binding sites, without local heating of the rigid helix bundles that connect these sites. This efficient energy transport mechanism enables the allosteric propagation of binding energy through the connecting helix structures.

  4. Allosteric Ligand Binding and Anisotropic Energy Flow in Albumin

    NASA Astrophysics Data System (ADS)

    Dyer, Brian

    2014-03-01

    Protein allostery usually involves propagation of local structural changes through the protein to a remote site. Coupling of structural changes at remote sites is thought to occur through anisotropic energy transport, but the nature of this process is poorly understood. We have studied the relationship between allosteric interactions of remote ligand binding sites of the protein and energy flow through the structure of bovine serum albumin (BSA). We applied ultrafast infrared spectroscopy to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic flow through the protein structure following input of thermal energy into the flexible ligand binding sites. We also observe anisotropic heat flow through the structure, without local heating of the rigid helix bundles that connect these sites. We will discuss the implications of this efficient energy transport mechanism with regard to the allosteric propagation of binding energy through the connecting helix structures.

  5. Common Internal Allosteric Network Links Anesthetic Binding Sites in a Pentameric Ligand-Gated Ion Channel.

    PubMed

    Joseph, Thomas T; Mincer, Joshua S

    2016-01-01

    General anesthetics bind reversibly to ion channels, modifying their global conformational distributions, but the underlying atomic mechanisms are not completely known. We examine this issue by way of the model protein Gloeobacter violaceous ligand-gated ion channel (GLIC) using computational molecular dynamics, with a coarse-grained model to enhance sampling. We find that in flooding simulations, both propofol and a generic particle localize to the crystallographic transmembrane anesthetic binding region, and that propofol also localizes to an extracellular region shared with the crystallographic ketamine binding site. Subsequent simulations to probe these binding modes in greater detail demonstrate that ligand binding induces structural asymmetry in GLIC. Consequently, we employ residue interaction correlation analysis to describe the internal allosteric network underlying the coupling of ligand and distant effector sites necessary for conformational change. Overall, the results suggest that the same allosteric network may underlie the actions of various anesthetics, regardless of binding site. PMID:27403526

  6. Common Internal Allosteric Network Links Anesthetic Binding Sites in a Pentameric Ligand-Gated Ion Channel

    PubMed Central

    Joseph, Thomas T.

    2016-01-01

    General anesthetics bind reversibly to ion channels, modifying their global conformational distributions, but the underlying atomic mechanisms are not completely known. We examine this issue by way of the model protein Gloeobacter violaceous ligand-gated ion channel (GLIC) using computational molecular dynamics, with a coarse-grained model to enhance sampling. We find that in flooding simulations, both propofol and a generic particle localize to the crystallographic transmembrane anesthetic binding region, and that propofol also localizes to an extracellular region shared with the crystallographic ketamine binding site. Subsequent simulations to probe these binding modes in greater detail demonstrate that ligand binding induces structural asymmetry in GLIC. Consequently, we employ residue interaction correlation analysis to describe the internal allosteric network underlying the coupling of ligand and distant effector sites necessary for conformational change. Overall, the results suggest that the same allosteric network may underlie the actions of various anesthetics, regardless of binding site. PMID:27403526

  7. Exploration of Gated Ligand Binding Recognizes an Allosteric Site for Blocking FABP4-Protein Interaction

    PubMed Central

    Li, Yan; Li, Xiang; Dong, Zigang

    2015-01-01

    Fatty acid binding protein 4 (FABP4), reversibly binding to fatty acids and other lipids with high affinities, is a potential target for treatment of cancers. The binding site of FABP4 is buried in an interior cavity and thereby ligand binding/unbinding is coupled with opening/closing of FABP4. It is a difficult task both experimentally and computationally to illuminate the entry or exit pathway, especially with the conformational gating. In this report we combine extensive computer simulations, clustering analysis, and Markov state model to investigate the binding mechanism of FABP4 and troglitazone. Our simulations capture spontaneous binding and unbinding events as well as the conformational transition of FABP4 between the open and closed states. An allosteric binding site on the protein surface is recognized for development of novel FABP4 inhibitors. The binding affinity is calculated and compared with the experimental value. The kinetic analysis suggests that ligand residence on the protein surface may delay the binding process. Overall, our results provide a comprehensive picture of ligand diffusion on the protein surface, ligand migration into the buried cavity, and the conformational change of FABP4 at an atomic level. PMID:26580122

  8. Allosteric modulation of ligand binding to [3H](+)pentazocine-defined sigma recognition sites by phenytoin.

    PubMed

    DeHaven-Hudkins, D L; Ford-Rice, F Y; Allen, J T; Hudkins, R L

    1993-01-01

    The allosteric modulation of sigma recognition sites by phenytoin (diphenylhydantoin) has been demonstrated by the ability of phenytoin to stimulate binding of various [3H] sigma ligands, as well as to slow dissociation from sigma sites and to shift sigma sites from a low- to a high-affinity state. Phenytoin stimulated the binding of the sigma 1- selective ligand [3H](+)pentazocine in a dose-dependent manner. Stimulation of binding at a final concentration of 250 microM phenytoin was associated with a decrease in the KD. The affinities of the sigma reference compounds caramiphen, dextromethorphan, dextrophan, (+)3-PPP and (+)SKF-10,047 were three- to eight-fold higher, while the affinities of benzetimide, BMY-14802, carbetapentane, DTG and haloperidol were unchanged in the presence of 250 microM phenytoin. The relative sensitivity of sigma compounds to allosteric modulation by phenytoin is not a property of all sigma ligands, and may provide an in vitro basis for distinguishing actions of sigma compounds and predicting sigma effects in vivo. PMID:8515681

  9. Evolution of oligomeric state through allosteric pathways that mimic ligand binding

    PubMed Central

    Perica, Tina; Kondo, Yasushi; Tiwari, Sandhya P.; McLaughlin, Stephen H.; Kemplen, Katherine R.; Zhang, Xiuwei; Steward, Annette; Reuter, Nathalie; Clarke, Jane; Teichmann, Sarah A.

    2015-01-01

    Evolution and design of protein complexes is almost always viewed through the lens of amino acid mutations at protein interfaces. We showed previously that residues not involved in the physical interaction between proteins make important contributions to oligomerisation by acting indirectly or allosterically. Here, we sought to investigate the mechanism by which allosteric mutations act using the example of the PyrR family of pyrimidine operon attenuators. In this family, a perfectly sequence-conserved helix that forms a tetrameric interface is exposed as solvent-accessible surface in dimeric orthologues. This means that mutations must be acting from a distance to destabilize the interface. We identified eleven key mutations controlling oligomeric state, all distant from the interfaces and outside ligand-binding pockets. Finally, we show that the key mutations introduce conformational changes equivalent to the conformational shift between the free versus the nucleotide-bound conformations of the proteins. PMID:25525255

  10. Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design

    PubMed Central

    Larsson, Andreas; Nordlund, Paer; Jansson, Anna; Anand, Ganesh S.

    2016-01-01

    A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, KD, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (KD ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD). PMID:27253209

  11. Allosterism at muscarinic receptors: ligands and mechanisms.

    PubMed

    Birdsall, N J M; Lazareno, S

    2005-06-01

    The evaluation of allosteric ligands at muscarinic receptors is discussed in terms of the ability of the experimental data to be interpreted by the allosteric ternary complex model. The compilation of useful SAR information of allosteric ligands is not simple, especially for muscarinic receptors, where there are multiple allosteric sites and complex interactions. PMID:15974931

  12. Homotropic cooperativity from the activation pathway of the allosteric ligand-responsive regulatory trp RNA-binding attenuation protein.

    PubMed

    Kleckner, Ian R; McElroy, Craig A; Kuzmic, Petr; Gollnick, Paul; Foster, Mark P

    2013-12-10

    The trp RNA-binding attenuation protein (TRAP) assembles into an 11-fold symmetric ring that regulates transcription and translation of trp-mRNA in bacilli via heterotropic allosteric activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic resonance studies have revealed that Trp-induced activation coincides with both microsecond to millisecond rigidification and local structural changes in TRAP, the pathway of binding of the 11 Trp ligands to the TRAP ring remains unclear. Moreover, because each of 11 bound Trp molecules is completely surrounded by protein, its release requires flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence to study the kinetics of Trp binding by Bacillus stearothermophilus TRAP over a range of temperatures and observed well-separated kinetic steps. These data were analyzed using nonlinear least-squares fitting of several two- and three-step models. We found that a model with two binding steps best describes the data, although the structural equivalence of the binding sites in TRAP implies a fundamental change in the time-dependent structure of the TRAP rings upon Trp binding. Application of the two-binding step model reveals that Trp binding is much slower than the diffusion limit, suggesting a gating mechanism that depends on the dynamics of apo TRAP. These data also reveal that dissociation of Trp from the second binding mode is much slower than after the first Trp binding mode, revealing insight into the mechanism for positive homotropic allostery, or cooperativity. Temperature-dependent analyses reveal that both binding modes imbue increases in bondedness and order toward a more compressed active state. These results provide insight into mechanisms of cooperative TRAP activation and underscore the importance of protein dynamics for ligand binding, ligand release, protein activation, and allostery. PMID:24224873

  13. A functional NMR for membrane proteins: dynamics, ligand binding, and allosteric modulation.

    PubMed

    Oxenoid, Kirill; Chou, James J

    2016-05-01

    By nature of conducting ions, transporting substrates and transducing signals, membrane channels, transporters and receptors are expected to exhibit intrinsic conformational dynamics. It is therefore of great interest and importance to understand the various properties of conformational dynamics acquired by these proteins, for example, the relative population of states, exchange rate, conformations of multiple states, and how small molecule ligands modulate the conformational exchange. Because small molecule binding to membrane proteins can be weak and/or dynamic, structural characterization of these effects is very challenging. This review describes several NMR studies of membrane protein dynamics, ligand-induced conformational rearrangements, and the effect of ligand binding on the equilibrium of conformational exchange. The functional significance of the observed phenomena is discussed. PMID:26928605

  14. LIBSA – A Method for the Determination of Ligand-Binding Preference to Allosteric Sites on Receptor Ensembles

    PubMed Central

    2015-01-01

    Incorporation of receptor flexibility into computational drug discovery through the relaxed complex scheme is well suited for screening against a single binding site. In the absence of a known pocket or if there are multiple potential binding sites, it may be necessary to do docking against the entire surface of the target (global docking). However no suitable and easy-to-use tool is currently available to rank global docking results based on the preference of a ligand for a given binding site. We have developed a protocol, termed LIBSA for LIgand Binding Specificity Analysis, that analyzes multiple docked poses against a single or ensemble of receptor conformations and returns a metric for the relative binding to a specific region of interest. By using novel filtering algorithms and the signal-to-noise ratio (SNR), the relative ligand-binding frequency at different pockets can be calculated and compared quantitatively. Ligands can then be triaged by their tendency to bind to a site instead of ranking by affinity alone. The method thus facilitates screening libraries of ligand cores against a large library of receptor conformations without prior knowledge of specific pockets, which is especially useful to search for hits that selectively target a particular site. We demonstrate the utility of LIBSA by showing that it correctly identifies known ligand binding sites and predicts the relative preference of a set of related ligands for different pockets on the same receptor. PMID:24437606

  15. Polypharmacology within CXCR4: Multiple binding sites and allosteric behavior

    NASA Astrophysics Data System (ADS)

    Planesas, Jesús M.; Pérez-Nueno, Violeta I.; Borrell, José I.; Teixidó, Jordi

    2014-10-01

    CXCR4 is a promiscuous receptor, which binds multiple diverse ligands. As usual in promiscuous proteins, CXCR4 has a large binding site, with multiple subsites, and high flexibility. Hence, it is not surprising that it is involved in the phenomenon of allosteric modulation. However, incomplete knowledge of allosteric ligand-binding sites has hampered an in-depth molecular understanding of how these inhibitors work. For example, it is known that lipidated fragments of intracellular GPCR loops, so called pepducins, such as pepducin ATI-2341, modulate CXCR4 activity using an agonist allosteric mechanism. Nevertheless, there are also examples of small organic molecules, such as AMD11070 and GSK812397, which may act as antagonist allosteric modulators. Here, we give new insights into this issue by proposing the binding interactions between the CXCR4 receptor and the above-mentioned allosteric modulators. We propose that CXCR4 has minimum two topographically different allosteric binding sites. One allosteric site would be in the intracellular loop 1 (ICL1) where pepducin ATI-2341 would bind to CXCR4, and the second one, in the extracellular side of CXCR4 in a subsite into the main orthosteric binding pocket, delimited by extracellular loops n° 1, 2, and the N-terminal end, where antagonists AMD11070 and GSK812397 would bind. Prediction of allosteric interactions between CXCR4 and pepducin ATI-2341 were studied first by rotational blind docking to determine the main binding region and a subsequent refinement of the best pose was performed using flexible docking methods and molecular dynamics. For the antagonists AMD11070 and GSK812397, the entire CXCR4 protein surface was explored by blind docking to define the binding region. A second docking analysis by subsites of the identified binding region was performed to refine the allosteric interactions. Finally, we identified the binding residues that appear to be essential for CXCR4 (agonists and antagonists) allosteric

  16. Allosteric regulation of pentameric ligand-gated ion channels

    PubMed Central

    Taly, Antoine; Hénin, Jérôme; Changeux, Jean-Pierre; Cecchini, Marco

    2014-01-01

    Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger—a neurotransmitter—into an ion flux through the postsynaptic membrane. They are oligomeric assemblies that provide prototypical examples of allosterically regulated integral membrane proteins. Here, we present an overview of the most recent advances on the signal transduction mechanism based on the X-ray structures of both prokaryotic and invertebrate eukaryotic pLGICs and on atomistic Molecular Dynamics simulations. The present results suggest that ion gating involves a large structural reorganization of the molecule mediated by two distinct quaternary transitions, a global twisting and the blooming of the extracellular domain, which can be modulated by ligand binding at the topographically distinct orthosteric and allosteric sites. The emerging model of gating is consistent with a wealth of functional studies and will boost the development of novel pharmacological strategies. PMID:25478624

  17. A Unified View of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gating: Combining the Allosterism of a Ligand-gated Channel with the Enzymatic Activity of an ATP-binding Cassette (ABC) Transporter*

    PubMed Central

    Kirk, Kevin L.; Wang, Wei

    2011-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ion channel in that its gating is coupled to an intrinsic enzymatic activity (ATP hydrolysis). This enzymatic activity derives from the evolutionary origin of CFTR as an ATP-binding cassette transporter. CFTR gating is distinct from that of a typical ligand-gated channel because its ligand (ATP) is usually consumed during the gating cycle. However, recent findings indicate that CFTR gating exhibits allosteric properties that are common to conventional ligand-gated channels (e.g. unliganded openings and constitutive mutations). Here, we provide a unified view of CFTR gating that combines the allosterism of a ligand-gated channel with its unique enzymatic activity. PMID:21296873

  18. Allosteric Modulation of G Protein Coupled Receptors by Cytoplasmic, Transmembrane and Extracellular Ligands

    PubMed Central

    Yanamala, Naveena; Klein-Seetharaman, Judith

    2010-01-01

    G protein coupled receptors (GPCRs) bind diverse classes of ligands, and depending on the receptor, these may bind in their transmembrane or the extracellular domains, demonstrating the principal ability of GPCRs to bind ligand in either domains. Most recently, it was also observed that small molecule ligands can bind in the cytoplasmic domain, and modulate binding and response to extracellular or transmembrane ligands. Thus, all three domains in GPCRs are potential sites for allosteric ligands, and whether a ligand is allosteric or orthosteric depends on the receptor. Here, we will review the evidence supporting the presence of putative binding pockets in all three domains of GPCRs and discuss possible pathways of communication between these pockets. PMID:24009470

  19. Molecular Recognition of the Catalytic Zinc(II) Ion in MMP-13: Structure-Based Evolution of an Allosteric Inhibitor to Dual Binding Mode Inhibitors with Improved Lipophilic Ligand Efficiencies

    PubMed Central

    Fischer, Thomas; Riedl, Rainer

    2016-01-01

    Matrix metalloproteinases (MMPs) are a class of zinc dependent endopeptidases which play a crucial role in a multitude of severe diseases such as cancer and osteoarthritis. We employed MMP-13 as the target enzyme for the structure-based design and synthesis of inhibitors able to recognize the catalytic zinc ion in addition to an allosteric binding site in order to increase the affinity of the ligand. Guided by molecular modeling, we optimized an initial allosteric inhibitor by addition of linker fragments and weak zinc binders for recognition of the catalytic center. Furthermore we improved the lipophilic ligand efficiency (LLE) of the initial inhibitor by adding appropriate zinc binding fragments to lower the clogP values of the inhibitors, while maintaining their potency. All synthesized inhibitors showed elevated affinity compared to the initial hit, also most of the novel inhibitors displayed better LLE. Derivatives with carboxylic acids as the zinc binding fragments turned out to be the most potent inhibitors (compound 3 (ZHAWOC5077): IC50 = 134 nM) whereas acyl sulfonamides showed the best lipophilic ligand efficiencies (compound 18 (ZHAWOC5135): LLE = 2.91). PMID:26938528

  20. Molecular Recognition of the Catalytic Zinc(II) Ion in MMP-13: Structure-Based Evolution of an Allosteric Inhibitor to Dual Binding Mode Inhibitors with Improved Lipophilic Ligand Efficiencies.

    PubMed

    Fischer, Thomas; Riedl, Rainer

    2016-01-01

    Matrix metalloproteinases (MMPs) are a class of zinc dependent endopeptidases which play a crucial role in a multitude of severe diseases such as cancer and osteoarthritis. We employed MMP-13 as the target enzyme for the structure-based design and synthesis of inhibitors able to recognize the catalytic zinc ion in addition to an allosteric binding site in order to increase the affinity of the ligand. Guided by molecular modeling, we optimized an initial allosteric inhibitor by addition of linker fragments and weak zinc binders for recognition of the catalytic center. Furthermore we improved the lipophilic ligand efficiency (LLE) of the initial inhibitor by adding appropriate zinc binding fragments to lower the clogP values of the inhibitors, while maintaining their potency. All synthesized inhibitors showed elevated affinity compared to the initial hit, also most of the novel inhibitors displayed better LLE. Derivatives with carboxylic acids as the zinc binding fragments turned out to be the most potent inhibitors (compound 3 (ZHAWOC5077): IC50 = 134 nM) whereas acyl sulfonamides showed the best lipophilic ligand efficiencies (compound 18 (ZHAWOC5135): LLE = 2.91). PMID:26938528

  1. Regulation of G Protein-Coupled Receptors by Allosteric Ligands

    PubMed Central

    2013-01-01

    Topographically distinct, druggable, allosteric sites may be present on all G protein-coupled receptors (GPCRs). As such, targeting these sites with synthetic small molecules offers an attractive approach to develop receptor-subtype selective chemical leads for the development of novel therapies. A crucial part of drug development is to understand the acute and chronic effects of such allosteric modulators at their corresponding GPCR target. Key regulatory processes including cell-surface delivery, endocytosis, recycling, and down-regulation tightly control the number of receptors at the surface of the cell. As many GPCR therapeutics will be administered chronically, understanding how such ligands modulate these regulatory pathways forms an essential part of the characterization of novel GPCR ligands. This is true for both orthosteric and allosteric ligands. In this Review, we summarize our current understanding of GPCR regulatory processes with a particular focus on the effects and implications of allosteric targeting of GPCRs. PMID:23398684

  2. Design and Synthesis of Photoaffinity Labeling Ligands of the l-Prolyl-l-leucyl-glycinmide Binding Site Involved in the Allosteric Modulation of the Dopamine Receptor

    PubMed Central

    Fisher, Abigail; Mann, Amandeep; Verma, Vaneeta; Thomas, Nancy; Mishra, Ram K.; Johnson, Rodney L.

    2008-01-01

    Pro-Leu-Gly-NH2 (PLG), in addition to its endocrine effects, possesses the ability to modulate dopamine D2 receptors within the CNS. However, the precise binding site of PLG is unknown. Potential photoaffinity labeling ligands of the PLG binding site were designed as tools to be used in the identification of the macromolecule that possesses this binding site. Six different photoaffinity labeling ligands were designed and synthesized based upon γ-lactam PLG peptidomimetic 1. The 4-azido-benzoyl and 4-azido-2-hydroxy-benzoyl photoaffinity labeling moieties were placed at opposite ends of PLG peptidomimetic 1 to generate a series of ligands that potentially could be used to map the PLG binding site. All of the compounds that were synthesized possessed activity comparable to or better than PLG in enhancing [3H]N-propylnorapomorphine agonist binding to dopamine receptors. Photoaffinity ligands that were cross-linked to the receptor preparation produced a modulatory effect that was either comparable to or greater than the increase in agonist binding produced by the respective ligands that were not cross-linked to the dopamine receptor. The results indicate that these photoaffinity labeling agents are binding at the same site as PLG and PLG peptidomimetic 1. PMID:16392815

  3. Interactions of orthosteric and allosteric ligands with [3H]dimethyl-W84 at the common allosteric site of muscarinic M2 receptors.

    PubMed

    Tränkle, Christian; Weyand, Oliver; Voigtländer, Uta; Mynett, Anita; Lazareno, Sebastian; Birdsall, Nigel J M; Mohr, Klaus

    2003-07-01

    An optimized assay for the binding of [3H]dimethyl-W84 to its allosteric site on M2 muscarinic receptors has been used to directly measure the affinities of allosteric ligands. Their potencies agree with those deduced indirectly by their modulation of the equilibrium binding and kinetics of [3H]N-methylscopolamine ([3H]NMS) binding to the orthosteric site. The affinities and cooperativities of orthosteric antagonists with [3H]dimethyl-W84 have also been quantitated. These affinities agree with those measured directly in a competition assay using [3H]NMS. All these data are compatible with the predictions of the allosteric ternary complex model. The association and dissociation kinetics of [3H]dimethyl-W84 are rapid but the estimate of its association rate constant is nevertheless comparable with that found for the orthosteric radioligand, [3H]NMS. This is unexpected, given that the allosteric site to which [3H]dimethyl-W84 binds is thought to be located on the external face of the receptor and above the [3H]NMS binding site that is buried within the transmembrane helices. The atypical allosteric ligands tacrine and 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3) inhibit [3H]dimethyl-W84 binding with the same potencies and comparably steep slope factors as found for inhibition of [3H]NMS binding. Tacrine and Duo3 decrease [3H]dimethyl-W84 affinity, not the number of binding sites. It is suggested that these atypical ligands either bind to the two known spatially separated allosteric sites on muscarinic receptors with positive cooperativity or their binding to the common allosteric site modulates receptor-receptor interactions such that homotropic positive cooperativity within a dimer or higher oligomer is generated. PMID:12815174

  4. Proposed Mode of Binding and Action of Positive Allosteric Modulators at Opioid Receptors.

    PubMed

    Shang, Yi; Yeatman, Holly R; Provasi, Davide; Alt, Andrew; Christopoulos, Arthur; Canals, Meritxell; Filizola, Marta

    2016-05-20

    Available crystal structures of opioid receptors provide a high-resolution picture of ligand binding at the primary ("orthosteric") site, that is, the site targeted by endogenous ligands. Recently, positive allosteric modulators of opioid receptors have also been discovered, but their modes of binding and action remain unknown. Here, we use a metadynamics-based strategy to efficiently sample the binding process of a recently discovered positive allosteric modulator of the δ-opioid receptor, BMS-986187, in the presence of the orthosteric agonist SNC-80, and with the receptor embedded in an explicit lipid-water environment. The dynamics of BMS-986187 were enhanced by biasing the potential acting on the ligand-receptor distance and ligand-receptor interaction contacts. Representative lowest-energy structures from the reconstructed free-energy landscape revealed two alternative ligand binding poses at an allosteric site delineated by transmembrane (TM) helices TM1, TM2, and TM7, with some participation of TM6. Mutations of amino acid residues at these proposed allosteric sites were found to either affect the binding of BMS-986187 or its ability to modulate the affinity and/or efficacy of SNC-80. Taken together, these combined experimental and computational studies provide the first atomic-level insight into the modulation of opioid receptor binding and signaling by allosteric modulators. PMID:26841170

  5. Structure-Based Ligand Discovery Targeting Orthosteric and Allosteric Pockets of Dopamine Receptors

    PubMed Central

    Lane, J. Robert; Chubukov, Pavel; Liu, Wei; Canals, Meritxell; Cherezov, Vadim; Abagyan, Ruben; Stevens, Raymond C.

    2013-01-01

    Small molecules targeting allosteric pockets of G protein–coupled receptors (GPCRs) have a great therapeutic potential for the treatment of neurologic and other chronic disorders. Here we performed virtual screening for orthosteric and putative allosteric ligands of the human dopamine D3 receptor (D3R) using two optimized crystal-structure–based models: the receptor with an empty binding pocket (D3RAPO), and the receptor complex with dopamine (D3RDopa). Subsequent biochemical and functional characterization revealed 14 novel ligands with a binding affinity of better than 10 μM in the D3RAPO candidate list (56% hit rate), and 8 novel ligands in the D3RDopa list (32% hit rate). Most ligands in the D3RAPO model span both orthosteric and extended pockets and behave as antagonists at D3R, with compound 7 showing the highest potency of dopamine inhibition (IC50 = 7 nM). In contrast, compounds identified by the D3RDopa model are predicted to occupy an allosteric site at the extracellular extension of the pocket, and they all lack the anchoring amino group. Compounds targeting the allosteric site display a variety of functional activity profiles, where behavior of at least two compounds (23 and 26) is consistent with noncompetitive allosteric modulation of dopamine signaling in the extracellular signal-regulated kinase 1 and 2 phosphorylation and β-arrestin recruitment assays. The high affinity and ligand efficiency of the chemically diverse hits identified in this study suggest utility of structure-based screening targeting allosteric sites of GPCRs. PMID:24021214

  6. Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

    PubMed Central

    Wang, Ningkun; Lodge, Jean M.; Fierke, Carol A.; Mapp, Anna K.

    2014-01-01

    Allosteric binding events play a critical role in the formation and stability of transcriptional activator–coactivator complexes, perhaps in part due to the often intrinsically disordered nature of one or more of the constituent partners. The kinase-inducible domain interacting (KIX) domain of the master coactivator CREB binding protein/p300 is a conformationally dynamic domain that complexes with transcriptional activators at two discrete binding sites in allosteric communication. The complexation of KIX with the transcriptional activation domain of mixed-lineage leukemia protein leads to an enhancement of binding by the activation domain of CREB (phosphorylated kinase-inducible domain of CREB) to the second site. A transient kinetic analysis of the ternary complex formation aided by small molecule ligands that induce positive or negative cooperative binding reveals that positive cooperativity is largely governed by stabilization of the bound complex as indicated by a decrease in koff. Thus, this suggests the increased binding affinity for the second ligand is not due to an allosteric creation of a more favorable binding interface by the first ligand. This is consistent with data from us and from others indicating that the on rates of conformationally dynamic proteins approach the limits of diffusion. In contrast, negative cooperativity is manifested by alterations in both kon and koff, suggesting stabilization of the binary complex. PMID:25049401

  7. Kinetic analysis of ligand binding to the Ehrlich cell nucleoside transporter: Pharmacological characterization of allosteric interactions with the sup 3 Hnitrobenzylthioinosine binding site

    SciTech Connect

    Hammond, J.R. )

    1991-06-01

    Kinetic analysis of the binding of {sup 3}Hnitrobenzylthioinosine ({sup 3}H NBMPR) to Ehrlich ascites tumor cell plasma membranes was conducted in the presence and absence of a variety of nucleoside transport inhibitors and substrates. The association of {sup 3}H NBMPR with Ehrlich cell membranes occurred in two distinct phases, possibly reflecting functional conformation changes in the {sup 3}HNBMPR binding site/nucleoside transporter complex. Inhibitors of the equilibrium binding of {sup 3}HNBMPR, tested at submaximal inhibitory concentrations, generally decreased the rate of association of {sup 3}HNBMPR, but the magnitude of this effect varied significantly with the agent tested. Adenosine and diazepam had relatively minor effects on the association rate, whereas dipyridamole and mioflazine slowed the rate dramatically. Inhibitors of nucleoside transport also decreased the rate of dissociation of {sup 3}HNBMPR, with an order of potency significantly different from their relative potencies as inhibitors of the equilibrium binding of {sup 3}HNBMPR. Dilazep, dipyridamole, and mioflazine were effective inhibitors of both {sup 3}HNBMPR dissociation and equilibrium binding. The lidoflazine analogue R75231, on the other hand, had no effect on the rate of dissociation of {sup 3}HNBMPR at concentrations below 300 microM, even though it was one of the most potent inhibitors of {sup 3}HNBMPR binding tested (Ki less than 100 nM). In contrast, a series of natural substrates for the nucleoside transport system enhanced the rate of dissociation of {sup 3}HNBMPR with an order of effectiveness that paralleled their relative affinities for the permeant site of the transporter. The most effective enhancers of {sup 3}HNBMPR dissociation, however, were the benzodiazepines diazepam, chlordiazepoxide, and triazolam.

  8. Molecular mechanism of allosteric modulation at GPCRs: insight from a binding kinetics study at the human A1 adenosine receptor

    PubMed Central

    Guo, Dong; Venhorst, Suzanne N; Massink, Arnault; van Veldhoven, Jacobus P D; Vauquelin, Georges; IJzerman, Adriaan P; Heitman, Laura H

    2014-01-01

    Background and Purpose Many GPCRs can be allosterically modulated by small-molecule ligands. This modulation is best understood in terms of the kinetics of the ligand–receptor interaction. However, many current kinetic assays require at least the (radio)labelling of the orthosteric ligand, which is impractical for studying a range of ligands. Here, we describe the application of a so-called competition association assay at the adenosine A1 receptor for this purpose. Experimental Approach We used a competition association assay to examine the binding kinetics of several unlabelled orthosteric agonists of the A1 receptor in the absence or presence of two allosteric modulators. We also tested three bitopic ligands, in which an orthosteric and an allosteric pharmacophore were covalently linked with different spacer lengths. The relevance of the competition association assay for the binding kinetics of the bitopic ligands was also explored by analysing simulated data. Key Results The binding kinetics of an unlabelled orthosteric ligand were affected by the addition of an allosteric modulator and such effects were probe- and concentration-dependent. Covalently linking the orthosteric and allosteric pharmacophores into one bitopic molecule had a substantial effect on the overall on- or off-rate. Conclusion and Implications The competition association assay is a useful tool for exploring the allosteric modulation of the human adenosine A1 receptor. This assay may have general applicability to study allosteric modulation at other GPCRs as well. PMID:25040887

  9. Virtual screening with AutoDock Vina and the common pharmacophore engine of a low diversity library of fragments and hits against the three allosteric sites of HIV integrase: participation in the SAMPL4 protein-ligand binding challenge.

    PubMed

    Perryman, Alexander L; Santiago, Daniel N; Forli, Stefano; Santos-Martins, Diogo; Olson, Arthur J

    2014-04-01

    To rigorously assess the tools and protocols that can be used to understand and predict macromolecular recognition, and to gain more structural insight into three newly discovered allosteric binding sites on a critical drug target involved in the treatment of HIV infections, the Olson and Levy labs collaborated on the SAMPL4 challenge. This computational blind challenge involved predicting protein-ligand binding against the three allosteric sites of HIV integrase (IN), a viral enzyme for which two drugs (that target the active site) have been approved by the FDA. Positive control cross-docking experiments were utilized to select 13 receptor models out of an initial ensemble of 41 different crystal structures of HIV IN. These 13 models of the targets were selected using our new "Rank Difference Ratio" metric. The first stage of SAMPL4 involved using virtual screens to identify 62 active, allosteric IN inhibitors out of a set of 321 compounds. The second stage involved predicting the binding site(s) and crystallographic binding mode(s) for 57 of these inhibitors. Our team submitted four entries for the first stage that utilized: (1) AutoDock Vina (AD Vina) plus visual inspection; (2) a new common pharmacophore engine; (3) BEDAM replica exchange free energy simulations, and a Consensus approach that combined the predictions of all three strategies. Even with the SAMPL4's very challenging compound library that displayed a significantly lower amount of structural diversity than most libraries that are conventionally employed in prospective virtual screens, these approaches produced hit rates of 24, 25, 34, and 27 %, respectively, on a set with 19 % declared binders. Our only entry for the second stage challenge was based on the results of AD Vina plus visual inspection, and it ranked third place overall according to several different metrics provided by the SAMPL4 organizers. The successful results displayed by these approaches highlight the utility of the computational

  10. Engineering and optimization of an allosteric biosensor protein for peroxisome proliferator-activated receptor γ ligands.

    PubMed

    Li, Jingjing; Gierach, Izabela; Gillies, Alison R; Warden, Charles D; Wood, David W

    2011-11-15

    The peroxisome proliferator-activated receptor gamma (PPARγ or PPARG) belongs to the nuclear receptor superfamily, and is a potential drug target for a variety of diseases. In this work, we constructed a series of bacterial biosensors for the identification of functional PPARγ ligands. These sensors entail modified Escherichia coli cells carrying a four-domain fusion protein, comprised of the PPARγ ligand binding domain (LBD), an engineered mini-intein domain, the E. coli maltose binding protein (MBD), and a thymidylate synthase (TS) reporter enzyme. E. coli cells expressing this protein exhibit hormone ligand-dependent growth phenotypes. Unlike our published estrogen (ER) and thyroid receptor (TR) biosensors, the canonical PPARγ biosensor cells displayed pronounced growth in the absence of ligand. They were able to distinguish agonists and antagonists, however, even in the absence of agonist. To improve ligand sensitivity of this sensor, we attempted to engineer and optimize linker peptides flanking the PPARγ LBD insertion point. Truncation of the original linkers led to decreased basal growth and significantly enhanced ligand sensitivity of the PPARγ sensor, while substitution of the native linkers with optimized G(4)S (Gly-Gly-Gly-Gly-Ser) linkers further increased the sensitivity. Our studies demonstrate that the properties of linkers, especially the C-terminal linker, greatly influence the efficiency and fidelity of the allosteric signal induced by ligand binding. Our work also suggests an approach to increase allosteric behavior in this multidomain sensor protein, without modification of the functional LBD. PMID:21893405

  11. Nucleation of an Allosteric Response via Ligand-induced Loop Folding

    PubMed Central

    Naganathan, Saranga; Beckett, Dorothy

    2009-01-01

    The Escherichia coli biotin repressor, BirA, is an allosteric transcription regulatory protein to which binding of the small ligand corepressor, biotinyl-5’-AMP, promotes homodimerization and subsequent DNA binding. Structural data indicate that the apo- or unliganded repressor is characterized by four partially disordered loops that are ordered in the ligand-bound dimer. While three of these loops participate directly in the dimerization, the fourth, consisting of residues 212-234 is distal to the interface. This loop, which is ordered around the adenine ring of the adenylate in the BirA adenylate structure, is referred to as the adenylate binding loop or ABL. Although residues in the loop do not directly interact with the ligand, a hydrophobic cluster consisting of a tryptophan and two valine side chains assembles over the adenine base. Results of previous measurements suggest that folding of the ABL is integral to the allosteric response. This idea and the role of the hydrophobic cluster in the process were investigated by systematic replacement of each side chain in the cluster with alanine and analysis of the variant proteins for small ligand binding and dimerization. Isothermal titration calorimetry measurements indicate defects in adenylate binding for all ABL variants. Additionally, sedimentation equilibrium measurements reveal that coupling between adenylate binding and dimerization is compromised in each mutant. Partial proteolysis measurements indicate that the mutants are defective in ligand-linked folding of the ABL. These results indicate that the hydrophobic cluster is critical to the ligand-induced disorder-to-order transition in the ABL and that this transition is integral to the allosteric response in the biotin repressor. PMID:17765263

  12. Computational predictions suggest that structural similarity in viral polymerases may lead to comparable allosteric binding sites.

    PubMed

    Brown, Jodian A; Espiritu, Marie V; Abraham, Joel; Thorpe, Ian F

    2016-08-15

    The identification of ligand-binding sites is often the first step in drug targeting and design. To date there are numerous computational tools available to predict ligand binding sites. These tools can guide or mitigate the need for experimental methods to identify binding sites, which often require significant resources and time. Here, we evaluate four ligand-binding site predictor (LBSP) tools for their ability to predict allosteric sites within the Hepatitis C Virus (HCV) polymerase. Our results show that the LISE LBSP is able to identify all three target allosteric sites within the HCV polymerase as well as a known allosteric site in the Coxsackievirus polymerase. LISE was then employed to identify novel binding sites within the polymerases of the Dengue, West Nile, and Foot-and-mouth Disease viruses. Our results suggest that all three viral polymerases have putative sites that share structural or chemical similarities with allosteric pockets of the HCV polymerase. Thus, these binding locations may represent an evolutionarily conserved structural feature of several viral polymerases that could be exploited for the development of small molecule therapeutics. PMID:27262620

  13. Elucidation of direct competition and allosteric modulation of small-molecular-weight protein ligands using surface plasmon resonance methods.

    PubMed

    Huber, Walter; Sinopoli, Alessandro; Kohler, Josiane; Hug, Melanie; Ruf, Armin; Huber, Sylwia

    2015-08-01

    The present work introduces a surface plasmon resonance-based method for the discrimination of direct competition and allosteric effects that occur in ternary systems comprising a receptor protein and two small-molecular-weight ligands that bind to it. Fatty acid binding protein 4, fructose-1,6-bisphosphatase and human serum albumin were used as model receptor molecules to demonstrate the performance of the method. For each of the receptor molecules, pairs of ligand molecules were selected for which either direct competition or an allosteric effect had already been determined by other methods. The method of discrimination introduced here is based on the surface plasmon resonance responses observed at equilibrium when an immobilized receptor protein is brought into contact with binary mixtures of interacting ligands. These experimentally determined responses are compared with the responses calculated using a theoretical model that considers both direct competition and allosteric ligand interaction modes. This study demonstrates that the allosteric ternary complex model, which enables calculation of the fractional occupancy of the protein by each ligand in such ternary systems, is well suited for the theoretical calculation of these types of responses. For all of the ternary systems considered in this work, the experimental and calculated responses in the chosen concentration ratio range were identical within a five-σ confidence interval when the calculations considered the correct interaction mode of the ligands (direct competition or different types of allosteric regulation), and in case of allosteric modulation, also the correct strength of this effect. This study also demonstrates that the allosteric ternary complex model-based calculations are well suited to predict the ideal concentration ratio range or even single concentration ratios that can serve as hot spots for discrimination, and such hot spots can drastically reduce the numbers of measurements needed

  14. Clickable Photoaffinity Ligands for Metabotropic Glutamate Receptor 5 Based on Select Acetylenic Negative Allosteric Modulators.

    PubMed

    Gregory, Karen J; Velagaleti, Ranganadh; Thal, David M; Brady, Ryan M; Christopoulos, Arthur; Conn, P Jeffrey; Lapinsky, David J

    2016-07-15

    G protein-coupled receptors (GPCRs) represent the largest class of current drug targets. In particular, small-molecule allosteric modulators offer substantial potential for selectively "tuning" GPCR activity. However, there remains a critical need for experimental strategies that unambiguously determine direct allosteric ligand-GPCR interactions, to facilitate both chemical biology studies and rational structure-based drug design. We now report the development and use of first-in-class clickable allosteric photoprobes for a GPCR based on metabotropic glutamate receptor 5 (mGlu5) negative allosteric modulator (NAM) chemotypes. Select acetylenic mGlu5 NAM lead compounds were rationally modified to contain either a benzophenone or an aryl azide as a photoreactive functional group, enabling irreversible covalent attachment to mGlu5 via photoactivation. Additionally, a terminal alkyne or an aliphatic azide was incorporated as a click chemistry handle, allowing chemoselective attachment of fluorescent moieties to the irreversibly mGlu5-bound probe via tandem photoaffinity labeling-bioorthogonal conjugation. These clickable photoprobes retained submicromolar affinity for mGlu5 and negative cooperativity with glutamate, interacted with the "common allosteric-binding site," displayed slow binding kinetics, and could irreversibly label mGlu5 following UV exposure. We depleted the number of functional mGlu5 receptors using an irreversibly bound NAM to elucidate and delineate orthosteric agonist affinity and efficacy. Finally, successful conjugation of fluorescent dyes via click chemistry was demonstrated for each photoprobe. In the future, these clickable photoprobes are expected to aid our understanding of the structural basis of mGlu5 allosteric modulation. Furthermore, tandem photoaffinity labeling-bioorthogonal conjugation is expected to be a broadly applicable experimental strategy across the entire GPCR superfamily. PMID:27115427

  15. Reciprocal Allosteric Modulation of Carbon Monoxide and Warfarin Binding to Ferrous Human Serum Heme-Albumin

    PubMed Central

    Bocedi, Alessio; De Sanctis, Giampiero; Ciaccio, Chiara; Tundo, Grazia R.; Di Masi, Alessandra; Fanali, Gabriella; Nicoletti, Francesco P.; Fasano, Mauro; Smulevich, Giulietta; Ascenzi, Paolo; Coletta, Massimo

    2013-01-01

    Human serum albumin (HSA), the most abundant protein in human plasma, could be considered as a prototypic monomeric allosteric protein, since the ligand-dependent conformational adaptability of HSA spreads beyond the immediate proximity of the binding site(s). As a matter of fact, HSA is a major transport protein in the bloodstream and the regulation of the functional allosteric interrelationships between the different binding sites represents a fundamental information for the knowledge of its transport function. Here, kinetics and thermodynamics of the allosteric modulation: (i) of carbon monoxide (CO) binding to ferrous human serum heme-albumin (HSA-heme-Fe(II)) by warfarin (WF), and (ii) of WF binding to HSA-heme-Fe(II) by CO are reported. All data were obtained at pH 7.0 and 25°C. Kinetics of CO and WF binding to the FA1 and FA7 sites of HSA-heme-Fe(II), respectively, follows a multi-exponential behavior (with the same relative percentage for the two ligands). This can be accounted for by the existence of multiple conformations and/or heme-protein axial coordination forms of HSA-heme-Fe(II). The HSA-heme-Fe(II) populations have been characterized by resonance Raman spectroscopy, indicating the coexistence of different species characterized by four-, five- and six-coordination of the heme-Fe atom. As a whole, these results suggest that: (i) upon CO binding a conformational change of HSA-heme-Fe(II) takes place (likely reflecting the displacement of an endogenous ligand by CO), and (ii) CO and/or WF binding brings about a ligand-dependent variation of the HSA-heme-Fe(II) population distribution of the various coordinating species. The detailed thermodynamic and kinetic analysis here reported allows a quantitative description of the mutual allosteric effect of CO and WF binding to HSA-heme-Fe(II). PMID:23555601

  16. Identification of an allosteric binding site for RORγt inhibition

    PubMed Central

    Scheepstra, Marcel; Leysen, Seppe; van Almen, Geert C.; Miller, J. Richard; Piesvaux, Jennifer; Kutilek, Victoria; van Eenennaam, Hans; Zhang, Hongjun; Barr, Kenneth; Nagpal, Sunil; Soisson, Stephen M.; Kornienko, Maria; Wiley, Kristen; Elsen, Nathaniel; Sharma, Sujata; Correll, Craig C.; Trotter, B. Wesley; van der Stelt, Mario; Oubrie, Arthur; Ottmann, Christian; Parthasarathy, Gopal; Brunsveld, Luc

    2015-01-01

    RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors. PMID:26640126

  17. Atypical muscarinic allosteric modulation: cooperativity between modulators and their atypical binding topology in muscarinic M2 and M2/M5 chimeric receptors.

    PubMed

    Tränkle, Christian; Dittmann, Andreas; Schulz, Uwe; Weyand, Oliver; Buller, Stefan; Jöhren, Kirstin; Heller, Eberhard; Birdsall, Nigel J M; Holzgrabe, Ulrike; Ellis, John; Höltje, Hans Dieter; Mohr, Klaus

    2005-12-01

    The binding and function of muscarinic acetylcholine receptors can be modulated allosterically. Some allosteric muscarinic ligands are "atypical", having steep concentration-effect curves and not interacting competitively with "typical" allosteric modulators. For atypical agents, a second allosteric site has been proposed. Different approaches have been used to gain further insight into the interaction with M2 receptors of two atypical agents, tacrine and the bispyridinium compound 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bispyridinium dibromide (Duo3). Interaction studies, using radioligand binding assays and the allosteric ligands obidoxime, Mg2+, and the new tool hexamethonium to antagonize the allosteric actions of the atypical ligands, showed different modes of interaction for tacrine and Duo3 at M2 receptors. A negatively cooperative interaction was observed between hexamethonium and tacrine (but not Duo3). A tacrine dimer that exhibited increased allosteric potency relative to tacrine but behaved like a typical allosteric modulator was competitively inhibited by hexamethonium. M2/M5-receptor mutants revealed a dependence of tacrine and Duo3 affinity on different receptor epitopes. This was confirmed by docking simulations using a three-dimensional model of the M2 receptor. These showed that the allosteric site could accommodate two molecules of tacrine simultaneously but only one molecule of Duo3, which binds in different mode from typical allosteric agents. Therefore, the atypical actions of tacrine and Duo3 involve different modes of receptor interaction, but their sites of attachment seem to be the "common" allosteric binding domain at the entrance to the orthosteric ligand binding pocket of the M2-receptor. Additional complex behavior may be rationalized by allosteric interactions transmitted within a receptor dimer. PMID:16157694

  18. Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct, Highly Flexible Allosteric Bicarbonate Binding Pocket

    PubMed Central

    Saalau-Bethell, Susanne M; Berdini, Valerio; Cleasby, Anne; Congreve, Miles; Coyle, Joseph E; Lock, Victoria; Murray, Christopher W; O'Brien, M Alistair; Rich, Sharna J; Sambrook, Tracey; Vinkovic, Mladen; Yon, Jeff R; Jhoti, Harren

    2014-01-01

    Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β-methylene adenosine 5′-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein. PMID:24616449

  19. Bithionol Potently Inhibits Human Soluble Adenylyl Cyclase through Binding to the Allosteric Activator Site.

    PubMed

    Kleinboelting, Silke; Ramos-Espiritu, Lavoisier; Buck, Hannes; Colis, Laureen; van den Heuvel, Joop; Glickman, J Fraser; Levin, Lonny R; Buck, Jochen; Steegborn, Clemens

    2016-04-29

    The signaling molecule cAMP regulates functions ranging from bacterial transcription to mammalian memory. In mammals, cAMP is synthesized by nine transmembrane adenylyl cyclases (ACs) and one soluble AC (sAC). Despite similarities in their catalytic domains, these ACs differ in regulation. Transmembrane ACs respond to G proteins, whereas sAC is uniquely activated by bicarbonate. Via bicarbonate regulation, sAC acts as a physiological sensor for pH/bicarbonate/CO2, and it has been implicated as a therapeutic target, e.g. for diabetes, glaucoma, and a male contraceptive. Here we identify the bisphenols bithionol and hexachlorophene as potent, sAC-specific inhibitors. Inhibition appears mostly non-competitive with the substrate ATP, indicating that they act via an allosteric site. To analyze the interaction details, we solved a crystal structure of an sAC·bithionol complex. The structure reveals that the compounds are selective for sAC because they bind to the sAC-specific, allosteric binding site for the physiological activator bicarbonate. Structural comparison of the bithionol complex with apo-sAC and other sAC·ligand complexes along with mutagenesis experiments reveals an allosteric mechanism of inhibition; the compound induces rearrangements of substrate binding residues and of Arg(176), a trigger between the active site and allosteric site. Our results thus provide 1) novel insights into the communication between allosteric regulatory and active sites, 2) a novel mechanism for sAC inhibition, and 3) pharmacological compounds targeting this allosteric site and utilizing this mode of inhibition. These studies provide support for the future development of sAC-modulating drugs. PMID:26961873

  20. Engineering an allosteric transcription factor to respond to new ligands

    PubMed Central

    Taylor, Noah D; Garruss, Alexander S; Moretti, Rocco; Chan, Sum; Arbing, Mark A; Cascio, Duilio; Rogers, Jameson K; Isaacs, Farren J; Kosuri, Sriram; Baker, David; Fields, Stanley; Church, George M; Raman, Srivatsan

    2016-01-01

    Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol or sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits. PMID:26689263

  1. Engineering an allosteric transcription factor to respond to new ligands.

    PubMed

    Taylor, Noah D; Garruss, Alexander S; Moretti, Rocco; Chan, Sum; Arbing, Mark A; Cascio, Duilio; Rogers, Jameson K; Isaacs, Farren J; Kosuri, Sriram; Baker, David; Fields, Stanley; Church, George M; Raman, Srivatsan

    2016-02-01

    Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits. PMID:26689263

  2. Allosteric role of the large-scale domain opening in biological catch-binding

    NASA Astrophysics Data System (ADS)

    Pereverzev, Yuriy V.; Prezhdo, Oleg V.; Sokurenko, Evgeni V.

    2009-05-01

    The proposed model demonstrates the allosteric role of the two-domain region of the receptor protein in the increased lifetimes of biological receptor/ligand bonds subjected to an external force. The interaction between the domains is represented by a bounded potential, containing two minima corresponding to the attached and separated conformations of the two protein domains. The dissociative potential with a single minimum describing receptor/ligand binding fluctuates between deep and shallow states, depending on whether the domains are attached or separated. A number of valuable analytic expressions are derived and are used to interpret experimental data for two catch bonds. The P-selectin/P-selectin-glycoprotein-ligand-1 (PSGL-1) bond is controlled by the interface between the epidermal growth factor (EGF) and lectin domains of P-selectin, and the type 1 fimbrial adhesive protein (FimH)/mannose bond is governed by the interface between the lectin and pilin domains of FimH. Catch-binding occurs in these systems when the external force stretches the receptor proteins and increases the interdomain distance. The allosteric effect is supported by independent measurements, in which the domains are kept separated by attachment of another ligand. The proposed model accurately describes the experimentally observed anomalous behavior of the lifetimes of the P-selectin/PSGL-1 and FimH/mannose complexes as a function of applied force and provides valuable insights into the mechanism of catch-binding.

  3. FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase

    PubMed Central

    Tiraidis, Costas; Alexacou, Kyra-Melinda; Zographos, Spyros E.; Leonidas, Demetres D.; Gimisis, Thanasis; Oikonomakos, Nikos G.

    2007-01-01

    FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b–FR258900 complex and refined it to 2.2 Å resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45′ (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy-phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site. PMID:17600143

  4. FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase.

    PubMed

    Tiraidis, Costas; Alexacou, Kyra-Melinda; Zographos, Spyros E; Leonidas, Demetres D; Gimisis, Thanasis; Oikonomakos, Nikos G

    2007-08-01

    FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b-FR258900 complex and refined it to 2.2 A resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45' (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy-phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site. PMID:17600143

  5. Role of Heme Pocket Water in Allosteric Regulation of Ligand Reactivity in Human Hemoglobin.

    PubMed

    Esquerra, Raymond M; Bibi, Bushra M; Tipgunlakant, Pooncharas; Birukou, Ivan; Soman, Jayashree; Olson, John S; Kliger, David S; Goldbeck, Robert A

    2016-07-26

    Water molecules can enter the heme pockets of unliganded myoglobins and hemoglobins, hydrogen bond with the distal histidine, and introduce steric barriers to ligand binding. The spectrokinetics of photodissociated CO complexes of human hemoglobin and its isolated α and β chains were analyzed for the effect of heme hydration on ligand rebinding. A strong coupling was observed between heme hydration and quaternary state. This coupling may contribute significantly to the 20-60-fold difference between the R- and T-state bimolecular CO binding rate constants and thus to the modulation of ligand reactivity that is the hallmark of hemoglobin allostery. Heme hydration proceeded over the course of several kinetic phases in the tetramer, including the R to T quaternary transition. An initial 150 ns hydration phase increased the R-state distal pocket water occupancy, nw(R), to a level similar to that of the isolated α (∼60%) and β (∼10%) chains, resulting in a modest barrier to ligand binding. A subsequent phase, concurrent with the first step of the R → T transition, further increased the level of heme hydration, increasing the barrier. The final phase, concurrent with the final step of the allosteric transition, brought the water occupancy of the T-state tetramer, nw(T), even higher and close to full occupancy in both the α and β subunits (∼90%). This hydration level could present an even larger barrier to ligand binding and contribute significantly to the lower iron reactivity of the T state toward CO. PMID:27355904

  6. Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1α*

    PubMed Central

    Jiang, Jason Y.; Nagaraju, Mulpuri; Meyer, Rebecca C.; Zhang, Li; Hamelberg, Donald; Hall, Randy A.; Brown, Edward M.; Conn, P. Jeffrey; Yang, Jenny J.

    2014-01-01

    Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca2+. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca2+-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca2+-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca2+ enhanced mGluR1α-mediated intracellular Ca2+ responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca2+ diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca2+, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca2+ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca2+. These studies reveal that binding of extracellular Ca2+ to the predicted Ca2+-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α. PMID:24280223

  7. Light-activated DNA binding in a designed allosteric protein

    SciTech Connect

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R.

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  8. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    PubMed Central

    Yanamala, Naveena; Tirupula, Kalyan C; Klein-Seetharaman, Judith

    2008-01-01

    Metabotropic glutamate receptors (mGluRs) are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%), the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%), the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86%) and 8/9 (89%) for ArgusLab and 10/14 (71%) and 7/9 (78%) for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by photochemical isomerization

  9. The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein*

    PubMed Central

    Townsend, Philip D.; Rodgers, Thomas L.; Glover, Laura C.; Korhonen, Heidi J.; Richards, Shane A.; Colwell, Lucy J.; Pohl, Ehmke; Wilson, Mark R.; Hodgson, David R. W.; McLeish, Tom C. B.; Cann, Martin J.

    2015-01-01

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. PMID:26187469

  10. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    NASA Astrophysics Data System (ADS)

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-04-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.

  11. Molecular Dynamics Simulations Reveal the Mechanisms of Allosteric Activation of Hsp90 by Designed Ligands

    PubMed Central

    Vettoretti, Gerolamo; Moroni, Elisabetta; Sattin, Sara; Tao, Jiahui; Agard, David A.; Bernardi, Anna; Colombo, Giorgio

    2016-01-01

    Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators. PMID:27032695

  12. Al(+)-ligand binding energies

    NASA Technical Reports Server (NTRS)

    Sodupe, M.; Bauschlicher, Charles W., Jr.

    1991-01-01

    Ab initio calculations are used to optimize the structure and determine the binding energies of Al(+) to a series of ligands. For Al(+)-CN, the bonding was found to have a large covalent component. For the remaining ligands, the bonding is shown to be electrostatic in origin. The results obtained for Al(+) are compared with those previously reported for Mg(+).

  13. Development of M1 mAChR Allosteric and Bitopic Ligands: Prospective Therapeutics for the Treatment of Cognitive Deficits

    PubMed Central

    2013-01-01

    Since the cholinergic hypothesis of memory dysfunction was first reported, extensive research efforts have focused on elucidating the mechanisms by which this intricate system contributes to the regulation of processes such as learning, memory, and higher executive function. Several cholinergic therapeutic targets for the treatment of cognitive deficits, psychotic symptoms, and the underlying pathophysiology of neurodegenerative disorders, such as Alzheimer’s disease and schizophrenia, have since emerged. Clinically approved drugs now exist for some of these targets; however, they all may be considered suboptimal therapeutics in that they produce undesirable off-target activity leading to side effects, fail to address the wide variety of symptoms and underlying pathophysiology that characterize these disorders, and/or afford little to no therapeutic effect in subsets of patient populations. A promising target for which there are presently no approved therapies is the M1 muscarinic acetylcholine receptor (M1 mAChR). Despite avid investigation, development of agents that selectively activate this receptor via the orthosteric site has been hampered by the high sequence homology of the binding site between the five muscarinic receptor subtypes and the wide distribution of this receptor family in both the central nervous system (CNS) and the periphery. Hence, a plethora of ligands targeting less structurally conserved allosteric sites of the M1 mAChR have been investigated. This Review aims to explain the rationale behind allosterically targeting the M1 mAChR, comprehensively summarize and critically evaluate the M1 mAChR allosteric ligand literature to date, highlight the challenges inherent in allosteric ligand investigation that are impeding their clinical advancement, and discuss potential methods for resolving these issues. PMID:23659787

  14. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding

    PubMed Central

    Ting, Pamela Y.; Johnson, Christian W.; Fang, Cong; Cao, Xiaoqing; Graeber, Thomas G.; Mattos, Carla; Colicelli, John

    2015-01-01

    RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr137. Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr137 phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr137 is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRASY137F and HRASY137E revealed conformation changes radiating from the mutated residue. Although consistent with Tyr137 participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr137 phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRASG12V with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr137 allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.—Ting, P. Y., Johnson, C. W., Fang, C., Cao, X., Graeber, T. G., Mattos, C., Colicelli, J. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding. PMID:25999467

  15. Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

    PubMed Central

    Spurny, Radovan; Debaveye, Sarah; Farinha, Ana; Veys, Ken; Vos, Ann M.; Gossas, Thomas; Atack, John; Bertrand, Sonia; Bertrand, Daniel; Danielson, U. Helena; Tresadern, Gary; Ulens, Chris

    2015-01-01

    The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential. PMID:25918415

  16. Allosteric interaction of trimebutine maleate with dihydropyridine binding sites.

    PubMed

    Nagasaki, M; Kurosawa, H; Naito, K; Tamaki, H

    1990-07-31

    The effects of trimebutine maleate on [3H]nitrendipine binding to guinea-pig ileal smooth muscle membranes and Ca2(+)-induced contraction of the taenia cecum were studied. Specific binding of [3H]nitrendipine to smooth muscle membranes was saturable, with a KD value and maximum number of binding sites (Bmax) of 0.16 nM and 1070 fmol/mg protein, respectively. Trimebutine inhibited [3H]nitrendipine binding in a concentration-dependent manner with a Ki value of 9.3 microM. In the presence of trimebutine (10 microM), Scatchard analysis indicated a competitive-like inhibition with a decrease in the binding affinity (0.31 nM) without a change in Bmax (1059 fmol/mg protein). However, a dissociation experiment using trimebutine (10 or 100 microM) showed that the decreased affinity was due to an increase of the dissociation rate constant of [3H]nitrendipine binding to the membrane. In mechanical experiments using the taenia cecum, trimebutine (3-30 microM) caused a parallel rightward shift of the dose-response curve for the contractile response to a higher concentration range of Ca2+ under high-K+ conditions in a noncompetitive manner. These results suggest that trimebutine has negative allosteric interactions with 1,4-dihydropyridine binding sites on voltage-dependent Ca2+ channels and antagonizes Ca2+ influx, consequently inhibiting contractions of intestinal smooth muscle. PMID:2171963

  17. Bio-layer interferometry for measuring kinetics of protein-protein interactions and allosteric ligand effects.

    PubMed

    Shah, Naman B; Duncan, Thomas M

    2014-01-01

    We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit ε with the catalytic complex of Escherichia coli ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit ε could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of ε undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of ε's conformations is predominant. The assay measures kinetics of ε's binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound ε to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on ε's conformational changes. PMID:24638157

  18. Carbohydrate affinity for the glucose-galactose binding protein is regulated by allosteric domain motions.

    PubMed

    Ortega, Gabriel; Castaño, David; Diercks, Tammo; Millet, Oscar

    2012-12-01

    Protein function, structure, and dynamics are intricately correlated, but studies on structure-activity relationships are still only rarely complemented by a detailed analysis of dynamics related to function (functional dynamics). Here, we have applied NMR to investigate the functional dynamics in two homologous periplasmic sugar binding proteins with bidomain composition: Escherichia coli glucose/galactose (GGBP) and ribose (RBP) binding proteins. In contrast to their structural and functional similarity, we observe a remarkable difference in functional dynamics: For RBP, the absence of segmental motions allows only for isolated structural adaptations upon carbohydrate binding in line with an induced fit mechanism; on the other hand, GGBP shows extensive segmental mobility in both apo and holo states, enabling selection of the most favorable conformation upon carbohydrate binding in line with a population shift mechanism. Collective segmental motions are controlled by the hinge composition: by swapping two identified key residues between RBP and GGBP we also interchange their segmental hinge mobility, and the doubly mutated GGBP* no longer experiences changes in conformational entropy upon ligand binding while the complementary RBP* shows the segmental dynamics observed in wild-type GGBP. Most importantly, the segmental interdomain dynamics always increase the apparent substrate affinity and thus, are functional, underscoring the allosteric control that the hinge region exerts on ligand binding. PMID:23148479

  19. The Allosteric Binding Sites of Sulfotransferase 1A1

    PubMed Central

    Cook, Ian; Wang, Ting; Falany, Charles N.

    2015-01-01

    Human sulfotransferases (SULTs) comprise a small, 13-member enzyme family that regulates the activities of thousands of compounds—endogenous metabolites, drugs, and other xenobiotics. SULTs transfer the sulfuryl-moiety (–SO3) from a nucleotide donor, PAPS (3′-phosphoadenosine 5′-phosphosulfate), to the hydroxyls and primary amines of acceptors. SULT1A1, a progenitor of the family, has evolved to sulfonate compounds that are remarkably structurally diverse. SULT1A1, which is found in many tissues, is the predominant SULT in liver, where it is a major component of phase II metabolism. Early work demonstrated that catechins and nonsteroidal anti-inflammatory drugs inhibit SULT1A1 and suggested that the inhibition was not competitive versus substrates. Here, the mechanism of inhibition of a single, high affinity representative from each class [epigallocatechin gallate (EGCG) and mefenamic acid] is determined using initial-rate and equilibrium-binding studies. The findings reveal that the inhibitors bind at sites separate from those of substrates, and at saturation turnover of the enzyme is reduced to a nonzero value. Further, the EGCG inhibition patterns suggest a molecular explanation for its isozyme specificity. Remarkably, the inhibitors bind at sites that are separate from one another, and binding at one site does not affect affinity at the other. For the first time, it is clear that SULT1A1 is allosterically regulated, and that it contains at least two, functionally distinct allosteric sites, each of which responds to a different class of compounds. PMID:25534770

  20. Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

    PubMed

    DeVree, Brian T; Mahoney, Jacob P; Vélez-Ruiz, Gisselle A; Rasmussen, Soren G F; Kuszak, Adam J; Edwald, Elin; Fung, Juan-Jose; Manglik, Aashish; Masureel, Matthieu; Du, Yang; Matt, Rachel A; Pardon, Els; Steyaert, Jan; Kobilka, Brian K; Sunahara, Roger K

    2016-07-01

    G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR–G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity. PMID:27362234

  1. 5'-Substituted Amiloride Derivatives as Allosteric Modulators Binding in the Sodium Ion Pocket of the Adenosine A2A Receptor.

    PubMed

    Massink, Arnault; Louvel, Julien; Adlere, Ilze; van Veen, Corine; Huisman, Berend J H; Dijksteel, Gabrielle S; Guo, Dong; Lenselink, Eelke B; Buckley, Benjamin J; Matthews, Hayden; Ranson, Marie; Kelso, Michael; IJzerman, Adriaan P

    2016-05-26

    The sodium ion site is an allosteric site conserved among many G protein-coupled receptors (GPCRs). Amiloride 1 and 5-(N,N-hexamethylene)amiloride 2 (HMA) supposedly bind in this sodium ion site and can influence orthosteric ligand binding. The availability of a high-resolution X-ray crystal structure of the human adenosine A2A receptor (hA2AAR), in which the allosteric sodium ion site was elucidated, makes it an appropriate model receptor for investigating the allosteric site. In this study, we report the synthesis and evaluation of novel 5'-substituted amiloride derivatives as hA2AAR allosteric antagonists. The potency of the amiloride derivatives was assessed by their ability to displace orthosteric radioligand [(3)H]4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol ([(3)H]ZM-241,385) from both the wild-type and sodium ion site W246A mutant hA2AAR. 4-Ethoxyphenethyl-substituted amiloride 12l was found to be more potent than both amiloride and HMA, and the shift in potency between the wild-type and mutated receptor confirmed its likely binding to the sodium ion site. PMID:27124340

  2. The kinetics of effector binding to phosphofructokinase. The influence of effectors on the allosteric conformational transition.

    PubMed Central

    Roberts, D; Kellett, G L

    1980-01-01

    1. The extent of the allosteric transition from the R into the T conformation of rabbit skeletal muscle phosphofructokinase induced by Mg2+-1,N6-etheno-ATP was determined by stopped-flow fluorimetry from the amplitude of the slow phase of the Mg2+-1,N6-etheno-ATP fluorescence enhancement [Roberts & Kellet (1979) Biochem. J. 183, 349--360]. 2. The amplitude of the slow phase was decreased by low concentrations of the activators cyclic AMP and fructose 1,6-bisphosphate, but increased in a complex manner by the inhibitor citrate. 3. Mg2+-1,N6-etheno-ATP and Mg2+-ATP are unable to induce the T conformation to a detectable extent in the presence of saturating cyclic AMP, but can do so readily in the presence of saturating fructose 1,6-bisphosphate. 4. The conformational transitions induced in enzyme alone by different ligands were observed by changes in intrinsic protein fluorescence. In general, an R-type conformation has diminished protein fluorescence compared with a T-type conformation. 5. Mg2+-ATP exerts a complex effect on protein fluorescence; both the enhancement at low concentrations and the quenching at high concentrations of Mg2+-ATP result from the binding of Mg2+-ATP to the inhibitory site and the ensuing allosteric transition. Enhancement reflects the extent of the allosteric transition and involves both tyrosine and tryptophan, probably in the region of the active site; quenching reflects occupation of the inhibitory site and involves tyrosine at the inhibitory site. 6. The mechanism of the allosteric transition from the R into the T conformation induced by Mg2+-1,N6-etheno-ATP at low concentrations occurs predominantly by a 'prior-isomerization' pathway; at higher concentrations a limited contribution from a 'substrate-guided' pathway occurs. 7. The allosteric behaviour of phosphofructokinase with respect to Mg2+-ATP and Mg2+-1,N6-ethenol-ATP binding may be accounted for in terms of the simple, concerted model. Images Fig. 1. Fig. 5. Fig. 6. PMID:6260084

  3. Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

    PubMed

    Huang, Xi-Ping; Karpiak, Joel; Kroeze, Wesley K; Zhu, Hu; Chen, Xin; Moy, Sheryl S; Saddoris, Kara A; Nikolova, Viktoriya D; Farrell, Martilias S; Wang, Sheng; Mangano, Thomas J; Deshpande, Deepak A; Jiang, Alice; Penn, Raymond B; Jin, Jian; Koller, Beverly H; Kenakin, Terry; Shoichet, Brian K; Roth, Bryan L

    2015-11-26

    At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs. PMID:26550826

  4. Identification and Pharmacological Characterization of Multiple Allosteric Binding Sites on the Free Fatty Acid 1 Receptor

    PubMed Central

    Lin, Daniel C.-H.; Guo, Qi; Luo, Jian; Zhang, Jane; Nguyen, Kathy; Chen, Michael; Tran, Thanh; Dransfield, Paul J.; Brown, Sean P.; Houze, Jonathan; Vimolratana, Marc; Jiao, Xian Yun; Wang, Yingcai; Birdsall, Nigel J. M.

    2012-01-01

    Activation of FFA1 (GPR40), a member of G protein-coupling receptor family A, is mediated by medium- and long-chain fatty acids and leads to amplification of glucose-stimulated insulin secretion, suggesting a potential role for free fatty acid 1 (FFA1) as a target for type 2 diabetes. It was assumed previously that there is a single binding site for fatty acids and synthetic FFA1 agonists. However, using members of two chemical series of partial and full agonists that have been identified, radioligand binding interaction studies revealed that the full agonists do not bind to the same site as the partial agonists but exhibit positive heterotropic cooperativity. Analysis of functional data reveals positive functional cooperativity between the full agonists and partial agonists in various functional assays (in vitro and ex vivo) and also in vivo. Furthermore, the endogenous fatty acid docosahexaenoic acid (DHA) shows negative or neutral cooperativity with members of both series of agonists in binding assays but displays positive cooperativity in functional assays. Another synthetic agonist is allosteric with members of both agonist series, but apparently competitive with DHA. Therefore, there appear to be three allosterically linked binding sites on FFA1 with agonists specific for each of these sites. Activation of free fatty acid 1 receptor (FFAR1) by each of these agonists is differentially affected by mutations of two arginine residues, previously found to be important for FFAR1 binding and activation. These ligands with their high potencies and strong positive functional cooperativity with endogenous fatty acids, demonstrated in vitro and in vivo, have the potential to deliver therapeutic benefits. PMID:22859723

  5. Identification and pharmacological characterization of multiple allosteric binding sites on the free fatty acid 1 receptor.

    PubMed

    Lin, Daniel C-H; Guo, Qi; Luo, Jian; Zhang, Jane; Nguyen, Kathy; Chen, Michael; Tran, Thanh; Dransfield, Paul J; Brown, Sean P; Houze, Jonathan; Vimolratana, Marc; Jiao, Xian Yun; Wang, Yingcai; Birdsall, Nigel J M; Swaminath, Gayathri

    2012-11-01

    Activation of FFA1 (GPR40), a member of G protein-coupling receptor family A, is mediated by medium- and long-chain fatty acids and leads to amplification of glucose-stimulated insulin secretion, suggesting a potential role for free fatty acid 1 (FFA1) as a target for type 2 diabetes. It was assumed previously that there is a single binding site for fatty acids and synthetic FFA1 agonists. However, using members of two chemical series of partial and full agonists that have been identified, radioligand binding interaction studies revealed that the full agonists do not bind to the same site as the partial agonists but exhibit positive heterotropic cooperativity. Analysis of functional data reveals positive functional cooperativity between the full agonists and partial agonists in various functional assays (in vitro and ex vivo) and also in vivo. Furthermore, the endogenous fatty acid docosahexaenoic acid (DHA) shows negative or neutral cooperativity with members of both series of agonists in binding assays but displays positive cooperativity in functional assays. Another synthetic agonist is allosteric with members of both agonist series, but apparently competitive with DHA. Therefore, there appear to be three allosterically linked binding sites on FFA1 with agonists specific for each of these sites. Activation of free fatty acid 1 receptor (FFAR1) by each of these agonists is differentially affected by mutations of two arginine residues, previously found to be important for FFAR1 binding and activation. These ligands with their high potencies and strong positive functional cooperativity with endogenous fatty acids, demonstrated in vitro and in vivo, have the potential to deliver therapeutic benefits. PMID:22859723

  6. Altering residues N125 and D149 impacts sugar effector binding and allosteric parameters in Escherichia coli lactose repressor.

    PubMed

    Xu, Jia; Liu, Shirley; Chen, Mingzhi; Ma, Jianpeng; Matthews, Kathleen S

    2011-10-25

    Lactose repressor protein (LacI), a negative transcriptional regulator in Escherichia coli, relies on an allosteric conformational change for its function. The LacI effector isopropyl-β,D-thiogalactoside (IPTG) promotes this allosteric response and engages the side chains of residues N125 and D149 based on the crystallographic structure of LacI·IPTG. Targeted molecular dynamics (TMD) simulations have indicated involvement of these side chains during the protein structural changes in response to inducer binding. To examine this region further, we applied stochastic boundary molecular dynamics (SBMD) simulation and identified a transient interaction between residues N125 and D149. On the basis of these data, we introduced substitutions for either/both residues and analyzed their impact on protein function. The substitutions utilized were alanine to preclude hydrogen bonding or cysteine to allow disulfide bond formation, which was not observed for N125C/D149C. Minimal impacts were observed on operator affinity for all substitutions, but D149C, N125A/D149A, and N125C/D149C bound to IPTG with 5-8-fold lower affinity than wild-type LacI, and exhibited decreased allosteric amplitude (K(RI/O)/K(R/O)). Of interest, the double mutants did not exhibit an allosteric response to an alternate inducer, 2-phenylethyl-β,D-galactoside (PhEG), despite demonstration of PhEG binding. Further, the presence of the anti-inducer, o-nitrophenyl-β,D-fucoside (ONPF), enhanced operator affinity for wild-type LacI and all other mutant proteins examined, but behaved as an inducer for N125A/D149A, decreasing operator binding affinity. These results confirm the role of residues 125 and 149 in ligand binding and allosteric response and illustrate how readily the function of a regulatory protein can be altered. PMID:21928765

  7. Genetically encoded photo-cross-linkers map the binding site of an allosteric drug on a G protein-coupled receptor.

    PubMed

    Grunbeck, Amy; Huber, Thomas; Abrol, Ravinder; Trzaskowski, Bartosz; Goddard, William A; Sakmar, Thomas P

    2012-06-15

    G protein-coupled receptors (GPCRs) are dynamic membrane proteins that bind extracellular molecules to transduce signals. Although GPCRs represent the largest class of therapeutic targets, only a small percentage of their ligand-binding sites are precisely defined. Here we describe the novel application of targeted photo-cross-linking using unnatural amino acids to obtain structural information about the allosteric binding site of a small molecule drug, the CCR5-targeted HIV-1 co-receptor blocker maraviroc. PMID:22455376

  8. Mg(+)-ligand binding energies

    NASA Technical Reports Server (NTRS)

    Bauschlicher, Charles W., Jr.; Partridge, Harry

    1991-01-01

    Ab initio calculations are used to optimize the structures and determine the binding energies of Mg(+) to a series of ligands. Mg(+) bonds electrostatically with benzene, acetone, H2, CO, and NH3 and a self-consistent-field treatment gives a good description of the bonding. The bonding in MgCN(+) and MgCH3(+) is largely covalent and a correlated treatment is required.

  9. Docking of noncompetitive inhibitors into dengue virus type 2 protease: understanding the interactions with allosteric binding sites.

    PubMed

    Othman, Rozana; Kiat, Tan Siew; Khalid, Norzulaani; Yusof, Rohana; Newhouse, E Irene; Newhouse, James S; Alam, Masqudul; Rahman, Noorsaadah Abdul

    2008-08-01

    A group of flavanones and their chalcones, isolated from Boesenbergia rotunda L., were previously reported to show varying degrees of noncompetitive inhibitory activities toward Dengue virus type 2 (Den2) protease. Results obtained from automated docking studies are in agreement with experimental data in which the ligands were shown to bind to sites other than the active site of the protease. The calculated K(i) values are very small, indicating that the ligands bind quite well to the allosteric binding site. Greater inhibition by pinostrobin, compared to the other compounds, can be explained by H-bonding interaction with the backbone carbonyl of Lys74, which is bonded to Asp75 (one of the catalytic triad residues). In addition, structure-activity relationship analysis yields structural information that may be useful for designing more effective therapeutic drugs against dengue virus infections. PMID:18656912

  10. Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors.

    PubMed

    Jakubík, J; Bacáková, L; El-Fakahany, E E; Tucek, S

    1997-07-01

    It is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M1-M4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [3H]N-methylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (-)-eburnamonine on the M2 and M4 receptors and by brucine on the M1 and M3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M1, M3, and M4 receptors, for pentylthio-TZTP on the M2 receptors, and for arecoline on the M3 receptors. (-)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M2 receptors and for pilocarpine on the M4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (-)-eburnamonine and pilocarpine and (-)-eburnamonine and oxotremorine-M on the M2 receptors (25- and 7-fold increases in

  11. Are all regions of folded proteins that undergo ligand-dependent order-disorder transitions targets for allosteric peptide mimetics?†

    PubMed Central

    Fenton, Aron W.

    2013-01-01

    Although the classical view of how proteins function relied on well folded structures, it is now recognized that the function of many proteins is dependent on being intrinsically disordered. The primary consideration in this work is the intermediate group of proteins that are overall well folded, but which contain small regions that undergo order/disorder transitions. In particular, the current focus is on those order/disorder transitions that are energetically coupled to ligand binding. As exemplified by the case of human liver pyruvate kinase (hL-PYK), peptides that mimic the sequence of the order/disorder region can be used as allosteric regulators of the enzyme. Based on this example and others reported in the literature, we propose that a similar use of peptides that mimic protein regions that experience ligand-dependent order-disorder transitions can be a generalized initiation point for the development of allosteric drugs. PMID:23520021

  12. Ligand Binding and Structural Changes Associated with Allostery in Yeast NAD+-specific Isocitrate Dehydrogenase

    PubMed Central

    Lee, McAlister-Henn

    2011-01-01

    Yeast NAD+-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four each of regulatory IDH1 and catalytic IDH2 subunits that share 42% sequence identity. IDH2 contains catalytic isocitrate/Mg2+ and NAD+ binding sites whereas IDH1 contains homologous binding sites, respectively, for cooperative binding of isocitrate and for allosteric binding of AMP. Ligand binding is highly ordered in vitro, and IDH exhibits the unusual property of half-site binding for all ligands. The structures of IDH solved in the absence or presence of ligands have shown: (a) a heterodimer to be the basic structural/functional unit of the enzyme, (b) the organization of heterodimers to form tetramer and octamer structures, (c) structural differences that may underlie cooperative and allosteric regulatory mechanisms, and (d) the possibility for formation of a disulfide bond that could reduce catalytic activity. In vivo analyses of mutant enzymes have elucidated the physiological importance of catalytic activity and allosteric regulation of this tricarboxylic acid cycle enzyme. Other studies have established the importance of a disulfide bond in regulation of IDH activity in vivo, as well as contributions of this bond to the property of half-site ligand binding exhibited by the wild-type enzyme. PMID:22008468

  13. An atomistic view of Hsp70 allosteric crosstalk: from the nucleotide to the substrate binding domain and back

    PubMed Central

    Chiappori, Federica; Merelli, Ivan; Milanesi, Luciano; Colombo, Giorgio; Morra, Giulia

    2016-01-01

    The Hsp70 is an allosterically regulated family of molecular chaperones. They consist of two structural domains, NBD and SBD, connected by a flexible linker. ATP hydrolysis at the NBD modulates substrate recognition at the SBD, while peptide binding at the SBD enhances ATP hydrolysis. In this study we apply Molecular Dynamics (MD) to elucidate the molecular determinants underlying the allosteric communication from the NBD to the SBD and back. We observe that local structural and dynamical modulation can be coupled to large-scale rearrangements, and that different combinations of ligands at NBD and SBD differently affect the SBD domain mobility. Substituting ADP with ATP in the NBD induces specific structural changes involving the linker and the two NBD lobes. Also, a SBD-bound peptide drives the linker docking by increasing the local dynamical coordination of its C-terminal end: a partially docked DnaK structure is achieved by combining ATP in the NBD and peptide in the SBD. We propose that the MD-based analysis of the inter domain dynamics and structure modulation could be used as a tool to computationally predict the allosteric behaviour and functional response of Hsp70 upon introducing mutations or binding small molecules, with potential applications for drug discovery. PMID:27025773

  14. Nanopharmacological Force Sensing to Reveal Allosteric Coupling in Transporter Binding Sites.

    PubMed

    Zhu, Rong; Sinwel, Doris; Hasenhuetl, Peter S; Saha, Kusumika; Kumar, Vivek; Zhang, Peng; Rankl, Christian; Holy, Marion; Sucic, Sonja; Kudlacek, Oliver; Karner, Andreas; Sandtner, Walter; Stockner, Thomas; Gruber, Hermann J; Freissmuth, Michael; Hauck Newman, Amy; Sitte, Harald H; Hinterdorfer, Peter

    2016-01-26

    Controversy regarding the number and function of ligand binding sites in neurotransmitter/sodium symporters arose from conflicting data in crystal structures and molecular pharmacology. Here, we have designed novel tools for atomic force microscopy that directly measure the interaction forces between the serotonin transporter (SERT) and the S- and R-enantiomers of citalopram on the single molecule level. This approach is based on force spectroscopy, which allows for the extraction of dynamic information under physiological conditions thus inaccessible via X-ray crystallography. Two distinct populations of characteristic binding strengths of citalopram to SERT were revealed in Na(+)-containing buffer. In contrast, in Li(+) -containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT when accessed in a physiological context. Competition experiments revealed that these two sites are allosterically coupled and exert reciprocal modulation. PMID:26695726

  15. Structural and Functional Analysis of the Allosteric Inhibition of IRE1α with ATP-Competitive Ligands.

    PubMed

    Feldman, Hannah C; Tong, Michael; Wang, Likun; Meza-Acevedo, Rosa; Gobillot, Theodore A; Lebedev, Ivan; Gliedt, Micah J; Hari, Sanjay B; Mitra, Arinjay K; Backes, Bradley J; Papa, Feroz R; Seeliger, Markus A; Maly, Dustin J

    2016-08-19

    The accumulation of unfolded proteins under endoplasmic reticulum (ER) stress leads to the activation of the multidomain protein sensor IRE1α as part of the unfolded protein response (UPR). Clustering of IRE1α lumenal domains in the presence of unfolded proteins promotes kinase trans-autophosphorylation in the cytosol and subsequent RNase domain activation. Interestingly, there is an allosteric relationship between the kinase and RNase domains of IRE1α, which allows ATP-competitive inhibitors to modulate the activity of the RNase domain. Here, we use kinase inhibitors to study how ATP-binding site conformation affects the activity of the RNase domain of IRE1α. We find that diverse ATP-competitive inhibitors of IRE1α promote dimerization and activation of RNase activity despite blocking kinase autophosphorylation. In contrast, a subset of ATP-competitive ligands, which we call KIRAs, allosterically inactivate the RNase domain through the kinase domain by stabilizing monomeric IRE1α. Further insight into how ATP-competitive inhibitors are able to divergently modulate the RNase domain through the kinase domain was gained by obtaining the first structure of apo human IRE1α in the RNase active back-to-back dimer conformation. Comparison of this structure with other existing structures of IRE1α and integration of our extensive structure activity relationship (SAR) data has led us to formulate a model to rationalize how ATP-binding site ligands are able to control the IRE1α oligomeric state and subsequent RNase domain activity. PMID:27227314

  16. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    PubMed Central

    Jakubík, Jan; El-Fakahany, Esam E.

    2010-01-01

    An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity) or decrease (negative cooperativity) in the binding or action of an orthosteric agonist (e.g., acetylcholine). Since the identification of gallamine as the first allosteric modulator of muscarinic receptors in 1976, this unique mode of receptor modulation has been intensively studied by many groups. This review summarizes over 30 years of research on the molecular mechanisms of allosteric interactions of drugs with the receptor and for new allosteric modulators of muscarinic receptors with potential therapeutic use. Identification of positive modulators of acetylcholine binding and function that enhance neurotransmission and the discovery of highly selective allosteric modulators are mile-stones on the way to novel therapeutic agents for the treatment of schizophrenia, Alzheimer’s disease and other disorders involving impaired cognitive function.

  17. Towards the identification of the allosteric Phe-binding site in phenylalanine hydroxylase.

    PubMed

    Carluccio, Carla; Fraternali, Franca; Salvatore, Francesco; Fornili, Arianna; Zagari, Adriana

    2016-03-01

    The enzyme phenylalanine hydroxylase (PAH) is defective in the inherited disorder phenylketonuria. PAH, a tetrameric enzyme, is highly regulated and displays positive cooperativity for its substrate, Phe. Whether Phe binds to an allosteric site is a matter of debate, despite several studies worldwide. To address this issue, we generated a dimeric model for Phe-PAH interactions, by performing molecular docking combined with molecular dynamics simulations on human and rat wild-type sequences and also on a human G46S mutant. Our results suggest that the allosteric Phe-binding site lies at the dimeric interface between the regulatory and the catalytic domains of two adjacent subunits. The structural and dynamical features of the site were characterized in depth and described. Interestingly, our findings provide evidence for lower allosteric Phe-binding ability of the G46S mutant than the human wild-type enzyme. This also explains the disease-causing nature of this mutant. PMID:26479306

  18. A dynamically coupled allosteric network underlies binding cooperativity in Src kinase

    NASA Astrophysics Data System (ADS)

    Foda, Zachariah H.; Shan, Yibing; Kim, Eric T.; Shaw, David E.; Seeliger, Markus A.

    2015-01-01

    Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

  19. A dynamically coupled allosteric network underlies binding cooperativity in Src kinase

    PubMed Central

    Foda, Zachariah H.; Shan, Yibing; Kim, Eric T.; Shaw, David E.; Seeliger, Markus A.

    2015-01-01

    Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity. PMID:25600932

  20. A dynamically coupled allosteric network underlies binding cooperativity in Src kinase.

    PubMed

    Foda, Zachariah H; Shan, Yibing; Kim, Eric T; Shaw, David E; Seeliger, Markus A

    2015-01-01

    Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity. PMID:25600932

  1. Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-coupled Receptor.

    PubMed

    Bock, Andreas; Bermudez, Marcel; Krebs, Fabian; Matera, Carlo; Chirinda, Brian; Sydow, Dominique; Dallanoce, Clelia; Holzgrabe, Ulrike; De Amici, Marco; Lohse, Martin J; Wolber, Gerhard; Mohr, Klaus

    2016-07-29

    G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site. PMID:27298318

  2. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases.

    PubMed

    Buey, Rubén M; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M; Revuelta, José L

    2015-01-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches. PMID:26558346

  3. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    NASA Astrophysics Data System (ADS)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  4. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    PubMed Central

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-01-01

    Inosine-5′-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches. PMID:26558346

  5. Agonist binding and function at the human alpha(2A)-adrenoceptor: allosteric modulation by amilorides.

    PubMed

    Leppik, R A; Birdsall, N J

    2000-11-01

    It has been found previously that amilorides act via an allosteric site on the alpha(2A)-adrenergic receptor to strongly inhibit antagonist binding. In this study, allosteric modulation of agonist binding and function at the alpha(2A)-adrenergic receptor was explored. The dissociation rate of the agonist [(3)H]UK14304 from alpha(2A)-receptors was decreased by the amilorides in a concentration-dependent manner. This contrasts with the increases in (3)H-antagonist dissociation rate found previously. The agonist-amiloride analog interaction data could be fitted to equations derived from the ternary complex allosteric model. The calculated log affinities of the amilorides at the [(3)H]UK14304-occupied receptor increased with the size of the 5-N-alkyl side chain and ranged from 2.4 for amiloride to 4.2 for 5-(N,N-hexamethylene)-amiloride. The calculated negative cooperativities cover a narrow range, in sharp contrast to the broad range found for antagonist-amiloride analog interactions. The effects of the amilorides on the agonist actions of UK14304, epinephrine, and norepinephrine were explored using a [(35)S]GTPgammaS functional assay, and the parameters calculated for the cooperativities and affinities of the UK14304-amiloride analog interactions, using the equation derived from the ternary complex allosteric model, were in good agreement with those derived from the kinetic studies. Therefore both the binding and functional data provide further support for the existence of a well defined allosteric site on the human alpha(2A)-adrenergic receptor. The binding mode of the amilorides at the agonist-occupied and antagonist-occupied receptor differs markedly but, within each group, the structure of either the agonist or the antagonist examined has only a slight effect on the allosteric interactions. PMID:11040058

  6. Allosteric Sensing of Fatty Acid Binding by NMR: Application to Human Serum Albumin.

    PubMed

    Jafari, Naeimeh; Ahmed, Rashik; Gloyd, Melanie; Bloomfield, Jonathon; Britz-McKibbin, Philip; Melacini, Giuseppe

    2016-08-25

    Human serum albumin (HSA) serves not only as a physiological oncotic pressure regulator and a ligand carrier but also as a biomarker for pathologies ranging from ischemia to diabetes. Moreover, HSA is a biopharmaceutical with a growing repertoire of putative clinical applications from hypovolemia to Alzheimer's disease. A key determinant of the physiological, diagnostic, and therapeutic functions of HSA is the amount of long chain fatty acids (LCFAs) bound to HSA. Here, we propose to utilize (13)C-oleic acid for the NMR-based assessment of albumin-bound LCFA concentration (CONFA). (13)C-Oleic acid primes HSA for a LCFA-dependent allosteric transition that modulates the frequency separation between the two main (13)C NMR peaks of HSA-bound oleic acid (ΔνAB). On the basis of ΔνAB, the overall [(12)C-LCFA]Tot/[HSA]Tot ratio is reproducibly estimated in a manner that is only minimally sensitive to glycation, albumin concentration, or redox potential, unlike other methods to quantify HSA-bound LCFAs such as the albumin-cobalt binding assay. PMID:27429126

  7. Ligand binding to WW tandem domains of YAP2 transcriptional regulator is under negative cooperativity.

    PubMed

    Schuchardt, Brett J; Mikles, David C; Hoang, Lawrence M; Bhat, Vikas; McDonald, Caleb B; Sudol, Marius; Farooq, Amjad

    2014-12-01

    YES-associated protein 2 (YAP2) transcriptional regulator drives a multitude of cellular processes, including the newly discovered Hippo tumor suppressor pathway, by virtue of the ability of its WW domains to bind and recruit PPXY-containing ligands to specific subcellular compartments. Herein, we employ an array of biophysical tools to investigate allosteric communication between the WW tandem domains of YAP2. Our data show that the WW tandem domains of YAP2 negatively cooperate when binding to their cognate ligands. Moreover, the molecular origin of such negative cooperativity lies in an unfavorable entropic contribution to the overall free energy relative to ligand binding to isolated WW domains. Consistent with this notion, the WW tandem domains adopt a fixed spatial orientation such that the WW1 domain curves outwards and stacks onto the binding groove of the WW2 domain, thereby sterically hindering ligand binding to both itself and its tandem partner. Although ligand binding to both WW domains disrupts such interdomain stacking interaction, they reorient themselves and adopt an alternative fixed spatial orientation in the liganded state by virtue of their ability to engage laterally so as to allow their binding grooves to point outwards and away from each other. In short, while the ability of WW tandem domains to aid ligand binding is well documented, our demonstration that they may also be subject to negative binding cooperativity represents a paradigm shift in our understanding of the molecular action of this ubiquitous family of protein modules. PMID:25283809

  8. Ligand Binding to WW Tandem Domains of YAP2 Transcriptional Regulator Is Under Negative Cooperativity

    PubMed Central

    Schuchardt, Brett J.; Mikles, David C.; Hoang, Lawrence M.; Bhat, Vikas; McDonald, Caleb B.; Sudol, Marius; Farooq, Amjad

    2014-01-01

    YAP2 transcriptional regulator drives a multitude of cellular processes, including the newly discovered Hippo tumor suppressor pathway, by virtue of the ability of its WW domains to bind and recruit PPXY-containing ligands to specific subcellular compartments. Herein, we employ an array of biophysical tools to investigate allosteric communication between the WW tandem domains of YAP2. Our data show that the WW tandem domains of YAP2 negatively cooperate when binding to their cognate ligands. Moreover, the molecular origin of such negative cooperativity lies in an unfavorable entropic contribution to the overall free energy relative to ligand binding to isolated WW domains. Consistent with this notion, the WW tandem domains adopt a fixed spatial orientation such that the WW1 domain curves outwards and stacks onto the binding groove of WW2 domain, thereby sterically hindering ligand binding to both itself and its tandem partner. Although ligand binding to both WW domains disrupts such interdomain stacking interaction, they reorient themselves and adopt an alternative fixed spatial orientation in the liganded state by virtue of their ability to engage laterally so as to allow their binding grooves to point outwards and away from each other. In short, while the ability of WW tandem domains to aid ligand binding is well-documented, our demonstration that they may also be subject to negative binding cooperativity represents a paradigm shift in our understanding of the molecular action of this ubiquitous family of protein modules. PMID:25283809

  9. Overlapping binding sites drive allosteric agonism and positive cooperativity in type 4 metabotropic glutamate receptors.

    PubMed

    Rovira, Xavier; Malhaire, Fanny; Scholler, Pauline; Rodrigo, Jordi; Gonzalez-Bulnes, Patricia; Llebaria, Amadeu; Pin, Jean-Philippe; Giraldo, Jesús; Goudet, Cyril

    2015-01-01

    Type 4 metabotropic glutamate (mGlu4) receptors are emerging targets for the treatment of various disorders. Accordingly, numerous mGlu4-positive allosteric modulators (PAMs) have been identified, some of which also display agonist activity. To identify the structural bases for their allosteric action, we explored the relationship between the binding pockets of mGlu4 PAMs with different chemical scaffolds and their functional properties. By use of innovative mGlu4 biosensors and second-messenger assays, we show that all PAMs enhance agonist action on the receptor through different degrees of allosteric agonism and positive cooperativity. For example, whereas VU0155041 and VU0415374 display equivalent efficacies [log(τ(B)) = 1.15 ± 0.38 and 1.25 ± 0.44, respectively], they increase the ability of L-AP4 to stabilize the active conformation of the receptor by 4 and 39 times, respectively. Modeling and docking studies identify 2 overlapping binding pockets as follows: a first site homologous to the pocket of natural agonists of class A GPCRs linked to allosteric agonism and a second one pointing toward a site topographically homologous to the Na(+) binding pocket of class A GPCRs, occupied by PAMs exhibiting the strongest cooperativity. These results reveal that intrinsic efficacy and cooperativity of mGlu4 PAMs are correlated with their binding mode, and vice versa, integrating structural and functional knowledge from different GPCR classes. PMID:25342125

  10. Probing allosteric binding sites of the maize endosperm ADP-glucose pyrophosphorylase.

    PubMed

    Boehlein, Susan K; Shaw, Janine R; Hannah, L Curtis; Stewart, Jon D

    2010-01-01

    Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a nonnative, low-activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) reported previously by others revealed that several sulfate ions bind to each enzyme. These sites are also believed to interact with allosteric regulators such as inorganic phosphate and 3-phosphoglycerate (3-PGA). Several arginine (Arg) side chains contact the bound sulfate ions in the potato structure and likely play important roles in allosteric effector binding. Alanine-scanning mutagenesis was applied to the corresponding Arg residues in both the small and large subunits of maize endosperm AGPase to determine their roles in allosteric regulation and thermal stability. Steady-state kinetic and regulatory parameters were measured for each mutant. All of the Arg mutants examined--in both the small and large subunits--bound 3-PGA more weakly than the wild type (A(50) increased by 3.5- to 20-fold). By contrast, the binding of two other maize AGPase allosteric activators (fructose-6-phosphate and glucose-6-phosphate) did not always mimic the changes observed for 3-PGA. In fact, compared to 3-PGA, fructose-6-phosphate is a more efficient activator in two of the Arg mutants. Phosphate binding was also affected by Arg substitutions. The combined data support a model for the binding interactions associated with 3-PGA in which allosteric activators and inorganic phosphate compete directly. PMID:19889875

  11. Membranes serve as allosteric activators of phospholipase A2, enabling it to extract, bind, and hydrolyze phospholipid substrates

    PubMed Central

    Mouchlis, Varnavas D.; Bucher, Denis; McCammon, J. Andrew; Dennis, Edward A.

    2015-01-01

    Defining the molecular details and consequences of the association of water-soluble proteins with membranes is fundamental to understanding protein–lipid interactions and membrane functioning. Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that compose the membrane bilayers, provide the ideal system for studying protein–lipid interactions. Our study focuses on understanding the catalytic cycle of two different human PLA2s: the cytosolic Group IVA cPLA2 and calcium-independent Group VIA iPLA2. Computer-aided techniques guided by deuterium exchange mass spectrometry data, were used to create structural complexes of each enzyme with a single phospholipid substrate molecule, whereas the substrate extraction process was studied using steered molecular dynamics simulations. Molecular dynamic simulations of the enzyme–substrate–membrane systems revealed important information about the mechanisms by which these enzymes associate with the membrane and then extract and bind their phospholipid substrate. Our data support the hypothesis that the membrane acts as an allosteric ligand that binds at the allosteric site of the enzyme’s interfacial surface, shifting its conformation from a closed (inactive) state in water to an open (active) state at the membrane interface. PMID:25624474

  12. Steric and allosteric effects of fatty acids on the binding of warfarin to human serum albumin revealed by molecular dynamics and free energy calculations.

    PubMed

    Fujiwara, Shin-Ichi; Amisaki, Takashi

    2011-01-01

    Human serum albumin (HSA) binds with drugs and fatty acids (FAs). This study was initiated to elucidate the relationship between the warfarin binding affinity of HSA and the positions of bound FA molecules. Molecular dynamics simulations of 11 HSA-warfarin-myristate complexes were performed. HSA-warfarin binding free energy was then calculated for each of the complexes by the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. The results indicated that the magnitude of the binding free energy was smaller in HSA-warfarin complexes that had 4 or more myristate molecules than in complexes with no myristate molecules. The unfavorable effect on the HSA-warfarin binding affinity was caused sterically by the binding of a myristate molecule to the FA binding site closest to the warfarin binding site. On the other hand, the magnitude of HSA-warfarin binding free energy was largest when 3 myristate molecules were bound to the high-affinity sites. The strongest HSA-warfarin binding was attributable to favorable entropic contribution related to larger atomic fluctuations of the amino acid residues at the warfarin binding site. In the binding of 2 myristate molecules to the sites with the highest and second-highest affinities, allosteric modulation that enhanced electrostatic interactions between warfarin and some of the amino acid residues around the warfarin binding site was observed. This study clarified the structural and energetic properties of steric/allosteric effects of FAs on the HSA-warfarin binding affinity and illustrated the approach to analyze protein-ligand interactions in situations such that multiple ligands bind to the other sites of the protein. PMID:21720037

  13. Mapping Cannabinoid 1 Receptor Allosteric Site(s): Critical Molecular Determinant and Signaling Profile of GAT100, a Novel, Potent, and Irreversibly Binding Probe.

    PubMed

    Laprairie, Robert B; Kulkarni, Abhijit R; Kulkarni, Pushkar M; Hurst, Dow P; Lynch, Diane; Reggio, Patricia H; Janero, David R; Pertwee, Roger G; Stevenson, Lesley A; Kelly, Melanie E M; Denovan-Wright, Eileen M; Thakur, Ganesh A

    2016-06-15

    agonism associated with Org27569 and PSNCBAM-1. Computational docking studies implicate C7.38(382) as a key feature of GAT100 ligand-binding motif. These data help inform the engineering of newer-generation, druggable CB1R allosteric modulators and demonstrate the utility of GAT100 as a covalent probe for mapping structure-function correlates characteristic of the druggable CB1R allosteric space. PMID:27046127

  14. Allosteric Modulation of Chemoattractant Receptors

    PubMed Central

    Allegretti, Marcello; Cesta, Maria Candida; Locati, Massimo

    2016-01-01

    Chemoattractants control selective leukocyte homing via interactions with a dedicated family of related G protein-coupled receptor (GPCR). Emerging evidence indicates that the signaling activity of these receptors, as for other GPCR, is influenced by allosteric modulators, which interact with the receptor in a binding site distinct from the binding site of the agonist and modulate the receptor signaling activity in response to the orthosteric ligand. Allosteric modulators have a number of potential advantages over orthosteric agonists/antagonists as therapeutic agents and offer unprecedented opportunities to identify extremely selective drug leads. Here, we resume evidence of allosterism in the context of chemoattractant receptors, discussing in particular its functional impact on functional selectivity and probe/concentration dependence of orthosteric ligands activities. PMID:27199992

  15. Two disparate ligand binding sites in the human P2Y1 receptor

    PubMed Central

    Zhang, Dandan; Gao, Zhan-Guo; Zhang, Kaihua; Kiselev, Evgeny; Crane, Steven; Wang, Jiang; Paoletta, Silvia; Yi, Cuiying; Ma, Limin; Zhang, Wenru; Han, Gye Won; Liu, Hong; Cherezov, Vadim; Katritch, Vsevolod; Jiang, Hualiang; Stevens, Raymond C.; Jacobson, Kenneth A.; Zhao, Qiang; Wu, Beili

    2015-01-01

    In response to adenosine 5′-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7Å resolution, and with a non-nucleotide antagonist BPTU at 2.2Å resolution. The structures reveal two distinct ligand binding sites, providing atomic details of P2Y1R’s unique ligand binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which, however, is different in shape and location from the nucleotide binding site in previously determined P2Y12R structure. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects. PMID:25822790

  16. Extracellular allosteric Na(+) binding to the Na(+),K(+)-ATPase in cardiac myocytes.

    PubMed

    Garcia, Alvaro; Fry, Natasha A S; Karimi, Keyvan; Liu, Chia-chi; Apell, Hans-Jürgen; Rasmussen, Helge H; Clarke, Ronald J

    2013-12-17

    Whole-cell patch-clamp measurements of the current, Ip, produced by the Na(+),K(+)-ATPase across the plasma membrane of rabbit cardiac myocytes show an increase in Ip over the extracellular Na(+) concentration range 0-50 mM. This is not predicted by the classical Albers-Post scheme of the Na(+),K(+)-ATPase mechanism, where extracellular Na(+) should act as a competitive inhibitor of extracellular K(+) binding, which is necessary for the stimulation of enzyme dephosphorylation and the pumping of K(+) ions into the cytoplasm. The increase in Ip is consistent with Na(+) binding to an extracellular allosteric site, independent of the ion transport sites, and an increase in turnover via an acceleration of the rate-determining release of K(+) to the cytoplasm, E2(K(+))2 → E1 + 2K(+). At normal physiological concentrations of extracellular Na(+) of 140 mM, it is to be expected that binding of Na(+) to the allosteric site would be nearly saturated. Its purpose would seem to be simply to optimize the enzyme's ion pumping rate under its normal physiological conditions. Based on published crystal structures, a possible location of the allosteric site is within a cleft between the α- and β-subunits of the enzyme. PMID:24359741

  17. Principles of Ligand Binding within a Completely Buried Cavity in HIF2[alpha] PAS-B

    SciTech Connect

    Key, Jason; Scheuermann, Thomas H.; Anderson, Peter C.; Daggett, Valerie; Gardner, Kevin H.

    2010-04-19

    Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors responsible for the metazoan hypoxia response and promote tumor growth, metastasis, and resistance to cancer treatment. The C-terminal Per-ARNT-Sim (PAS) domain of HIF2{alpha} (HIF2{alpha} PAS-B) contains a preformed solvent-inaccessible cavity that binds artificial ligands that allosterically perturb the formation of the HIF heterodimer. To better understand how small molecules bind within this domain, we examined the structures and equilibrium and transition-state thermodynamics of HIF2{alpha} PAS-B with several artificial ligands using isothermal titration calorimetry, NMR exchange spectroscopy, and X-ray crystallography. Rapid association rates reveal that ligand binding is not dependent upon a slow conformational change in the protein to permit ligand access, despite the closed conformation observed in the NMR and crystal structures. Compensating enthalpic and entropic contributions to the thermodynamic barrier for ligand binding suggest a binding-competent transition state characterized by increased structural disorder. Finally, molecular dynamics simulations reveal conversion between open and closed conformations of the protein and pathways of ligand entry into the binding pocket.

  18. Exploiting protein flexibility to predict the location of allosteric sites

    PubMed Central

    2012-01-01

    Background Allostery is one of the most powerful and common ways of regulation of protein activity. However, for most allosteric proteins identified to date the mechanistic details of allosteric modulation are not yet well understood. Uncovering common mechanistic patterns underlying allostery would allow not only a better academic understanding of the phenomena, but it would also streamline the design of novel therapeutic solutions. This relatively unexplored therapeutic potential and the putative advantages of allosteric drugs over classical active-site inhibitors fuel the attention allosteric-drug research is receiving at present. A first step to harness the regulatory potential and versatility of allosteric sites, in the context of drug-discovery and design, would be to detect or predict their presence and location. In this article, we describe a simple computational approach, based on the effect allosteric ligands exert on protein flexibility upon binding, to predict the existence and position of allosteric sites on a given protein structure. Results By querying the literature and a recently available database of allosteric sites, we gathered 213 allosteric proteins with structural information that we further filtered into a non-redundant set of 91 proteins. We performed normal-mode analysis and observed significant changes in protein flexibility upon allosteric-ligand binding in 70% of the cases. These results agree with the current view that allosteric mechanisms are in many cases governed by changes in protein dynamics caused by ligand binding. Furthermore, we implemented an approach that achieves 65% positive predictive value in identifying allosteric sites within the set of predicted cavities of a protein (stricter parameters set, 0.22 sensitivity), by combining the current analysis on dynamics with previous results on structural conservation of allosteric sites. We also analyzed four biological examples in detail, revealing that this simple coarse

  19. Predicting target-ligand interactions using protein ligand-binding site and ligand substructures

    PubMed Central

    2015-01-01

    Background Cell proliferation, differentiation, Gene expression, metabolism, immunization and signal transduction require the participation of ligands and targets. It is a great challenge to identify rules governing molecular recognition between chemical topological substructures of ligands and the binding sites of the targets. Methods We suppose that the ligand-target interactions are determined by ligand substructures as well as the physical-chemical properties of the binding sites. Therefore, we propose a fragment interaction model (FIM) to describe the interactions between ligands and targets, with the purpose of facilitating the chemical interpretation of ligand-target binding. First we extract target-ligand complexes from sc-PDB database, based on which, we get the target binding sites and the ligands. Then we represent each binding site as a fragment vector based on a target fragment dictionary that is composed of 199 clusters (denoted as fragements in this work) obtained by clustering 4200 trimers according to their physical-chemical properties. And then, we represent each ligand as a substructure vector based on a dictionary containing 747 substructures. Finally, we build the FIM by generating the interaction matrix M (representing the fragment interaction network), and the FIM can later be used for predicting unknown ligand-target interactions as well as providing the binding details of the interactions. Results The five-fold cross validation results show that the proposed model can get higher AUC score (92%) than three prevalence algorithms CS-PD (80%), BLM-NII (85%) and RF (85%), demonstrating the remarkable predictive ability of FIM. We also show that the ligand binding sites (local information) overweight the sequence similarities (global information) in ligand-target binding, and introducing too much global information would be harmful to the predictive ability. Moreover, The derived fragment interaction network can provide the chemical insights on

  20. Development of allosteric modulators of GPCRs for treatment of CNS disorders

    PubMed Central

    Nickols, Hilary Highfield; Conn, P. Jeffrey

    2013-01-01

    The discovery of allosteric modulators of G protein-coupled receptors (GPCRs) provides a promising new strategy with potential for developing novel treatments for a variety of central nervous system (CNS) disorders. Traditional drug discovery efforts targeting GPCRs have focused on developing ligands for orthosteric sites which bind endogenous ligands. Allosteric modulators target a site separate from the orthosteric site to modulate receptor function. These allosteric agents can either potentiate (positive allosteric modulator, PAM) or inhibit (negative allosteric modulator, NAM) the receptor response and often provide much greater subtype selectivity than do orthosteric ligands for the same receptors. Experimental evidence has revealed more nuanced pharmacological modes of action of allosteric modulators, with some PAMs showing allosteric agonism in combination with positive allosteric modulation in response to endogenous ligand (ago-potentiators) as well as “bitopic” ligands that interact with both the allosteric and orthosteric sites. Drugs targeting the allosteric site allow for increased drug selectivity and potentially decreased adverse side effects. Promising evidence has demonstrated potential utility of a number of allosteric modulators of GPCRs in multiple CNS disorders, including neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, as well as psychiatric or neurobehavioral diseases such as anxiety, schizophrenia, and addiction. PMID:24076101

  1. Chemical, target, and bioactive properties of allosteric modulation.

    PubMed

    van Westen, Gerard J P; Gaulton, Anna; Overington, John P

    2014-04-01

    Allosteric modulators are ligands for proteins that exert their effects via a different binding site than the natural (orthosteric) ligand site and hence form a conceptually distinct class of ligands for a target of interest. Here, the physicochemical and structural features of a large set of allosteric and non-allosteric ligands from the ChEMBL database of bioactive molecules are analyzed. In general allosteric modulators are relatively smaller, more lipophilic and more rigid compounds, though large differences exist between different targets and target classes. Furthermore, there are differences in the distribution of targets that bind these allosteric modulators. Allosteric modulators are over-represented in membrane receptors, ligand-gated ion channels and nuclear receptor targets, but are underrepresented in enzymes (primarily proteases and kinases). Moreover, allosteric modulators tend to bind to their targets with a slightly lower potency (5.96 log units versus 6.66 log units, p<0.01). However, this lower absolute affinity is compensated by their lower molecular weight and more lipophilic nature, leading to similar binding efficiency and surface efficiency indices. Subsequently a series of classifier models are trained, initially target class independent models followed by finer-grained target (architecture/functional class) based models using the target hierarchy of the ChEMBL database. Applications of these insights include the selection of likely allosteric modulators from existing compound collections, the design of novel chemical libraries biased towards allosteric regulators and the selection of targets potentially likely to yield allosteric modulators on screening. All data sets used in the paper are available for download. PMID:24699297

  2. Dynamics and allostery of the ionotropic glutamate receptors and the ligand binding domain.

    PubMed

    Tobi, Dror

    2016-02-01

    The dynamics of the ligand-binding domain (LBD) and the intact ionotropic glutamate receptor (iGluR) were studied using Gaussian Network Model (GNM) analysis. The dynamics of LBDs with various allosteric modulators is compared using a novel method of multiple alignment of GNM modes of motion. The analysis reveals that allosteric effectors change the dynamics of amino acids at the upper lobe interface of the LBD dimer as well as at the hinge region between the upper- and lower- lobes. For the intact glutamate receptor the analysis show that the clamshell-like movement of the LBD upper and lower lobes is coupled to the bending of the trans-membrane domain (TMD) helices which may open the channel pore. The results offer a new insight on the mechanism of action of allosteric modulators on the iGluR and support the notion of TMD helices bending as a possible mechanism for channel opening. In addition, the study validates the methodology of multiple GNM modes alignment as a useful tool to study allosteric effect and its relation to proteins dynamics. PMID:26677170

  3. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    PubMed Central

    Gregory, Karen J; Sexton, Patrick M; Christopoulos, Arthur

    2007-01-01

    Muscarinic acetylcholine receptors (mAChRs) are prototypical Family A G protein coupled-receptors. The five mAChR subtypes are widespread throughout the periphery and the central nervous system and, accordingly, are widely involved in a variety of both physiological and pathophysiological processes. There currently remains an unmet need for better therapeutic agents that can selectively target a given mAChR subtype to the relative exclusion of others. The main reason for the lack of such selective mAChR ligands is the high sequence homology within the acetylcholine-binding site (orthosteric site) across all mAChRs. However, the mAChRs possess at least one, and likely two, extracellular allosteric binding sites that can recognize small molecule allosteric modulators to regulate the binding and function of orthosteric ligands. Extensive studies of prototypical mAChR modulators, such as gallamine and alcuronium, have provided strong pharmacological evidence, and associated structure-activity relationships (SAR), for a “common” allosteric site on all five mAChRs. These studies are also supported by mutagenesis experiments implicating the second extracellular loop and the interface between the third extracellular loop and the top of transmembrane domain 7 as contributing to the common allosteric site. Other studies are also delineating the pharmacology of a second allosteric site, recognized by compounds such as staurosporine. In addition, allosteric agonists, such as McN-A-343, AC-42 and N-desmethylclozapine, have also been identified. Current challenges to the field include the ability to effectively detect and validate allosteric mechanisms, and to quantify allosteric effects on binding affinity and signaling efficacy to inform allosteric modulator SAR. PMID:19305798

  4. Ligand configurational entropy and protein binding.

    PubMed

    Chang, Chia-en A; Chen, Wei; Gilson, Michael K

    2007-01-30

    The restriction of a small molecule's motion on binding to a protein causes a loss of configurational entropy, and thus a penalty in binding affinity. Some energy models used in computer-aided ligand design neglect this entropic penalty, whereas others account for it based on an expected drop in the number of accessible rotamers upon binding. However, the validity of the physical assumptions underlying the various approaches is largely unexamined. The present study addresses this issue by using Mining Minima calculations to analyze the association of amprenavir with HIV protease. The computed loss in ligand configurational entropy is large, contributing approximately 25 kcal/mol (4.184 kJ/kcal) to DeltaG degrees. Most of this loss results from narrower energy wells in the bound state, rather than a drop in the number of accessible rotamers. Coupling among rotation/translation and internal degrees of freedom complicates the decomposition of the entropy change into additive terms. The results highlight the potential to gain affinity by designing conformationally restricted ligands and have implications for the formulation of energy models for ligand scoring. PMID:17242351

  5. Ligand configurational entropy and protein binding

    PubMed Central

    Chang, Chia-en A.; Chen, Wei; Gilson, Michael K.

    2007-01-01

    The restriction of a small molecule's motion on binding to a protein causes a loss of configurational entropy, and thus a penalty in binding affinity. Some energy models used in computer-aided ligand design neglect this entropic penalty, whereas others account for it based on an expected drop in the number of accessible rotamers upon binding. However, the validity of the physical assumptions underlying the various approaches is largely unexamined. The present study addresses this issue by using Mining Minima calculations to analyze the association of amprenavir with HIV protease. The computed loss in ligand configurational entropy is large, contributing ∼25 kcal/mol (4.184 kJ/kcal) to ΔG°. Most of this loss results from narrower energy wells in the bound state, rather than a drop in the number of accessible rotamers. Coupling among rotation/translation and internal degrees of freedom complicates the decomposition of the entropy change into additive terms. The results highlight the potential to gain affinity by designing conformationally restricted ligands and have implications for the formulation of energy models for ligand scoring. PMID:17242351

  6. Orthosteric and Allosteric Ligands of Nicotinic Acetylcholine Receptors for Smoking Cessation

    PubMed Central

    Mohamed, Tasnim S.; Jayakar, Selwyn S.; Hamouda, Ayman K.

    2015-01-01

    Nicotine addiction, the result of tobacco use, leads to over six million premature deaths world-wide per year, a number that is expected to increase by a third within the next two decades. While more than half of smokers want and attempt to quit, only a small percentage of smokers are able to quit without pharmacological interventions. Therefore, over the past decades, researchers in academia and the pharmaceutical industry have focused their attention on the development of more effective smoking cessation therapies, which is now a growing 1.9 billion dollar market. Because the role of neuronal nicotinic acetylcholine receptors (nAChR) in nicotine addiction is well established, nAChR based therapeutics remain the leading strategy for smoking cessation. However, the development of neuronal nAChR drugs that are selective for a nAChR subpopulation is challenging, and only few neuronal nAChR drugs are clinically available. Among the many neuronal nAChR subtypes that have been identified in the brain, the α4β2 subtype is the most abundant and plays a critical role in nicotine addiction. Here, we review the role of neuronal nAChRs, especially the α4β2 subtype, in the development and treatment of nicotine addiction. We also compare available smoking cessation medications and other nAChR orthosteric and allosteric ligands that have been developed with emphasis on the difficulties faced in the development of clinically useful compounds with high nAChR subtype selectivity. PMID:26635524

  7. Time, the Forgotten Dimension of Ligand Binding Teaching

    ERIC Educational Resources Information Center

    Corzo, Javier

    2006-01-01

    Ligand binding is generally explained in terms of the equilibrium constant K[subscript d] for the protein-ligand complex dissociation. However, both theoretical considerations and experimental data point to the life span of the protein-ligand complex as an important, but generally overlooked, aspect of ligand binding by macromolecules. Short-lived…

  8. An external sodium ion binding site controls allosteric gating in TRPV1 channels.

    PubMed

    Jara-Oseguera, Andres; Bae, Chanhyung; Swartz, Kenton J

    2016-01-01

    TRPV1 channels in sensory neurons are integrators of painful stimuli and heat, yet how they integrate diverse stimuli and sense temperature remains elusive. Here, we show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation. In studying the underlying mechanism, we find that the temperature sensors in TRPV1 activate in two steps to favor opening, and that the binding of sodium to an extracellular site exerts allosteric control over temperature-sensor activation and opening of the pore. The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium. Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli. PMID:26882503

  9. An external sodium ion binding site controls allosteric gating in TRPV1 channels

    PubMed Central

    Jara-Oseguera, Andres; Bae, Chanhyung; Swartz, Kenton J

    2016-01-01

    TRPV1 channels in sensory neurons are integrators of painful stimuli and heat, yet how they integrate diverse stimuli and sense temperature remains elusive. Here, we show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation. In studying the underlying mechanism, we find that the temperature sensors in TRPV1 activate in two steps to favor opening, and that the binding of sodium to an extracellular site exerts allosteric control over temperature-sensor activation and opening of the pore. The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium. Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli. DOI: http://dx.doi.org/10.7554/eLife.13356.001 PMID:26882503

  10. Comparison of crystal and solution hemoglobin binding of selected antigelling agents and allosteric modifiers

    SciTech Connect

    Mehanna, A.S.; Abraham, D.J. )

    1990-04-24

    This paper details comprehensive binding studies (solution and X-ray) of human hemoglobin A with a group of halogenated carboxylic acids that were investigated as potential antisickling agents. It is, to our knowledge, the first study to compare solution and crystal binding for a series of compounds under similar high-salt conditions used for cocrystallization. The compounds include ((3,4-dichlorobenzyl)oxy)acetic acid, ((p-bromobenzyl)oxy)acetic acid, clofibric acid, and bezafibrate. The location and stereochemistry of binding sites have been established by X-ray crystallography, while the number of binding sites and affinity constants were measured by using equilibrium dialysis. The observed crystal structures are consistent with the binding observed in solution and that the number of binding sites is independent of salt concentration, while the binding constant increases with increasing salt concentration. The studies also reveal that relatively small changes in the chemical structure of a drug molecule can result in entirely different binding sites on the protein. Moreover, the X-ray studies provide a possible explanation for the multiplicity in function exhibited by these compounds as allosteric modulators and/or antisickling agents. Finally, the studies indicate that these compounds bind differently to the R and T states of hemoglobin, and observation of special significance to the original design of these agents.

  11. Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding

    NASA Astrophysics Data System (ADS)

    Beveridge, Rebecca; Migas, Lukasz G.; Payne, Karl A. P.; Scrutton, Nigel S.; Leys, David; Barran, Perdita E.

    2016-07-01

    Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM-MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM-MS data.

  12. Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding

    PubMed Central

    Beveridge, Rebecca; Migas, Lukasz G.; Payne, Karl A. P.; Scrutton, Nigel S.; Leys, David; Barran, Perdita E.

    2016-01-01

    Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM–MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in the presence of the cofactor and depopulated in its absence. These findings, supported by MD simulations, indicate a more open structure for the apo form. HDX-MS reveals that while the dominant structural changes occur proximal to the cofactor-binding site, rearrangements on cofactor binding are evident throughout the protein, predominantly attributable to allosteric conformational tightening, consistent with IM–MS data. PMID:27418477

  13. Ligand binding and hexacoordination in synechocystis hemoglobin.

    PubMed

    Hvitved, A N; Trent, J T; Premer, S A; Hargrove, M S

    2001-09-14

    A large and phylogenetically diverse group of organisms contain truncated hemoglobins, including the unicellular cyanobacterium Synechocystis (Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K., Ascenzi, P., Moens, L., and Bolognesi, M. (2000) EMBO J. 19, 2424-2434). Synechocystis hemoglobin is also hexacoordinate, with a heme pocket histidine that reversibly coordinates the ligand binding site. Hexacoordinate hemoglobins are ubiquitous in plants and are now being identified in a diverse array of organisms including humans (Arredondo-Peter, R., Hargrove, M. S., Moran, J. F., Sarath, G., and Klucas, R. V. (1998) Plant Physiol. 118, 1121-1125; Trent, J. T., III, Watts, R. A., and Hargrove, M. S. (2001) J. Biol. Chem. 276, 30106-30110). Rate constants for association and dissociation of the hexacoordinating amino acid side chain in Synechocystis hemoglobin have been measured along with bimolecular rate constants for association of oxygen and carbon monoxide following laser flash photolysis. These values were compared with ligand binding initiated by rapid mixing. Site-directed mutagenesis was used to determine the roles of several heme pocket amino acids in facilitating hexacoordination and stabilizing bound oxygen. It is demonstrated that Synechocystis hemoglobin contains a very reactive binding site and that ligand migration through the protein is rapid. Rate constants for hexacoordination by His(46) are also large and facilitated by other heme pocket amino acids including Gln(43). PMID:11438545

  14. Activation and Allosteric Modulation of Human μ Opioid Receptor in Molecular Dynamics.

    PubMed

    Bartuzi, Damian; Kaczor, Agnieszka A; Matosiuk, Dariusz

    2015-11-23

    Allosteric protein modulation has gained increasing attention in drug design. Its application as a mechanism of action could bring forth safer and more effective medicines. Targeting opioid receptors with allosteric modulators can result in better treatment of pain, depression, and respiratory and immune disorders. In this work we use recent reports on negative modulators of μ opioid receptor as a starting point for identification of allosteric sites and mechanisms of opioid receptor modulation using homology modeling and docking and molecular dynamics studies. An allosteric binding site description is presented. Results suggest a shared binding region for lipophilic allosteric ligands, reveal possible differences in the modulation mechanism between cannabinoids and salvinorin A, and show ambiguous properties of the latter. Also, they emphasize the importance of native-like environment in molecular dynamics simulations and uncover relationships between modulator and orthosteric ligand binding and receptor behavior. Relationships between ligands, transmission switch, and hydrophobic lock are analyzed. PMID:26517559

  15. Allostery Mediates Ligand Binding to WWOX Tumor Suppressor via a Conformational Switch

    PubMed Central

    Schuchardt, Brett J.; Mikles, David C.; Bhat, Vikas; McDonald, Caleb B.; Sudol, Marius; Farooq, Amjad

    2014-01-01

    While being devoid of the ability to recognize ligands itself, the WW2 domain is believed to aid ligand binding to WW1 domain in the context of WW1-WW2 tandem module of WWOX tumor suppressor. In an effort to test the generality of this hypothesis, we have undertaken here a detailed biophysical analysis of the binding of WW domains of WWOX alone and in the context of WW1-WW2 tandem module to an array of putative PPXY ligands. Our data show that while the WW1 domain of WWOX binds to all ligands in a physiologically-relevant manner, the WW2 domain does not. Moreover, ligand binding to WW1 domain in the context of WW1-WW2 tandem module is two-to-three-fold stronger than when treated alone. We also provide evidence that the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. Of particular note is the observation that the physical association of WW2 domain with WW1 blocks access to ligand. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the physical association of WW domains in the liganded conformation. Taken together, our study underscores a key role of allosteric communication in the ability of WW2 orphan domain to chaperone physiological action of WW1 domain within the context of the WW1-WW2 tandem module of WWOX. PMID:25703206

  16. Allostery mediates ligand binding to WWOX tumor suppressor via a conformational switch.

    PubMed

    Schuchardt, Brett J; Mikles, David C; Bhat, Vikas; McDonald, Caleb B; Sudol, Marius; Farooq, Amjad

    2015-04-01

    While being devoid of the ability to recognize ligands itself, the WW2 domain is believed to aid ligand binding to the WW1 domain in the context of a WW1-WW2 tandem module of WW domain-containing oxidoreductase (WWOX) tumor suppressor. In an effort to test the generality of this hypothesis, we have undertaken here a detailed biophysical analysis of the binding of WW domains of WWOX alone and in the context of the WW1-WW2 tandem module to an array of putative proline-proline-x-tyrosine (PPXY) ligands. Our data show that while the WW1 domain of WWOX binds to all ligands in a physiologically relevant manner, the WW2 domain does not. Moreover, ligand binding to the WW1 domain in the context of the WW1-WW2 tandem module is two-to-three-fold stronger than when treated alone. We also provide evidence that the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. Of particular note is the observation that the physical association of the WW2 domain with WW1 blocks access to ligands. Consequently, ligand binding to the WW1 domain not only results in the displacement of the WW2 lid but also disrupts the physical association of WW domains in the liganded conformation. Taken together, our study underscores a key role of allosteric communication in the ability of the WW2 orphan domain to chaperone physiological action of the WW1 domain within the context of the WW1-WW2 tandem module of WWOX. PMID:25703206

  17. Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

    PubMed

    Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

    2008-11-21

    Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS. PMID:18820259

  18. Follitropin receptors contain cryptic ligand binding sites.

    PubMed

    Lin, Win; Bernard, Michael P; Cao, Donghui; Myers, Rebecca V; Kerrigan, John E; Moyle, William R

    2007-01-01

    Human choriogonadotropin (hCG) and follitropin (hFSH) have been shown to contact different regions of the extracellular domains of G-protein coupled lutropin (LHR) and follitropin (FSHR) receptors. We report here that hCG and hFSH analogs interact with different regions of an FSHR/LHR chimera having only two unique LHR residues and that binds both hormones with high affinity. hCG and hFSH analogs dock with this receptor chimera in a manner similar to that in which they bind LHR and FSHR, respectively. This shows that although the FSHR does not normally bind hCG, it contains a cryptic lutropin binding site that has the potential to recognize hCG in a manner similar to the LHR. The presence of this cryptic site may explain why equine lutropins bind many mammalian FSHR and why mutations in the transmembrane domain distant from the extracellular domain enable the FSHR to bind hCG. The leucine-rich repeat domain (LRD) of the FSHR also appears to contain a cryptic FSH binding site that is obscured by other parts of the extracellular domain. This will explain why contacts seen in crystals of hFSH complexed with an LRD fragment of the human FSHR are hard to reconcile with the abilities of FSH analogs to interact with membrane G-protein coupled FSHR. We speculate that cryptic lutropin binding sites in the FSHR, which are also likely to be present in thyrotropin receptors (TSHR), permit the physiological regulation of ligand binding specificity. Cryptic FSH binding sites in the LRD may enable alternate spliced forms of the FSHR to interact with FSH. PMID:17059863

  19. Effects of ligand binding on the mechanical stability of protein GB1 studied by steered molecular dynamics simulation.

    PubMed

    Su, Ji-Guo; Zhao, Shu-Xin; Wang, Xiao-Feng; Li, Chun-Hua; Li, Jing-Yuan

    2016-08-01

    Regulation of the mechanical properties of proteins plays an important role in many biological processes, and sheds light on the design of biomaterials comprised of protein. At present, strategies to regulate protein mechanical stability focus mainly on direct modulation of the force-bearing region of the protein. Interestingly, the mechanical stability of GB1 can be significantly enhanced by the binding of Fc fragments of human IgG antibody, where the binding site is distant from the force-bearing region of the protein. The mechanism of this long-range allosteric control of protein mechanics is still elusive. In this work, the impact of ligand binding on the mechanical stability of GB1 was investigated using steered molecular dynamics simulation, and a mechanism underlying the enhanced protein mechanical stability is proposed. We found that the external force causes deformation of both force-bearing region and ligand binding site. In other words, there is a long-range coupling between these two regions. The binding of ligand restricts the distortion of the binding site and reduces the deformation of the force-bearing region through a long-range allosteric communication, which thus improves the overall mechanical stability of the protein. The simulation results are very consistent with previous experimental observations. Our studies thus provide atomic-level insights into the mechanical unfolding process of GB1, and explain the impact of ligand binding on the mechanical properties of the protein through long-range allosteric regulation, which should facilitate effective modulation of protein mechanical properties. PMID:27444879

  20. Landscape of protein-small ligand binding modes.

    PubMed

    Kasahara, Kota; Kinoshita, Kengo

    2016-09-01

    Elucidating the mechanisms of specific small-molecule (ligand) recognition by proteins is a long-standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein-ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all-against-all comparison of 20,040 protein-ligand complexes provided the landscape of the protein-ligand binding modes and its relationships with protein- and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R(2)  = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein-ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them. PMID:27327045

  1. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site.

    PubMed

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R; Phillips, Chris; Augustin, Martin A; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-05-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented. PMID:27139631

  2. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site

    PubMed Central

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R.; Phillips, Chris; Augustin, Martin A.; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-01-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5–inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented. PMID:27139631

  3. Allosteric Regulation of the Carbohydrate-binding Ability of a Novel Conger Eel Galectin by d-Mannoside

    PubMed Central

    Watanabe, Mizuki; Nakamura, Osamu; Muramoto, Koji; Ogawa, Tomohisa

    2012-01-01

    Conger eel has two galectins, termed congerins I and II (Con I and II), that function in mucus as biodefense molecules. Con I and II have acquired a novel protein fold via domain swapping and a new ligand-binding site by accelerated evolution, which enables recognition of some marine bacteria. In this study, we identified a new congerin isotype, congerin P (Con-P), from the peritoneal cells of conger eel. Although Con-P displayed obvious homology with galectins, we observed substitution of 7 out of 8 amino acid residues in the carbohydrate recognition domain that are conserved in all other known galectins. To understand the structure-function relationships of this unique galectin, recombinant Con-P was successfully expressed in Escherichia coli by using a Con II-tagged fusion protein system and subsequently characterized. In the presence of d-mannose, Con-P displayed 30-fold greater hemagglutinating activity than Con I; however, no activity was observed without mannose, indicating that d-mannoside can act as a modulator of Con-P. Frontal affinity chromatography analysis showed that activated Con-P, allosterically induced by mannose, displayed affinity for oligomannose-type sugars as well as N-acetyllactosamine-type β-galactosides. Thus, Con-P represents a new member of the galectin family with unique properties. PMID:22810239

  4. Insights on Structural Characteristics and Ligand Binding Mechanisms of CDK2

    PubMed Central

    Li, Yan; Zhang, Jingxiao; Gao, Weimin; Zhang, Lilei; Pan, Yanqiu; Zhang, Shuwei; Wang, Yonghua

    2015-01-01

    Cyclin-dependent kinase 2 (CDK2) is a crucial regulator of the eukaryotic cell cycle. However it is well established that monomeric CDK2 lacks regulatory activity, which needs to be aroused by its positive regulators, cyclins E and A, or be phosphorylated on the catalytic segment. Interestingly, these activation steps bring some dynamic changes on the 3D-structure of the kinase, especially the activation segment. Until now, in the monomeric CDK2 structure, three binding sites have been reported, including the adenosine triphosphate (ATP) binding site (Site I) and two non-competitive binding sites (Site II and III). In addition, when the kinase is subjected to the cyclin binding process, the resulting structural changes give rise to a variation of the ATP binding site, thus generating an allosteric binding site (Site IV). All the four sites are demonstrated as being targeted by corresponding inhibitors, as is illustrated by the allosteric binding one which is targeted by inhibitor ANS (fluorophore 8-anilino-1-naphthalene sulfonate). In the present work, the binding mechanisms and their fluctuations during the activation process attract our attention. Therefore, we carry out corresponding studies on the structural characterization of CDK2, which are expected to facilitate the understanding of the molecular mechanisms of kinase proteins. Besides, the binding mechanisms of CDK2 with its relevant inhibitors, as well as the changes of binding mechanisms following conformational variations of CDK2, are summarized and compared. The summary of the conformational characteristics and ligand binding mechanisms of CDK2 in the present work will improve our understanding of the molecular mechanisms regulating the bioactivities of CDK2. PMID:25918937

  5. Selective binding modes and allosteric inhibitory effects of lupane triterpenes on protein tyrosine phosphatase 1B.

    PubMed

    Jin, Tiantian; Yu, Haibo; Huang, Xu-Feng

    2016-01-01

    Protein Tyrosine Phosphatase 1B (PTP1B) has been recognized as a promising therapeutic target for treating obesity, diabetes, and certain cancers for over a decade. Previous drug design has focused on inhibitors targeting the active site of PTP1B. However, this has not been successful because the active site is positively charged and conserved among the protein tyrosine phosphatases. Therefore, it is important to develop PTP1B inhibitors with alternative inhibitory strategies. Using computational studies including molecular docking, molecular dynamics simulations, and binding free energy calculations, we found that lupane triterpenes selectively inhibited PTP1B by targeting its more hydrophobic and less conserved allosteric site. These findings were verified using two enzymatic assays. Furthermore, the cell culture studies showed that lupeol and betulinic acid inhibited the PTP1B activity stimulated by TNFα in neurons. Our study indicates that lupane triterpenes are selective PTP1B allosteric inhibitors with significant potential for treating those diseases with elevated PTP1B activity. PMID:26865097

  6. Selective binding modes and allosteric inhibitory effects of lupane triterpenes on protein tyrosine phosphatase 1B

    PubMed Central

    Jin, Tiantian; Yu, Haibo; Huang, Xu-Feng

    2016-01-01

    Protein Tyrosine Phosphatase 1B (PTP1B) has been recognized as a promising therapeutic target for treating obesity, diabetes, and certain cancers for over a decade. Previous drug design has focused on inhibitors targeting the active site of PTP1B. However, this has not been successful because the active site is positively charged and conserved among the protein tyrosine phosphatases. Therefore, it is important to develop PTP1B inhibitors with alternative inhibitory strategies. Using computational studies including molecular docking, molecular dynamics simulations, and binding free energy calculations, we found that lupane triterpenes selectively inhibited PTP1B by targeting its more hydrophobic and less conserved allosteric site. These findings were verified using two enzymatic assays. Furthermore, the cell culture studies showed that lupeol and betulinic acid inhibited the PTP1B activity stimulated by TNFα in neurons. Our study indicates that lupane triterpenes are selective PTP1B allosteric inhibitors with significant potential for treating those diseases with elevated PTP1B activity. PMID:26865097

  7. Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain.

    PubMed

    Kim, Duk-Joong; Choi, Chang-Ki; Lee, Chan-Soo; Park, Mee-Hee; Tian, Xizhe; Kim, Nam Doo; Lee, Kee-In; Choi, Joong-Kwon; Ahn, Jin Hee; Shin, Eun-Young; Shin, Injae; Kim, Eung-Gook

    2016-01-01

    p21-activated kinases (PAKs) are key regulators of actin dynamics, cell proliferation and cell survival. Deregulation of PAK activity contributes to the pathogenesis of various human diseases, including cancer and neurological disorders. Using an ELISA-based screening protocol, we identified naphtho(hydro)quinone-based small molecules that allosterically inhibit PAK activity. These molecules interfere with the interactions between the p21-binding domain (PBD) of PAK1 and Rho GTPases by binding to the PBD. Importantly, they inhibit the activity of full-length PAKs and are selective for PAK1 and PAK3 in vitro and in living cells. These compounds may potentially be useful for determining the details of the PAK signaling pathway and may also be used as lead molecules in the development of more selective and potent PAK inhibitors. PMID:27126178

  8. Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain

    PubMed Central

    Kim, Duk-Joong; Choi, Chang-Ki; Lee, Chan-Soo; Park, Mee-Hee; Tian, Xizhe; Kim, Nam Doo; Lee, Kee-In; Choi, Joong-Kwon; Ahn, Jin Hee; Shin, Eun-Young; Shin, Injae; Kim, Eung-Gook

    2016-01-01

    p21-activated kinases (PAKs) are key regulators of actin dynamics, cell proliferation and cell survival. Deregulation of PAK activity contributes to the pathogenesis of various human diseases, including cancer and neurological disorders. Using an ELISA-based screening protocol, we identified naphtho(hydro)quinone-based small molecules that allosterically inhibit PAK activity. These molecules interfere with the interactions between the p21-binding domain (PBD) of PAK1 and Rho GTPases by binding to the PBD. Importantly, they inhibit the activity of full-length PAKs and are selective for PAK1 and PAK3 in vitro and in living cells. These compounds may potentially be useful for determining the details of the PAK signaling pathway and may also be used as lead molecules in the development of more selective and potent PAK inhibitors. PMID:27126178

  9. Analysis of ligand binding and protein dynamics of human retinoid X receptor alpha ligand-binding domain by nuclear magnetic resonance.

    PubMed

    Lu, Jianyun; Cistola, David P; Li, Ellen

    2006-02-14

    Retinoid X receptors (RXRs) are nuclear receptors that can activate transcription as homodimers or as obligate heterodimeric partners of other nuclear receptors. While the crystal structures of the RXR ligand-binding domains (LBD) have been previously determined, the dynamics of activation is less well characterized at an atomic level. To probe the effect of ligand binding on RXR LBD dynamics, we initiated nuclear magnetic resonance studies of recombinant human RXRalpha LBD (T223-T462) with and without bound 9-cis-retinoic acid (9cRA). The 1HN, 15N, 13C(alpha), 13CO, and 13C(beta) resonance assignments were established for 164 of 240 residues in apo-RXRalpha LBD. Resonances corresponding to an additional 47 residues emerged upon 9cRA binding. These additional residues included those located in the vicinity of the ligand-binding pocket (helices H3, H5, and strands S1, S2), as well as residues located at the dimerization interface (helices H9 and H10). Thus 9cRA binding stabilized the ligand-binding pocket and had allosteric effects on the dimerization interface. Ligand-induced chemical shift perturbations outside the binding cavity were mapped to helix H3 and the AF-2 helix H12, indicating conformational changes in these regions. However, helix H11, a component of the tetramerization interface, and a large part of helix H10, a component of the dimerization interface, remained undetectable even after 9cRA binding. Although apo- and holo-hRXRalpha LBD existed predominantly as homodimers in solution, exchange between monomeric, dimeric, and tetrameric forms of the protein could have contributed to line broadening of cross-peaks corresponding to helices H10 and H11. 15N T1, T2, and steady-state {1H}-15N NOE data collected at 500 and 700 MHz static magnetic fields showed that the internal motions for the residues in the H1-H3 loop (K245-D263) were much less restricted than those in the protein core for both apo- and holo-forms. Significant exchange R(ex) contributions to

  10. Binding of cGMP to both allosteric sites of cGMP-binding cGMP-specific phosphodiesterase (PDE5) is required for its phosphorylation.

    PubMed Central

    Turko, I V; Francis, S H; Corbin, J D

    1998-01-01

    cGMP-binding phosphodiesterases contain two homologous allosteric cGMP-binding sites (sites a and b) that are arranged in tandem; they constitute a superfamily of mammalian cyclic nucleotide receptors distinct from the cyclic nucleotide-dependent protein kinases/cation channels family. The functional role of each of these two sites in the phosphodiesterases is not known. The cGMP-binding sites of one of these phosphodiesterases, the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE, PDE5), have been analysed by using site-directed mutagenesis. Mutations that affect cGMP binding to either one or both allosteric sites do not influence cGMP hydrolysis in the catalytic site under the conditions used. However, compared with wild-type enzyme, the D289A, D478A and D289A/D478A mutants, which are defective in cGMP binding to either site a or site b, or both allosteric sites, require much higher cGMP concentrations for the allosteric stimulation of phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. The cGMP effect is on the cGB-PDE rather than on the catalytic subunit of the protein kinase because the latter enzyme does not require cGMP for activity. The D289N mutant, which has higher binding affinity for cGMP than does the wild-type enzyme, is phosphorylated at lower concentrations of cGMP than is the wild-type enzyme. It is concluded that cGMP binding to the allosteric sites of cGB-PDE does not directly affect catalysis, but binding to both of these sites regulates phosphorylation of this enzyme. PMID:9445376

  11. Combining quantum mechanical ligand conformation analysis and protein modeling to elucidate GPCR-ligand binding modes.

    PubMed

    Schultes, Sabine; Engelhardt, Harald; Roumen, Luc; Zuiderveld, Obbe P; Haaksma, Eric E J; de Esch, Iwan J P; Leurs, Rob; de Graaf, Chris

    2013-01-01

    SAR beyond protein-ligand interactions: By combining structure-affinity relationships, protein-ligand modeling studies, and quantum mechanical calculations, we show that ligand conformational energies and basicity play critical roles in ligand binding to the histamine H4 receptor, a GPCR that plays a key role in inflammation. PMID:23161844

  12. Identification of a Highly Conserved Allosteric Binding Site on Mnk1 and Mnk2.

    PubMed

    Basnet, Sunita K C; Diab, Sarah; Schmid, Raffaella; Yu, Mingfeng; Yang, Yuchao; Gillam, Todd Alexander; Teo, Theodosia; Li, Peng; Peat, Tom; Albrecht, Hugo; Wang, Shudong

    2015-11-01

    Elevated levels of phosphorylated eukaryotic initiation factor 4E (eIF4E) have been implicated in many tumor types, and mitogen activated protein kinase-interacting kinases (Mnks) are the only known kinases that phosphorylate eIF4E at Ser209. The phosphorylation of eIF4E is essential for oncogenic transformation but is of no significance to normal growth and development. Pharmacological inhibition of Mnks therefore provides a nontoxic and effective strategy for cancer therapy. However, a lack of specific Mnk inhibitors has confounded pharmacological target validation and clinical development. Herein, we report the identification of a novel series of Mnk inhibitors and their binding modes. A systematic workflow has been established to distinguish between type III and type I/II inhibitors. A selection of 66 compounds was tested for Mnk1 and Mnk2 inhibition, and 9 out of 20 active compounds showed type III interaction with an allosteric site of the proteins. Most of the type III inhibitors exhibited dual Mnk1 and Mnk2 activities and demonstrated potent antiproliferative properties against the MV4-11 acute myeloid leukemia cell line. Interestingly, ATP-/substrate-competitive inhibitors were found to be highly selective for Mnk2, with little or no activity for Mnk1. Our study suggests that Mnk1 and Mnk2 share a common structure of the allosteric inhibitory binding site but possess different structural features of the ATP catalytic domain. The findings will assist in the future design and development of Mnk targeted anticancer therapeutics. PMID:26268528

  13. Fluorescence study of ligand binding to potato tuber pyrophosphate-dependent phosphofructokinase: evidence for competitive binding between fructose-1,6-bisphosphate and fructose-2,6-bisphosphate.

    PubMed

    Podestá, Florencio E; Plaxton, William C

    2003-06-01

    The intrinsic fluorescence of potato tuber pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) was used as an indicator of conformational changes due to ligand binding. Binding of the substrates and the allosteric activator fructose-2,6-bisphosphate was quantitatively compared to their respective kinetic effects on enzymatic activity. PFP exhibited a relatively high affinity for its isolated substrates, relative to the enzyme's respective K(m) (substrate) values. There are two distinct types of fructose-1,6-bisphosphate interaction with PFP, corresponding to catalytic and activatory binding. Activatory fructose-1,6-bisphosphate binding shares several characteristics with fructose-2,6-bisphosphate binding, indicating that both ligands compete for the same allosteric activator site. Activation by fructose-1,6-bisphosphate or fructose-2,6-bisphosphate was exerted primarily on the forward (glycolytic) reaction by greatly increasing the enzyme's affinity for fructose-6-phosphate. Binding of substrates and effectors to PFP and PFP kinetic properties were markedly influenced by assay pH. Results indicate an increased glycolytic role for PFP during cytosolic acidification that accompanies anoxia stress. PMID:12745260

  14. Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non-competitive allosteric inhibitor

    PubMed Central

    Bertini, R; Barcelos, LS; Beccari, AR; Cavalieri, B; Moriconi, A; Bizzarri, C; Di Benedetto, P; Di Giacinto, C; Gloaguen, I; Galliera, E; Corsi, MM; Russo, RC; Andrade, SP; Cesta, MC; Nano, G; Aramini, A; Cutrin, JC; Locati, M; Allegretti, M; Teixeira, MM

    2012-01-01

    BACKGROUND AND PURPOSE DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. EXPERIMENTAL APPROACH The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [35S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation. KEY RESULTS A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys99 on CXCR1 and the non-conserved residue Asp293 on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury. CONCLUSION AND IMPLICATIONS DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases. PMID:21718305

  15. Structural Basis of the Lactate-dependent Allosteric Regulation of Oxygen Binding in Arthropod Hemocyanin

    SciTech Connect

    Hirota, S.; Tanaka, N; Micetic, I; Di Muro, P; Nagao, S; Kitagishi, H; Magliozzo, R; Peisach, J; Beltramini, M; Bubacco, L

    2010-01-01

    Hemocyanin (Hc) is an oxygen carrier protein in which oxygen binding is regulated by allosteric effectors such as H{sup +} and L-lactate. Isothermal titration calorimetric measurements showed that L-lactate binds to dodecameric and heterohexameric Hc and to the CaeSS3 homohexamer but not to the CaeSS2 monomer. The binding of lactate caused no change in the optical absorption and x-ray absorption spectra of either oxy- or deoxy-Hc, suggesting that no structural rearrangement of the active site occurred. At pH 6.5, the oxygen binding rate constant k{sub obs} obtained by flash photolysis showed a significant increase upon addition of L-lactate, whereas L-lactate addition had little effect at pH 8.3. Lactate binding caused a concentration-dependent shift in the interhexameric distances at pH 6.5 based on small angle x-ray scattering measurements. These results show that L-lactate affects oxygen affinity at pH 6.5 by modulating the global structure of Hc without affecting its binuclear copper center (the active site). In contrast to this, the active site structure of deoxy-Hc is affected by changes in pH (Hirota, S., Kawahara, T., Beltramini, M., Di Muro, P., Magliozzo, R. S., Peisach, J., Powers, L. S., Tanaka, N., Nagao, S., and Bubacco, L. (2008) J. Biol. Chem. 283, 31941-31948). Upon addiction of lactate, the kinetic behavior of oxygen rebinding for Hc was heterogeneous under low oxygen concentrations at pH 6.5 due to changes in the T and R state populations, and the equilibrium was found to shift from the T toward the R state with addition of lactate.

  16. pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins

    PubMed Central

    Gorfe, Alemayehu A.

    2015-01-01

    Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation. PMID:26506102

  17. Allosteric communication between DNA-binding and light-responsive domains of diatom class I aureochromes

    PubMed Central

    Banerjee, Ankan; Herman, Elena; Serif, Manuel; Maestre-Reyna, Manuel; Hepp, Sebastian; Pokorny, Richard; Kroth, Peter G.; Essen, Lars-Oliver; Kottke, Tilman

    2016-01-01

    The modular architecture of aureochrome blue light receptors, found in several algal groups including diatoms, is unique by having the LOV-type photoreceptor domain fused to the C-terminus of its putative effector, an N-terminal DNA-binding bZIP module. The structural and functional understanding of aureochromes’ light-dependent signaling mechanism is limited, despite their promise as an optogenetic tool. We show that class I aureochromes 1a and 1c from the diatom Phaeodactylum tricornutum are regulated in a light-independent circadian rhythm. These aureochromes are capable to form functional homo- and heterodimers, which recognize the ACGT core sequence within the canonical ‘aureo box’, TGACGT, in a light-independent manner. The bZIP domain holds a more folded and less flexible but extended conformation in the duplex DNA-bound state. FT-IR spectroscopy in the absence and the presence of DNA shows light-dependent helix unfolding in the LOV domain, which leads to conformational changes in the bZIP region. The solution structure of DNA bound to aureochrome points to a tilted orientation that was further validated by molecular dynamics simulations. We propose that aureochrome signaling relies on an allosteric pathway from LOV to bZIP that results in conformational changes near the bZIP-DNA interface without major effects on the binding affinity. PMID:27179025

  18. Allosteric inhibition of a stem cell RNA-binding protein by an intermediary metabolite

    PubMed Central

    Clingman, Carina C; Deveau, Laura M; Hay, Samantha A; Genga, Ryan M; Shandilya, Shivender MD; Massi, Francesca; Ryder, Sean P

    2014-01-01

    Gene expression and metabolism are coupled at numerous levels. Cells must sense and respond to nutrients in their environment, and specialized cells must synthesize metabolic products required for their function. Pluripotent stem cells have the ability to differentiate into a wide variety of specialized cells. How metabolic state contributes to stem cell differentiation is not understood. In this study, we show that RNA-binding by the stem cell translation regulator Musashi-1 (MSI1) is allosterically inhibited by 18–22 carbon ω-9 monounsaturated fatty acids. The fatty acid binds to the N-terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. We identify stearoyl-CoA desaturase-1 as a MSI1 target, revealing a feedback loop between ω-9 fatty acid biosynthesis and MSI1 activity. We propose that other RRM proteins could act as metabolite sensors to couple gene expression changes to physiological state. DOI: http://dx.doi.org/10.7554/eLife.02848.001 PMID:24935936

  19. Allosteric communication between DNA-binding and light-responsive domains of diatom class I aureochromes.

    PubMed

    Banerjee, Ankan; Herman, Elena; Serif, Manuel; Maestre-Reyna, Manuel; Hepp, Sebastian; Pokorny, Richard; Kroth, Peter G; Essen, Lars-Oliver; Kottke, Tilman

    2016-07-01

    The modular architecture of aureochrome blue light receptors, found in several algal groups including diatoms, is unique by having the LOV-type photoreceptor domain fused to the C-terminus of its putative effector, an N-terminal DNA-binding bZIP module. The structural and functional understanding of aureochromes' light-dependent signaling mechanism is limited, despite their promise as an optogenetic tool. We show that class I aureochromes 1a and 1c from the diatom Phaeodactylum tricornutum are regulated in a light-independent circadian rhythm. These aureochromes are capable to form functional homo- and heterodimers, which recognize the ACGT core sequence within the canonical 'aureo box', TGACGT, in a light-independent manner. The bZIP domain holds a more folded and less flexible but extended conformation in the duplex DNA-bound state. FT-IR spectroscopy in the absence and the presence of DNA shows light-dependent helix unfolding in the LOV domain, which leads to conformational changes in the bZIP region. The solution structure of DNA bound to aureochrome points to a tilted orientation that was further validated by molecular dynamics simulations. We propose that aureochrome signaling relies on an allosteric pathway from LOV to bZIP that results in conformational changes near the bZIP-DNA interface without major effects on the binding affinity. PMID:27179025

  20. Diazepam-bound GABAA receptor models identify new benzodiazepine binding-site ligands

    PubMed Central

    Richter, Lars; de Graaf, Chris; Sieghart, Werner; Varagic, Zdravko; Mörzinger, Martina; de Esch, Iwan J P; Ecker, Gerhard F; Ernst, Margot

    2012-01-01

    Benzodiazepines exert their anxiolytic, anticonvulsant, muscle-relaxant and sedative-hypnotic properties by allosterically enhancing the action of GABA at GABAA receptors via their benzodiazepine-binding site. Although these drugs have been used clinically since 1960, the molecular basis of this interaction is still not known. By using multiple homology models and an un biased docking protocol, we identified a binding hypothesis for the diazepam-bound structure of the benzodiazepine site, which was confirmed by experimental evidence. Moreover, two independent virtual screening approaches based on this structure identified known benzodiazepine-site ligands from different structural classes and predicted potential new ligands for this site. Receptor-binding assays and electrophysiological studies on recombinant receptors confirmed these predictions and thus identified new chemotypes for the benzodiazepine-binding site. Our results support the validity of the diazepam-bound structure of the benzodiazepine-binding pocket, demonstrate its suitability for drug discovery and pave the way for structure-based drug design. PMID:22446838

  1. Plasmon resonance enhanced mechanical detection of ligand binding

    SciTech Connect

    Ariyaratne, Amila; Zocchi, Giovanni

    2015-01-05

    Small molecule binding to the active site of enzymes typically modifies the mechanical stiffness of the enzyme. We exploit this effect, in a setup which combines nano-mechanics and surface plasmon resonance (SPR) enhanced optics, for the label free detection of ligand binding to an enzyme. The large dynamic range of the signal allows to easily obtain binding curves for small ligands, in contrast to traditional SPR methods which rely on small changes in index of refraction. Enzyme mechanics, assessed by nano-rheology, thus emerges as an alternative to electronic and spin resonances, assessed by traditional spectroscopies, for detecting ligand binding.

  2. Paramagnetic Ligand Tagging To Identify Protein Binding Sites

    PubMed Central

    2015-01-01

    Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein–ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein–ligand systems with varying binding affinity. PMID:26289584

  3. An Allosteric Pathway Revealed in the Ribosome Binding Stress Factor BipA

    SciTech Connect

    Makanji, H.; deLivron, M; Robinson, V

    2009-01-01

    BipA is a highly conserved prokaryotic GTPase that functions as a master regulator of stress and virulence processes in bacteria. It is a member of the translational factor family of GTPases along with EF-G, IF-2 and LepA. Structural and biochemical data suggest that ribosome binding specificity for each member of this family lies in an effector domain. As with other bacterial GTPases, the ribosome binding and GTPase activities of this protein are tightly coupled. However, the mechanism by which this occurs is still unknown. A series of experiments have been designed to probe structural features of the protein to see if we can pinpoint specific areas of BipA, perhaps even individual residues, which are important to its association with the ribosome. Included in the list are the C-terminal effector domain of the protein, which is distinct to the BipA family of proteins, and amino acid residues in the switch I and II regions of the G domain. Using sucrose density gradients, we have shown that the C-terminal domain is required in order for BipA to bind to the ribosome. Moreover, deletion of this domain increases the GTP hydrolysis rates of the protein, likely through relief of inhibitory contacts. Additional evidence has revealed an allosteric connection between the conformationally flexible switch II region and the C-terminal domain of BipA. Site directed mutagenesis, sucrose gradients and malachite green assays are being used to elucidate the details of this coupling.

  4. Allosteric Coupling between the Intracellular Coupling Helix 4 and Regulatory Sites of the First Nucleotide-binding Domain of CFTR

    PubMed Central

    Dawson, Jennifer E.; Farber, Patrick J.; Forman-Kay, Julie D.

    2013-01-01

    Cystic fibrosis is caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator), leading to folding and processing defects and to chloride channel gating misfunction. CFTR is regulated by ATP binding to its cytoplasmic nucleotide-binding domains, NBD1 and NBD2, and by phosphorylation of the NBD1 regulatory insert (RI) and the regulatory extension (RE)/R region. These regulatory effects are transmitted to the rest of the channel via NBD interactions with intracellular domain coupling helices (CL), particularly CL4. Using a sensitive method for detecting inter-residue correlations between chemical shift changes in NMR spectra, an allosteric network was revealed within NBD1, with a construct lacking RI. The CL4-binding site couples to the RI-deletion site and the C-terminal residues of NBD1 that precede the R region in full-length CFTR. Titration of CL4 peptide into NBD1 perturbs the conformational ensemble in these sites with similar titration patterns observed in F508del, the major CF-causing mutant, and in suppressor mutants F494N, V510D and Q637R NBD1, as well as in a CL4-NBD1 fusion construct. Reciprocally, the C-terminal mutation, Q637R, perturbs dynamics in these three sites. This allosteric network suggests a mechanism synthesizing diverse regulatory NBD1 interactions and provides biophysical evidence for the allosteric coupling required for CFTR function. PMID:24058550

  5. X-ray crystallographic analyses of symmetrical allosteric effectors of hemoglobin: compounds designed to link primary and secondary binding sites.

    PubMed

    Safo, Martin K; Boyiri, Telih; Burnett, James C; Danso-Danquah, Richmond; Moure, Carmen M; Joshi, Gajanan S; Abraham, Donald J

    2002-04-01

    The rational design and X-ray crystallographic analyses of two symmetrical allosteric effectors of hemoglobin (Hb) are reported. Compound design was directed by the previously solved co-crystal structure of one of the most potent allosteric effectors of Hb, 2-[4-[(3,5-dichlorophenylcarbamoyl)-methyl]-phenoxy]-2-methylpropionic acid (RSR4), which revealed two distinct binding sites for this compound in the Hb central water cavity. The primary binding site has been observed for all compounds of this structural class, which stabilize deoxy Hb by engaging in inter-dimer contacts with three of the four protein subunits. Interactions at the secondary binding site of RSR4 occur primarily between the beta(1) and beta(2) subunits and serve to further constrain the deoxy state. Based on these observations, it was hypothesized that compounds with the ability to simultaneously span and link both of these sites would possess increased potency, but at a lower molar concentration than RSR4. Two symmetrical compounds were designed and synthesized based on this hypothesis. The symmetrical effector approach was taken to minimize the number of compound orientations needed to successfully bind at either of the distinct allosteric sites. X-ray crystallographic analyses of these two effectors in complex with Hb revealed that they successfully spanned the RSR4 primary and secondary binding sites. However, the designed compounds interacted with the secondary binding site in such a way that intra-dimer, as opposed to inter-dimer, interactions were generated. In agreement with these observations, in vitro evaluation of the symmetrical effectors in Hb solution indicated that neither compound possessed the potency of RSR4. A detailed analysis of symmetrical effector-Hb contacts and comparisons with the binding contacts of RSR4 are discussed. PMID:11914488

  6. Structures of Two Melanoma-Associated Antigens Suggest Allosteric Regulation of Effector Binding

    PubMed Central

    Roos, Anette K.; Aitkenhead, Hazel; Oppermann, Udo C. T.; Cho, Hearn J.; Osman, Roman; Gileadi, Opher

    2016-01-01

    The MAGE (melanoma associated antigen) protein family are tumour-associated proteins normally present only in reproductive tissues such as germ cells of the testis. The human genome encodes over 60 MAGE genes of which one class (containing MAGE-A3 and MAGE-A4) are exclusively expressed in tumours, making them an attractive target for the development of targeted and immunotherapeutic cancer treatments. Some MAGE proteins are thought to play an active role in driving cancer, modulating the activity of E3 ubiquitin ligases on targets related to apoptosis. Here we determined the crystal structures of MAGE-A3 and MAGE-A4. Both proteins crystallized with a terminal peptide bound in a deep cleft between two tandem-arranged winged helix domains. MAGE-A3 (but not MAGE-A4), is predominantly dimeric in solution. Comparison of MAGE-A3 and MAGE-A3 with a structure of an effector-bound MAGE-G1 suggests that a major conformational rearrangement is required for binding, and that this conformational plasticity may be targeted by allosteric binders. PMID:26910052

  7. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

    PubMed

    Marsh, Lorraine

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function. PMID:26064949

  8. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites

    PubMed Central

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where “nonspecific” interactions contribute to biological function. PMID:26064949

  9. Exchange Kinetics of a Hydrophobic Ligand Binding Protein

    NASA Astrophysics Data System (ADS)

    Vaughn, Jeff; Stone, Martin

    2002-03-01

    Conformational fluctuations of proteins are thought to be important for determining the functional roles in biological activity. In some cases, the rates of these conformational changes may be directly correlated to, for example, the rates of catalysis or ligand binding. We are studying the role of conformational fluctuations in the binding of small volatile hydrophobic pheromones by the mouse major urinary proteins (MUPs). Communication among mice occurs, in part, with the MUP-1 protein. This urinary protein binds pheromones as a way to increase the longevity of the pheromone in an extracellular environment. Of interest is that the crystal structure of MUP-1 with a pheromone ligand shows the ligand to be completely occluded from the solvent with no obvious pathway to enter or exit. This suggests that conformational exchange of the protein may be required for ligand binding and release to occur. We hypothesize that the rate of conformational exchange may be a limiting factor determining the rate of ligand association and dissociation. By careful measurement of the on- and off-rates of ligand binding and the rates of conformational changes of the protein, a more defined picture of the interplay between protein structure and function can be obtained. To this end, heteronuclear saturation transfer, ^15N-exchange and ^15N dynamics experiments have been employed to probe the kinetics of ligand binding to MUP-1.

  10. Structural evidence for asymmetric ligand binding to transthyretin.

    PubMed

    Cianci, Michele; Folli, Claudia; Zonta, Francesco; Florio, Paola; Berni, Rodolfo; Zanotti, Giuseppe

    2015-08-01

    Human transthyretin (TTR) represents a notable example of an amyloidogenic protein, and several compounds that are able to stabilize its native state have been proposed as effective drugs in the therapy of TTR amyloidosis. The two thyroxine (T4) binding sites present in the TTR tetramer display negative binding cooperativity. Here, structures of TTR in complex with three natural polyphenols (pterostilbene, quercetin and apigenin) have been determined, in which this asymmetry manifests itself as the presence of a main binding site with clear ligand occupancy and related electron density and a second minor site with a much lower ligand occupancy. The results of an analysis of the structural differences between the two binding sites are consistent with such a binding asymmetry. The different ability of TTR ligands to saturate the two T4 binding sites of the tetrameric protein can be ascribed to the different affinity of ligands for the weaker binding site. In comparison, the high-affinity ligand tafamidis, co-crystallized under the same experimental conditions, was able to fully saturate the two T4 binding sites. This asymmetry is characterized by the presence of small but significant differences in the conformation of the cavity of the two binding sites. Molecular-dynamics simulations suggest the presence of even larger differences in solution. Competition binding assays carried out in solution revealed the presence of a preferential binding site in TTR for the polyphenols pterostilbene and quercetin that was different from the preferential binding site for T4. The TTR binding asymmetry could possibly be exploited for the therapy of TTR amyloidosis by using a cocktail of two drugs, each of which exhibits preferential binding for a distinct binding site, thus favouring saturation of the tetrameric protein and consequently its stabilization. PMID:26249340

  11. Development of a Highly Selective Allosteric Antagonist Radioligand for the Type 1 Cholecystokinin Receptor and Elucidation of Its Molecular Basis of Binding

    PubMed Central

    Dong, Maoqing; Vattelana, Ashton M.; Lam, Polo C.-H.; Orry, Andrew J.; Abagyan, Ruben; Christopoulos, Arthur; Sexton, Patrick M.; Haines, David R.

    2015-01-01

    Understanding the molecular basis of ligand binding to receptors provides insights useful for rational drug design. This work describes development of a new antagonist radioligand of the type 1 cholecystokinin receptor (CCK1R), (2-fluorophenyl)-2,3-dihydro-3-[(3-isoquinolinylcarbonyl)amino]-6-methoxy-2-oxo-l-H-indole-3-propanoate (T-0632), and exploration of the molecular basis of its binding. This radioligand bound specifically with high affinity within an allosteric pocket of CCK1R. T-0632 fully inhibited binding and action of CCK at this receptor, while exhibiting no saturable binding to the closely related type 2 cholecystokinin receptor (CCK2R). Chimeric CCK1R/CCK2R constructs were used to explore the molecular basis of T-0632 binding. Exchanging exonic regions revealed the functional importance of CCK1R exon 3, extending from the bottom of transmembrane segment (TM) 3 to the top of TM5, including portions of the intramembranous pocket as well as the second extracellular loop region (ECL2). However, CCK1R mutants in which each residue facing the pocket was changed to that present in CCK2R had no negative impact on T-0632 binding. Extending the chimeric approach to ECL2 established the importance of its C-terminal region, and site-directed mutagenesis of each nonconserved residue in this region revealed the importance of Ser208 at the top of TM5. A molecular model of T-0632-occupied CCK1R was consistent with these experimental determinants, also identifying Met121 in TM3 and Arg336 in TM6 as important. Although these residues are conserved in CCK2R, mutating them had a distinct impact on the two closely related receptors, suggesting differential orientation. This establishes the molecular basis of binding of a highly selective nonpeptidyl allosteric antagonist of CCK1R, illustrating differences in docking that extend beyond determinants attributable to distinct residues lining the intramembranous pocket in the two receptor subtypes. PMID:25319540

  12. Development of a highly selective allosteric antagonist radioligand for the type 1 cholecystokinin receptor and elucidation of its molecular basis of binding.

    PubMed

    Dong, Maoqing; Vattelana, Ashton M; Lam, Polo C-H; Orry, Andrew J; Abagyan, Ruben; Christopoulos, Arthur; Sexton, Patrick M; Haines, David R; Miller, Laurence J

    2015-01-01

    Understanding the molecular basis of ligand binding to receptors provides insights useful for rational drug design. This work describes development of a new antagonist radioligand of the type 1 cholecystokinin receptor (CCK1R), (2-fluorophenyl)-2,3-dihydro-3-[(3-isoquinolinylcarbonyl)amino]-6-methoxy-2-oxo-l-H-indole-3-propanoate (T-0632), and exploration of the molecular basis of its binding. This radioligand bound specifically with high affinity within an allosteric pocket of CCK1R. T-0632 fully inhibited binding and action of CCK at this receptor, while exhibiting no saturable binding to the closely related type 2 cholecystokinin receptor (CCK2R). Chimeric CCK1R/CCK2R constructs were used to explore the molecular basis of T-0632 binding. Exchanging exonic regions revealed the functional importance of CCK1R exon 3, extending from the bottom of transmembrane segment (TM) 3 to the top of TM5, including portions of the intramembranous pocket as well as the second extracellular loop region (ECL2). However, CCK1R mutants in which each residue facing the pocket was changed to that present in CCK2R had no negative impact on T-0632 binding. Extending the chimeric approach to ECL2 established the importance of its C-terminal region, and site-directed mutagenesis of each nonconserved residue in this region revealed the importance of Ser(208) at the top of TM5. A molecular model of T-0632-occupied CCK1R was consistent with these experimental determinants, also identifying Met(121) in TM3 and Arg(336) in TM6 as important. Although these residues are conserved in CCK2R, mutating them had a distinct impact on the two closely related receptors, suggesting differential orientation. This establishes the molecular basis of binding of a highly selective nonpeptidyl allosteric antagonist of CCK1R, illustrating differences in docking that extend beyond determinants attributable to distinct residues lining the intramembranous pocket in the two receptor subtypes. PMID:25319540

  13. Exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production.

    PubMed

    Geng, Feng; Chen, Zhen; Zheng, Ping; Sun, Jibin; Zeng, An-Ping

    2013-03-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyzes the first committed reaction of L-lysine biosynthesis in bacteria and plants and is allosterically regulated by L-lysine. In previous studies, DHDPSs from different species were proved to have different sensitivity to L-lysine inhibition. In this study, we investigated the key determinants of feedback regulation between two industrially important DHDPSs, the L-lysine-sensitive DHDPS from Escherichia coli and L-lysine-insensitive DHDPS from Corynebacterium glutamicum, by sequence and structure comparisons and site-directed mutation. Feedback inhibition of E. coli DHDPS was successfully alleviated after substitution of the residues around the inhibitor's binding sites with those of C. glutamicum DHDPS. Interestingly, mutagenesis of the lysine binding sites of C. glutamicum DHDPS according to E. coli DHDPS did not recover the expected feedback inhibition but an activation of DHDPS by L-lysine, probably due to differences in the allosteic signal transduction in the DHDPS of these two organisms. Overexpression of L-lysine-insensitive E. coli DHDPS mutants in E. coli MG1655 resulted in an improvement of L-lysine production yield by 46 %. PMID:22644522

  14. Regulation of the extracellular ligand binding activity of integrins.

    PubMed

    Fernandez, C; Clark, K; Burrows, L; Schofield, N R; Humphries, M J

    1998-07-01

    Integrins are a large heterodimeric family of cell surface adhesion receptors that bind extracellular matrix and cell surface ligands. The extracellular ligand binding activity of integrins is a dynamic and highly regulated event involving the induction of conformational changes within the integrin structure. The adhesive properties of integrins can be controlled by altering the activation state of the integrin, either through conformational change or receptor clustering, using mechanisms that are regulated by intracellular proteins. In this review, we will discuss what is currently known about integrin structure and the ligand binding sites present within the receptor. In addition, the mechanisms by which the ligand binding event is regulated through conformational change will be addressed, and the potential role of intracellular cytoplasmic proteins will be discussed. PMID:9637803

  15. A Correlation between Protein Function and Ligand Binding Profiles

    PubMed Central

    Shortridge, Matthew D.; Bokemper, Michael; Copeland, Jennifer C.; Stark, Jaime L.; Powers, Robert

    2011-01-01

    We report that proteins with the same function bind the same set of small molecules from a standardized chemical library. This observation led to a quantifiable and rapidly adaptable method for protein functional analysis using experimentally-derived ligand binding profiles. Ligand binding is measured using a high-throughput NMR ligand affinity screen with a structurally diverse chemical library. The method was demonstrated using a set of 19 proteins with a range of functions. A statistically significant similarity in ligand binding profiles was only observed between the two functionally identical albumins and between the five functionally similar amylases. This new approach is independent of sequence, structure or evolutionary information, and therefore, extends our ability to analyze and functionally annotate novel genes. PMID:21366353

  16. Identification of Soft Matter Binding Peptide Ligands Using Phage Display.

    PubMed

    Günay, Kemal Arda; Klok, Harm-Anton

    2015-10-21

    Phage display is a powerful tool for the selection of highly affine, short peptide ligands. While originally primarily used for the identification of ligands to proteins, the scope of this technique has significantly expanded over the past two decades. Phage display nowadays is also increasingly applied to identify ligands that selectively bind with high affinity to a broad range of other substrates including natural and biological polymers as well as a variety of low-molecular-weight organic molecules. Such peptides are of interest for various reasons. The ability to selectively and with high affinity bind to the substrate of interest allows the conjugation or immobilization of, e.g., nanoparticles or biomolecules, or generally, facilitates interactions at materials interfaces. On the other hand, presentation of peptide ligands that selectively bind to low-molecular-weight organic materials is of interest for the development of sensor surfaces. The aim of this article is to highlight the opportunities provided by phage display for the identification of peptide ligands that bind to synthetic or natural polymer substrates or to small organic molecules. The article will first provide an overview of the different peptide ligands that have been identified by phage display that bind to these "soft matter" targets. The second part of the article will discuss the different characterization techniques that allow the determination of the affinity of the identified ligands to the respective substrates. PMID:26275106

  17. Prediction of allosteric sites on protein surfaces with an elastic-network-model-based thermodynamic method

    NASA Astrophysics Data System (ADS)

    Su, Ji Guo; Qi, Li Sheng; Li, Chun Hua; Zhu, Yan Ying; Du, Hui Jing; Hou, Yan Xue; Hao, Rui; Wang, Ji Hua

    2014-08-01

    Allostery is a rapid and efficient way in many biological processes to regulate protein functions, where binding of an effector at the allosteric site alters the activity and function at a distant active site. Allosteric regulation of protein biological functions provides a promising strategy for novel drug design. However, how to effectively identify the allosteric sites remains one of the major challenges for allosteric drug design. In the present work, a thermodynamic method based on the elastic network model was proposed to predict the allosteric sites on the protein surface. In our method, the thermodynamic coupling between the allosteric and active sites was considered, and then the allosteric sites were identified as those where the binding of an effector molecule induces a large change in the binding free energy of the protein with its ligand. Using the proposed method, two proteins, i.e., the 70 kD heat shock protein (Hsp70) and GluA2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, were studied and the allosteric sites on the protein surface were successfully identified. The predicted results are consistent with the available experimental data, which indicates that our method is a simple yet effective approach for the identification of allosteric sites on proteins.

  18. Prediction of allosteric sites on protein surfaces with an elastic-network-model-based thermodynamic method.

    PubMed

    Su, Ji Guo; Qi, Li Sheng; Li, Chun Hua; Zhu, Yan Ying; Du, Hui Jing; Hou, Yan Xue; Hao, Rui; Wang, Ji Hua

    2014-08-01

    Allostery is a rapid and efficient way in many biological processes to regulate protein functions, where binding of an effector at the allosteric site alters the activity and function at a distant active site. Allosteric regulation of protein biological functions provides a promising strategy for novel drug design. However, how to effectively identify the allosteric sites remains one of the major challenges for allosteric drug design. In the present work, a thermodynamic method based on the elastic network model was proposed to predict the allosteric sites on the protein surface. In our method, the thermodynamic coupling between the allosteric and active sites was considered, and then the allosteric sites were identified as those where the binding of an effector molecule induces a large change in the binding free energy of the protein with its ligand. Using the proposed method, two proteins, i.e., the 70 kD heat shock protein (Hsp70) and GluA2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, were studied and the allosteric sites on the protein surface were successfully identified. The predicted results are consistent with the available experimental data, which indicates that our method is a simple yet effective approach for the identification of allosteric sites on proteins. PMID:25215770

  19. UMP kinase from Streptococcus pneumoniae: evidence for co-operative ATP binding and allosteric regulation

    PubMed Central

    2004-01-01

    UMP kinase catalyses the phosphorylation of UMP by ATP to yield UDP and ADP. In prokaryotes, the reaction is carried out by a hexameric enzyme, activated by GTP and inhibited by UTP. In the present study, Streptococcus pneumoniae UMP kinase was studied as a target for antibacterial research and its interest was confirmed by the demonstration of the essentiality of the gene for cell growth. In the presence of MnCl2 or MgCl2, the saturation kinetics of recombinant purified UMP kinase was hyperbolic for UMP (Km=0.1 mM) and sigmoidal for ATP (the substrate concentration at half-saturation S0.5=9.4±0.7 mM and n=1.9±0.1 in the presence of MgCl2). GTP increased the affinity for ATP and decreased the Hill coefficient (n). UTP decreased the affinity for ATP and only slightly increased the Hill coefficient. The kcat (175±13 s−1 in the presence of MgCl2) was not affected by the addition of GTP or UTP, whose binding site was shown to be different from the active site. The hydrodynamic radius of the protein similarly decreased in the presence of ATP or GTP. There was a shift in the pH dependence of the activity when the ATP concentration was switched from low to high. These results support the hypothesis of an allosteric transition from a conformation with low affinity for ATP to a form with high affinity, which would be induced by the presence of ATP or GTP. PMID:15324307

  20. DISTINCT ROLES OF β1 MIDAS, ADMIDAS AND LIMBS CATION-BINDING SITES IN LIGAND RECOGNITION BY INTEGRIN α2β1*

    PubMed Central

    Valdramidou, Dimitra; Humphries, Martin J.; Mould, A. Paul

    2012-01-01

    Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as α2β1, ligand recognition takes place exclusively at the α subunit I domain. However, activation of the αI domain depends on its interaction with a structurally similar domain in the β subunit known as the I-like or βI domain. The top face of the βI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS) and LIMBS (ligand-associated metal binding site). The role of these sites in controlling ligand binding to the αI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to α2β1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating mAb TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between αI and βI whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of βI. An activating mutation in the α2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca2+, Mg2+ and Mn2+ on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn2+ stimulates ligand binding, whereas the LIMBS is a stimulatory Ca2+-binding site, occupancy of which increases the affinity of Mg2+ for the MIDAS. PMID:18820259

  1. Metal ion and ligand binding of integrin α5β1

    PubMed Central

    Xia, Wei; Springer, Timothy A.

    2014-01-01

    Integrin α5β1 binds to an Arg–Gly–Asp (RGD) motif in its ligand fibronectin. We report high-resolution crystal structures of a four-domain α5β1 headpiece fragment, alone or with RGD peptides soaked into crystals, and RGD peptide affinity measurements. The headpiece crystallizes in a closed conformation essentially identical to that seen previously for α5β1 complexed with a Fab that allosterically inhibits ligand binding by stabilizing the closed conformation. Soaking experiments show that binding of cyclic RGD peptide with 20-fold higher affinity than a linear RGD peptide induces conformational change in the β1-subunit βI domain to a state that is intermediate between closed (low affinity) and open (high affinity). In contrast, binding of a linear RGD peptide induces no shape shifting. However, linear peptide binding induces shape shifting when Ca2+ is depleted during soaking. Ca2+ bound to the adjacent to metal ion-dependent adhesion site (ADMIDAS), at the locus of shape shifting, moves and decreases in occupancy, correlating with an increase in affinity for RGD measured when Ca2+ is depleted. The results directly demonstrate that Ca2+ binding to the ADMIDAS stabilizes integrins in the low-affinity, closed conformation. Comparisons in affinity between four-domain and six-domain headpiece constructs suggest that flexible integrin leg domains contribute to conformational equilibria. High-resolution views of the hybrid domain interface with the plexin–semaphorin–integrin (PSI) domain in different orientations show a ball-and-socket joint with a hybrid domain Arg side chain that rocks in a PSI domain socket lined with carbonyl oxygens. PMID:25475857

  2. Metal ion and ligand binding of integrin α5β1.

    PubMed

    Xia, Wei; Springer, Timothy A

    2014-12-16

    Integrin α5β1 binds to an Arg-Gly-Asp (RGD) motif in its ligand fibronectin. We report high-resolution crystal structures of a four-domain α5β1 headpiece fragment, alone or with RGD peptides soaked into crystals, and RGD peptide affinity measurements. The headpiece crystallizes in a closed conformation essentially identical to that seen previously for α5β1 complexed with a Fab that allosterically inhibits ligand binding by stabilizing the closed conformation. Soaking experiments show that binding of cyclic RGD peptide with 20-fold higher affinity than a linear RGD peptide induces conformational change in the β1-subunit βI domain to a state that is intermediate between closed (low affinity) and open (high affinity). In contrast, binding of a linear RGD peptide induces no shape shifting. However, linear peptide binding induces shape shifting when Ca(2+) is depleted during soaking. Ca(2+) bound to the adjacent to metal ion-dependent adhesion site (ADMIDAS), at the locus of shape shifting, moves and decreases in occupancy, correlating with an increase in affinity for RGD measured when Ca(2+) is depleted. The results directly demonstrate that Ca(2+) binding to the ADMIDAS stabilizes integrins in the low-affinity, closed conformation. Comparisons in affinity between four-domain and six-domain headpiece constructs suggest that flexible integrin leg domains contribute to conformational equilibria. High-resolution views of the hybrid domain interface with the plexin-semaphorin-integrin (PSI) domain in different orientations show a ball-and-socket joint with a hybrid domain Arg side chain that rocks in a PSI domain socket lined with carbonyl oxygens. PMID:25475857

  3. Persistent Binding of Ligands to the Aryl Hydrocarbon Receptor

    PubMed Central

    Bohonowych, Jessica E.; Denison, Michael S.

    2010-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxic effects of halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), and other structurally diverse ligands. While HAHs are several orders of magnitude more potent in producing AhR-dependent biochemical effects than PAHs or other AhR agonists, only the HAHs have been observed to produce AhR-dependent toxicity in vivo. Here we have characterized the dissociation of a prototypical HAH ligand ([3H] 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) and PAH-like ligand ([3H] β-naphthoflavone [βNF]) from the guinea pig, hamster, mouse, and rat hepatic cytosolic AhR in order to elucidate the relationship between the apparent ligand-binding affinities and the divergent potency of these chemicals. Both compounds dissociated very slowly from the AhR with the amount of specific binding remaining at 96 h ranging from 53% to 70% for [3H]TCDD and 26% to 85% for [3H] βNF, depending upon the species examined. The rate of ligand dissociation was unaffected by protein concentration or incubation temperature. Preincubation of cytosol with 2,3,7,8-tetrachlorodibenzofuran, carbaryl, or primaquine, prior to the addition of [3H]TCDD, shifted the apparent IC50 of these compounds as competitive AhR ligands by ∼10- to 50-fold. Our results support the need for reassessment of previous AhR ligand-binding affinity calculations and competitive binding analysis since these measurements are not carried out at equilibrium binding conditions. Our studies suggest that AhR binding affinity/occupancy has little effect on the observed differences in the persistence of gene expression by HAHs and PAHs. PMID:17431010

  4. Multiple allosteric sites on muscarinic receptors.

    PubMed

    Birdsall, N J; Lazareno, S; Popham, A; Saldanha, J

    2001-04-27

    Proteins and small molecules are capable of regulating the agonist binding and function of G-protein coupled receptors by multiple allosteric mechanisms. In the case of muscarinic receptors, there is the well-characterised allosteric site that binds, for example, gallamine and brucine. The protein kinase inhibitor, KT5720, has now been shown to bind to a second allosteric site and to regulate agonist and antagonist binding. The binding of brucine and gallamine does not affect KT5720 binding nor its effects on the dissociation of [3H]-N-methylscopolamine from M1 receptors. Therefore it is possible to have a muscarinic receptor with three small ligands bound simultaneously. A model of the M1 receptor, based on the recently determined structure of rhodopsin, has the residues that have been shown to be important for gallamine binding clustered within and to one side of a cleft in the extracellular face of the receptor. This cleft may represent the access route of acetylcholine to its binding site. PMID:11392621

  5. Analyzing Ligand Depletion in a Saturation Equilibrium Binding Experiment

    ERIC Educational Resources Information Center

    Claro, Enrique

    2006-01-01

    I present a proposal for a laboratory practice to generate and analyze data from a saturation equilibrium binding experiment addressed to advanced undergraduate students. [[superscript 3]H]Quinuclidinyl benzilate is a nonselective muscarinic ligand with very high affinity and very low nonspecific binding to brain membranes, which contain a high…

  6. The kinetics of effector binding to phosphofructokinase. The allosteric conformational transition induced by 1,N6-ethenoadenosine triphosphate.

    PubMed Central

    Roberts, D; Kellett, G L

    1979-01-01

    1. The fluorescent ATP analogue 1,N6-etheno-ATP is a good substrate and an efficient allosteric inhibitor of rabbit skeletal-muscle phosphofructokinase. 2. Fluorescence energy transfer occurs between bound 1,N6-etheno-ATP and phosphofructokinase. 1,N6-Etheno-ATP fluorescence is enhanced, intrinsic protein fluorescence is quenched, and the excitation spectrum of 1,N6-etheno-ATP fluorescence is characteristic of protein absorption. 3. The binding reaction of 1,N6-etheno-ATP observed by stopped-flow fluorimetry is biphasic. The fast phase results from binding to the catalytic site alone. The slow phase results from the allosteric transition of the R conformation into the T conformation induced by the binding of 1,N6-etheno-ATP to the regulatory site. 4. The fluorescence signal that allows the transition of the R conformation into the T conformation to be observed does not arise from 1,N6-etheno-ATP bound to the regulatory site. It arises instead from 1,N6-etheno-ATP bound to the catalytic site as a consequence of changes at the catalytic site caused by the transition of the R conformation into the T conformation. 5. In the presence of excess of Mg2+, the affinity of 1,N6-etheno-ATP for the regulatory site is very much greater in the T state than in the R state. Images Fig. 5. Fig. 8. PMID:160791

  7. Calculation of Mg(+)-ligand relative binding energies

    NASA Technical Reports Server (NTRS)

    Partridge, Harry; Bauschlicher, Charles W., Jr.

    1992-01-01

    The calculated relative binding energies of 16 organic molecules to Mg(+) are compared with experimental results where available. The geometries of the ligands and the Mg(+)-ligand complexes arc optimized at the self-consistent field level using a 6-31G* basis set. The Mg(+) binding energies are evaluated using second-order perturbation theory and basis sets of triple-sigma quality augmented with two sets of polarization functions. This level of theory is calibrated against higher levels of theory for selected systems. The computed binding energies are accurate to about 2 kcal/mol.

  8. Exploration of dimensions of estrogen potency: parsing ligand binding and coactivator binding affinities.

    PubMed

    Jeyakumar, M; Carlson, Kathryn E; Gunther, Jillian R; Katzenellenbogen, John A

    2011-04-15

    The estrogen receptors, ERα and ERβ, are ligand-regulated transcription factors that control gene expression programs in target tissues. The molecular events underlying estrogen action involve minimally two steps, hormone binding to the ER ligand-binding domain followed by coactivator recruitment to the ER·ligand complex; this ligand·receptor·coactivator triple complex then alters gene expression. Conceptually, the potency of an estrogen in activating a cellular response should reflect the affinities that characterize both steps involved in the assembly of the active ligand·receptor·coactivator complex. Thus, to better understand the molecular basis of estrogen potency, we developed a completely in vitro system (using radiometric and time-resolved FRET assays) to quantify independently three parameters: (a) the affinity of ligand binding to ER, (b) the affinity of coactivator binding to the ER·ligand complex, and (c) the potency of ligand recruitment of coactivator. We used this system to characterize the binding and potency of 12 estrogens with both ERα and ERβ. Some ligands showed good correlations between ligand binding affinity, coactivator binding affinity, and coactivator recruitment potency with both ERs, whereas others showed correlations with only one ER subtype or displayed discordant coactivator recruitment potencies. When ligands with low receptor binding affinity but high coactivator recruitment potencies to ERβ were evaluated in cell-based assays, elevation of cellular coactivator levels significantly and selectively improved their potency. Collectively, our results indicate that some low affinity estrogens may elicit greater cellular responses in those target cells that express higher levels of specific coactivators capable of binding to their ER complexes with high affinity. PMID:21321128

  9. Non-peptide ligand binding to the formyl peptide receptor FPR2--A comparison to peptide ligand binding modes.

    PubMed

    Stepniewski, Tomasz M; Filipek, Slawomir

    2015-07-15

    Ligands of the FPR2 receptor initiate many signaling pathways including activation of phospholipase C, protein kinase C, the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/protein kinase B pathway. The possible actions include also calcium flux, superoxide generation, as well as migration and proliferation of monocytes. FPR2 activation may induce a pro- and anti-inflammatory effect depending on the ligand type. It is also found that this receptor is involved in tumor growth. Most of currently known FPR2 ligands are agonists since they were designed based on N-formyl peptides, which are natural agonists of formyl receptors. Since the non-peptide drugs are indispensable for effective treatment strategies, we performed a docking study of such ligands employing a generated dual template homology model of the FPR2 receptor. The study revealed different binding modes of particular classes of these drugs. Based on the obtained docking poses we proposed a detailed location of three hydrophobic pockets in orthosteric binding site of FPR2. Our model emphasizes the importance of aromatic stacking, especially with regard to residues His102(3.29) and Phe257(6.51), for binding of FPR2 ligands. We also identified other residues important for non-peptide ligand binding in the binding site of FPR2. PMID:25882522

  10. ASD v3.0: unraveling allosteric regulation with structural mechanisms and biological networks

    PubMed Central

    Shen, Qiancheng; Wang, Guanqiao; Li, Shuai; Liu, Xinyi; Lu, Shaoyong; Chen, Zhongjie; Song, Kun; Yan, Junhao; Geng, Lv; Huang, Zhimin; Huang, Wenkang; Chen, Guoqiang; Zhang, Jian

    2016-01-01

    Allosteric regulation, the most direct and efficient way of regulating protein function, is induced by the binding of a ligand at one site that is topographically distinct from an orthosteric site. Allosteric Database (ASD, available online at http://mdl.shsmu.edu.cn/ASD) has been developed to provide comprehensive information featuring allosteric regulation. With increasing data, fundamental questions pertaining to allostery are currently receiving more attention from the mechanism of allosteric changes in an individual protein to the entire effect of the changes in the interconnected network in the cell. Thus, the following novel features were added to this updated version: (i) structural mechanisms of more than 1600 allosteric actions were elucidated by a comparison of site structures before and after the binding of an modulator; (ii) 261 allosteric networks were identified to unveil how the allosteric action in a single protein would propagate to affect downstream proteins; (iii) two of the largest human allosteromes, protein kinases and GPCRs, were thoroughly constructed; and (iv) web interface and data organization were completely redesigned for efficient access. In addition, allosteric data have largely expanded in this update. These updates are useful for facilitating the investigation of allosteric mechanisms, dynamic networks and drug discoveries. PMID:26365237

  11. ASD v3.0: unraveling allosteric regulation with structural mechanisms and biological networks.

    PubMed

    Shen, Qiancheng; Wang, Guanqiao; Li, Shuai; Liu, Xinyi; Lu, Shaoyong; Chen, Zhongjie; Song, Kun; Yan, Junhao; Geng, Lv; Huang, Zhimin; Huang, Wenkang; Chen, Guoqiang; Zhang, Jian

    2016-01-01

    Allosteric regulation, the most direct and efficient way of regulating protein function, is induced by the binding of a ligand at one site that is topographically distinct from an orthosteric site. Allosteric Database (ASD, available online at http://mdl.shsmu.edu.cn/ASD) has been developed to provide comprehensive information featuring allosteric regulation. With increasing data, fundamental questions pertaining to allostery are currently receiving more attention from the mechanism of allosteric changes in an individual protein to the entire effect of the changes in the interconnected network in the cell. Thus, the following novel features were added to this updated version: (i) structural mechanisms of more than 1600 allosteric actions were elucidated by a comparison of site structures before and after the binding of an modulator; (ii) 261 allosteric networks were identified to unveil how the allosteric action in a single protein would propagate to affect downstream proteins; (iii) two of the largest human allosteromes, protein kinases and GPCRs, were thoroughly constructed; and (iv) web interface and data organization were completely redesigned for efficient access. In addition, allosteric data have largely expanded in this update. These updates are useful for facilitating the investigation of allosteric mechanisms, dynamic networks and drug discoveries. PMID:26365237

  12. Ion-regulated allosteric binding of fullerenes (C60 and C70) by tetrathiafulvalene-calix[4]pyrroles.

    PubMed

    Davis, Christina M; Lim, Jong Min; Larsen, Karina R; Kim, Dong Sub; Sung, Young Mo; Lyons, Dani M; Lynch, Vincent M; Nielsen, Kent A; Jeppesen, Jan O; Kim, Dongho; Park, Jung Su; Sessler, Jonathan L

    2014-07-23

    The effect of ionic species on the binding of fullerenes (C60 and C70) by tetrathiafulvalene-calix[4]pyrrole (TTF-C4P) receptors and the nature of the resulting supramolecular complexes (TTF-C4P + fullerene + halide anion + tetraalkylammonium cation) was studied in the solid state through single crystal X-ray diffraction methods and in dichloromethane solution by means of continuous variation plots and UV-vis spectroscopic titrations. These analyses revealed a 1:1 stoichiometry between the anion-bound TTF-C4Ps and the complexed fullerenes. The latter guests are bound within the bowl-like cup of the C4P in a ball-and-socket binding mode. The interactions between the TTF-C4P receptors and the fullerene guests are highly influenced by both the nature of halide anions and their counter tetraalkylammonium cations. Three halides (F(-), Cl(-), and Br(-)) were studied. All three potentiate the binding of the two test fullerenes by inducing a conformational change from the 1,3-alternate to the cone conformer of the TTF-C4Ps, thus acting as positive heterotropic allosteric effectors. For a particular halide anion, the choice of tetraalkylammonium salts serves to modulate the strength of the TTF-C4P-fullerene host-guest binding interactions and, in conjunction with variations in the halide anion, can be exploited to alter the inherent selectivity of the host for a given fullerene. Differences in binding are reflected in the excited state optical properties. Overall, the present four-component system provides an illustration of how host-guest binding events involving appropriately designed artificial receptors can be fine-tuned via the addition of simple ionic species as allosteric modulators. PMID:24967663

  13. Trypsin-Ligand Binding Free Energy Calculation with AMOEBA

    PubMed Central

    Shi, Yue; Jiao, Dian; Schnieders, Michael J.; Ren, Pengyu

    2010-01-01

    The binding free energies of several benzamidine-like inhibitors to trypsin were examined using a polarizable potential. All the computed binding free energies are in good agreement with the experimental data. From free energy decomposition, electrostatic interaction was found to be the driving force for the binding. Structural analysis shows that the ligands form hydrogen bonds with residues and water molecules nearby in a competitive fashion. The dependence of binding free energy on molecular dipole moment and polarizability was also studied. While the binding free energy is independent on the dipole moment, it shows a negative correlation with the polarizability. PMID:19965178

  14. Spin exchange monitoring of the strong positive homotropic allosteric binding of a tetraradical by a synthetic receptor in water.

    PubMed

    Bardelang, David; Casano, Gilles; Poulhès, Florent; Karoui, Hakim; Filippini, Jessica; Rockenbauer, Antal; Rosas, Roselyne; Monnier, Valérie; Siri, Didier; Gaudel-Siri, Anouk; Ouari, Olivier; Tordo, Paul

    2014-12-17

    The flexible tetranitroxide 4T has been prepared and was shown to exhibit a nine line EPR spectrum in water, characteristic of significant through space spin exchange (J(ij)) between four electron spins interacting with four nitrogen nuclei (J(ij) ≫ a(N)). Addition of CB[8] to 4T decreases dramatically all the Jij couplings, and the nine line spectrum is replaced by the characteristic three line spectrum of a mononitroxide. The supramolecular association between 4T and CB[8] involves a highly cooperative asymmetric complexation by two CB[8] (K1 = 4027 M(-1); K2 = 202,800 M(-1); α = 201) leading to a rigid complex with remote nitroxide moieties. The remarkable enhancement for the affinity of the second CB[8] corresponds to an allosteric interaction energy of ≈13 kJ mol(-1), which is comparable to that of the binding of oxygen by hemoglobin. These results are confirmed by competition and reduction experiments, DFT and molecular dynamics calculations, mass spectrometry, and liquid state NMR of the corresponding reduced complex bearing hydroxylamine moieties. This study shows that suitably designed molecules can generate allosteric complexation with CB[8]. The molecule must (i) carry several recognizable groups for CB[8] and (ii) be folded so that the first binding event reorganizes the molecule (unfold) for a better subsequent recognition. The presence of accessible protonable amines and H-bond donors to fit with the second point are also further stabilizing groups of CB[8] complexation. In these conditions, the spin exchange coupling between four radicals has been efficiently and finely tuned and the resulting allosteric complexation induced a dramatic stabilization enhancement of the included paramagnetic moieties in highly reducing conditions through the formation of the supramolecular 4T@CB[8]2 complex. PMID:25418528

  15. An allosteric model for control of pore opening by substrate binding in the EutL microcompartment shell protein

    PubMed Central

    Thompson, Michael C; Cascio, Duilio; Leibly, David J; Yeates, Todd O

    2015-01-01

    The ethanolamine utilization (Eut) microcompartment is a protein-based metabolic organelle that is strongly associated with pathogenesis in bacteria that inhabit the human gut. The exterior shell of this elaborate protein complex is composed from a few thousand copies of BMC-domain shell proteins, which form a semi-permeable diffusion barrier that provides the interior enzymes with substrates and cofactors while simultaneously retaining metabolic intermediates. The ability of this protein shell to regulate passage of substrate and cofactor molecules is critical for microcompartment function, but the details of how this diffusion barrier can allow the passage of large cofactors while still retaining small intermediates remain unclear. Previous work has revealed two conformations of the EutL shell protein, providing substantial evidence for a gated pore that might allow the passage of large cofactors. Here we report structural and biophysical evidence to show that ethanolamine, the substrate of the Eut microcompartment, acts as a negative allosteric regulator of EutL pore opening. Specifically, a series of X-ray crystal structures of EutL from Clostridium perfringens, along with equilibrium binding studies, reveal that ethanolamine binds to EutL at a site that exists in the closed-pore conformation and which is incompatible with opening of the large pore for cofactor transport. The allosteric mechanism we propose is consistent with the cofactor requirements of the Eut microcompartment, leading to a new model for EutL function. Furthermore, our results suggest the possibility of redox modulation of the allosteric mechanism, opening potentially new lines of investigation. PMID:25752492

  16. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    PubMed

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  17. Using NMR to Develop New Allosteric and Allo-Network Drugs.

    PubMed

    Smith, Robert E; Tran, Kevin; Richards, Kristy M; Luo, Rensheng

    2015-01-01

    NMR is becoming an important tool for developing new allosteric and allo-network drugs that bind to allosteric sites on enzymes, partially inhibiting them and causing fewer side effects than drugs already developed that target active sites. This is based on systems thinking, in which active enzymes and other proteins are known to be flexible and interact with each other. In other words, proteins can exist in an ensemble of different conformations whose populations are tunable. NMR is being used to find the pathways through which the effects of binding of an allosteric ligand propagate. There are NMR screening assays for studying ligand binding. This includes determining the changes in the spin lattice relaxation due to changes in the mobility of atoms involved in the binding, measuring magnetization transfer from the protein to the ligand by saturation difference transfer NMR (STD-NMR) and the transfer of bulk magnetization to the ligand by water-Ligand Observed via Gradient Spectroscopy, or waterLOGSY. The chemical shifts of (1)H and (15)N of some of the atoms in amino acids change when an allosteric ligand binds to a protein. So, (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra can be used to identify key amino acids and ligand binding sites. The NMR chemical shifts of amino acids affected by ligand binding form a network that can be characterized. Allosteric networks can be identified by chemical shift covariance analysis (CHESCA). This approach has been used recently to study the binding of new molecular entities (NMEs) to potentially therapeutic drug targets. PMID:26577663

  18. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

    NASA Astrophysics Data System (ADS)

    Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

    2013-11-01

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  19. Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.

    PubMed

    Hamouda, Ayman K; Stewart, Deirdre S; Husain, S Shaukat; Cohen, Jonathan B

    2011-06-10

    Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 μM, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [(3)H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive

  20. Allosteric Coupling in the Bacterial Adhesive Protein FimH*

    PubMed Central

    Rodriguez, Victoria B.; Kidd, Brian A.; Interlandi, Gianluca; Tchesnokova, Veronika; Sokurenko, Evgeni V.; Thomas, Wendy E.

    2013-01-01

    The protein FimH is expressed by the majority of commensal and uropathogenic strains of Escherichia coli on the tips of type 1 fimbriae and mediates adhesion via a catch bond to its ligand mannose. Crystal structures of FimH show an allosteric conformational change, but it remains unclear whether all of the observed structural differences are part of the allosteric mechanism. Here we use the protein structural analysis tool RosettaDesign combined with human insight to identify and synthesize 10 mutations in four regions that we predicted would stabilize one of the conformations of that region. The function of each variant was characterized by measuring binding to the ligand mannose, whereas the allosteric state was determined using a conformation-specific monoclonal antibody. These studies demonstrated that each region investigated was indeed part of the FimH allosteric mechanism. However, the studies strongly suggested that some regions were more tightly coupled to mannose binding and others to antibody binding. In addition, we identified many FimH variants that appear locked in the low affinity state. Knowledge of regulatory sites outside the active and effector sites as well as the ability to make FimH variants locked in the low affinity state may be crucial to the future development of novel antiadhesive and antimicrobial therapies using allosteric regulation to inhibit FimH. PMID:23821547

  1. Structure of a small-molecule inhibitor complexed with GlmU from Haemophilus influenzae reveals an allosteric binding site

    SciTech Connect

    Mochalkin, Igor; Lightle, Sandra; Narasimhan, Lakshmi; Bornemeier, Dirk; Melnick, Michael; VanderRoest, Steven; McDowell, Laura

    2008-04-02

    N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and an attractive target for antibiotic drug discovery. GlmU catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolisaccharide biosynthesis in both Gram-negative and Gram-positive bacteria. Here we disclose a 1.9 {angstrom} resolution crystal structure of a synthetic small-molecule inhibitor of GlmU from Haemophilus influenzae (hiGlmU). The compound was identified through a high-throughput screening (HTS) configured to detect inhibitors that target the uridyltransferase active site of hiGlmU. The original HTS hit exhibited a modest micromolar potency (IC{sub 50} - 18 {mu}M in a racemic mixture) against hiGlmU and no activity against Staphylococcus aureus GlmU (saGlmU). The determined crystal structure indicated that the inhibitor occupies an allosteric site adjacent to the GlcNAc-1-P substrate-binding region. Analysis of the mechanistic model of the uridyltransferase reaction suggests that the binding of this allosteric inhibitor prevents structural rearrangements that are required for the enzymatic reaction, thus providing a basis for structure-guided design of a new class of mechanism-based inhibitors of GlmU.

  2. Application of BRET to monitor ligand binding to GPCRs

    PubMed Central

    Stoddart, Leigh A; Johnstone, Elizabeth K M; Wheal, Amanda J; Goulding, Joëlle; Robers, Matthew B; Machleidt, Thomas; Wood, Keith V

    2015-01-01

    Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a novel BRET approach to monitor ligand binding to G protein-coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a novel bioluminescent protein (NanoLuc) that can be readily expressed on the N-terminus of GPCRs. PMID:26030448

  3. Application of BRET to monitor ligand binding to GPCRs.

    PubMed

    Stoddart, Leigh A; Johnstone, Elizabeth K M; Wheal, Amanda J; Goulding, Joëlle; Robers, Matthew B; Machleidt, Thomas; Wood, Keith V; Hill, Stephen J; Pfleger, Kevin D G

    2015-07-01

    Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein-coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs. PMID:26030448

  4. Structural Diversity of Ligand-Binding Androgen Receptors Revealed by Microsecond Long Molecular Dynamics Simulations and Enhanced Sampling.

    PubMed

    Duan, Mojie; Liu, Na; Zhou, Wenfang; Li, Dan; Yang, Minghui; Hou, Tingjun

    2016-09-13

    Androgen receptor (AR) plays important roles in the development of prostate cancer (PCa). The antagonistic drugs, which suppress the activity of AR, are widely used in the treatment of PCa. However, the molecular mechanism of antagonism about how ligands affect the structures of AR remains elusive. To better understand the conformational variability of ARs bound with agonists or antagonists, we performed long time unbiased molecular dynamics (MD) simulations and enhanced sampling simulations for the ligand binding domain of AR (AR-LBD) in complex with various ligands. Based on the simulation results, we proposed an allosteric pathway linking ligands and helix 12 (H12) of AR-LBD, which involves the interactions among the ligands and the residues W741, H874, and I899. The interaction pathway provides an atomistic explanation of how ligands affect the structure of AR-LBD. A repositioning of H12 was observed, but it is facilitated by the C-terminal of H12, instead of by the loop between helix 11 (H11) and H12. The bias-exchange metadynamics simulations further demonstrated the above observations. More importantly, the free energy profiles constructed by the enhanced sampling simulations revealed the transition process between the antagonistic form and agonistic form of AR-LBD. Our results would be helpful for the design of more efficient antagonists of AR to combat PCa. PMID:27560203

  5. Synthesis and biological evaluation of negative allosteric modulators of the Kv11.1(hERG) channel.

    PubMed

    Yu, Zhiyi; van Veldhoven, Jacobus P D; 't Hart, Ingrid M E; Kopf, Adrian H; Heitman, Laura H; IJzerman, Adriaan P

    2015-12-01

    We synthesized and evaluated a series of compounds for their allosteric modulation at the Kv11.1 (hERG) channel. Most compounds were negative allosteric modulators of [(3)H]dofetilide binding to the channel, in particular 7f, 7h-j and 7p. Compounds 7f and 7p were the most potent negative allosteric modulators amongst all ligands, significantly increasing the dissociation rate of dofetilide in the radioligand kinetic binding assay, while remarkably reducing the affinities of dofetilide and astemizole in a competitive displacement assay. Additionally, both 7f and 7p displayed peculiar displacement characteristics with Hill coefficients significantly distinct from unity as shown by e.g., dofetilide, further indicative of their allosteric effects on dofetilide binding. Our findings in this investigation yielded several promising negative allosteric modulators for future functional and clinical research with respect to their antiarrhythmic propensities, either alone or in combination with known Kv11.1 blockers. PMID:26519929

  6. VEGFR-2 conformational switch in response to ligand binding

    PubMed Central

    Sarabipour, Sarvenaz; Ballmer-Hofer, Kurt; Hristova, Kalina

    2016-01-01

    VEGFR-2 is the primary regulator of angiogenesis, the development of new blood vessels from pre-existing ones. VEGFR-2 has been hypothesized to be monomeric in the absence of bound ligand, and to undergo dimerization and activation only upon ligand binding. Using quantitative FRET and biochemical analysis, we show that VEGFR-2 forms dimers also in the absence of ligand when expressed at physiological levels, and that these dimers are phosphorylated. Ligand binding leads to a change in the TM domain conformation, resulting in increased kinase domain phosphorylation. Inter-receptor contacts within the extracellular and TM domains are critical for the establishment of the unliganded dimer structure, and for the transition to the ligand-bound active conformation. We further show that the pathogenic C482R VEGFR-2 mutant, linked to infantile hemangioma, promotes ligand-independent signaling by mimicking the structure of the ligand-bound wild-type VEGFR-2 dimer. DOI: http://dx.doi.org/10.7554/eLife.13876.001 PMID:27052508

  7. Helix-helix interfaces and ligand binding.

    PubMed

    Kurochkina, Natalya; Choekyi, Tsering

    2011-08-21

    Helix-helix parallel interfaces can be characterized by certain combinations of amino acids, which repeatedly occur at core positions a and d (leucine zipper nomenclature) in homologous and nonhomologous proteins and influence interhelical angles. Applied for the prediction of interhelical angles in glutathione S-transferase, intracellular chloride channel and annexin molecules from various sources, correct results were achieved in 58 out of 62 proteins. Interhelical angles are found to correlate with the conformation of the glutathione S-transferase ligands glutathione, s-hexylglutathione, glutathione sulfonic acid, and glutathione-s-dinitrobenzene. PMID:21620863

  8. Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors.

    PubMed

    Ozoe, Yoshihisa; Kita, Tomo; Ozoe, Fumiyo; Nakao, Toshifumi; Sato, Kazuyuki; Hirase, Kangetsu

    2013-11-01

    γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [(3)H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [(3)H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [(3)H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [(3)H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [(3)H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B1a, were potent inhibitors of [(3)H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides

  9. Structural Basis of Cooperative Ligand Binding by the Glycine Riboswitch

    SciTech Connect

    E Butler; J Wang; Y Xiong; S Strobel

    2011-12-31

    The glycine riboswitch regulates gene expression through the cooperative recognition of its amino acid ligand by a tandem pair of aptamers. A 3.6 {angstrom} crystal structure of the tandem riboswitch from the glycine permease operon of Fusobacterium nucleatum reveals the glycine binding sites and an extensive network of interactions, largely mediated by asymmetric A-minor contacts, that serve to communicate ligand binding status between the aptamers. These interactions provide a structural basis for how the glycine riboswitch cooperatively regulates gene expression.

  10. Mechanistic insight into ligand binding to G-quadruplex DNA

    PubMed Central

    Di Leva, Francesco Saverio; Novellino, Ettore; Cavalli, Andrea; Parrinello, Michele; Limongelli, Vittorio

    2014-01-01

    Specific guanine-rich regions in human genome can form higher-order DNA structures called G-quadruplexes, which regulate many relevant biological processes. For instance, the formation of G-quadruplex at telomeres can alter cellular functions, inducing apoptosis. Thus, developing small molecules that are able to bind and stabilize the telomeric G-quadruplexes represents an attractive strategy for antitumor therapy. An example is 3-(benzo[d]thiazol-2-yl)-7-hydroxy-8-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-2H-chromen-2-one (compound 1), recently identified as potent ligand of the G-quadruplex [d(TGGGGT)]4 with promising in vitro antitumor activity. The experimental observations are suggestive of a complex binding mechanism that, despite efforts, has defied full characterization. Here, we provide through metadynamics simulations a comprehensive understanding of the binding mechanism of 1 to the G-quadruplex [d(TGGGGT)]4. In our calculations, the ligand explores all the available binding sites on the DNA structure and the free-energy landscape of the whole binding process is computed. We have thus disclosed a peculiar hopping binding mechanism whereas 1 is able to bind both to the groove and to the 3’ end of the G-quadruplex. Our results fully explain the available experimental data, rendering our approach of great value for further ligand/DNA studies. PMID:24753420

  11. Evidence for chemoreceptors with bimodular ligand-binding regions harboring two signal-binding sites

    PubMed Central

    Pineda-Molina, Estela; Reyes-Darias, José-Antonio; Lacal, Jesús; Ramos, Juan L.; García-Ruiz, Juan Manuel; Gavira, Jose A.; Krell, Tino

    2012-01-01

    Chemoreceptor-based signaling is a central mechanism in bacterial signal transduction. Receptors are classified according to the size of their ligand-binding region. The well-studied cluster I proteins have a 100- to 150-residue ligand-binding region that contains a single site for chemoattractant recognition. Cluster II receptors, which contain a 220- to 300-residue ligand-binding region and which are almost as abundant as cluster I receptors, remain largely uncharacterized. Here, we report high-resolution structures of the ligand-binding region of the cluster II McpS chemotaxis receptor (McpS-LBR) of Pseudomonas putida KT2440 in complex with different chemoattractants. The structure of McpS-LBR represents a small-molecule binding domain composed of two modules, each able to bind different signal molecules. Malate and succinate were found to bind to the membrane-proximal module, whereas acetate binds to the membrane-distal module. A structural alignment of the two modules revealed that the ligand-binding sites could be superimposed and that amino acids involved in ligand recognition are conserved in both binding sites. Ligand binding to both modules was shown to trigger chemotactic responses. Further analysis showed that McpS-like receptors were found in different classes of proteobacteria, indicating that this mode of response to different carbon sources may be universally distributed. The physiological relevance of the McpS architecture may lie in its capacity to respond with high sensitivity to the preferred carbon sources malate and succinate and, at the same time, mediate lower sensitivity responses to the less preferred but very abundant carbon source acetate. PMID:23112148

  12. Structural Dynamics of the Cereblon Ligand Binding Domain

    PubMed Central

    Hartmann, Marcus D.; Boichenko, Iuliia; Coles, Murray; Lupas, Andrei N.; Hernandez Alvarez, Birte

    2015-01-01

    Cereblon, a primary target of thalidomide and its derivatives, has been characterized structurally from both bacteria and animals. Especially well studied is the thalidomide binding domain, CULT, which shows an invariable structure across different organisms and in complex with different ligands. Here, based on a series of crystal structures of a bacterial representative, we reveal the conformational flexibility and structural dynamics of this domain. In particular, we follow the unfolding of large fractions of the domain upon release of thalidomide in the crystalline state. Our results imply that a third of the domain, including the thalidomide binding pocket, only folds upon ligand binding. We further characterize the structural effect of the C-terminal truncation resulting from the mental-retardation linked R419X nonsense mutation in vitro and offer a mechanistic hypothesis for its irresponsiveness to thalidomide. At 1.2Å resolution, our data provide a view of thalidomide binding at atomic resolution. PMID:26024445

  13. Cloud Computing for Protein-Ligand Binding Site Comparison

    PubMed Central

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery. PMID:23762824

  14. Cloud computing for protein-ligand binding site comparison.

    PubMed

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery. PMID:23762824

  15. NMR Mapping of the IFNAR1-EC binding site on IFNα2 reveals allosteric changes in the IFNAR2-EC binding site

    PubMed Central

    Akabayov, Sabine Ruth; Biron, Zohar; Lamken, Peter; Piehler, Jacob; Anglister, Jacob

    2010-01-01

    All type I interferons (IFNs) bind to a common cell-surface receptor consisting of two subunits. IFNs initiate intracellular signal transduction cascades by simultaneous interaction with the extracellular domains of its receptor subunits IFNAR1 and IFNAR2. In this study we mapped the surface of IFNα2 interacting with the extracellular domain of IFNAR1 (IFNAR1-EC) by following changes in or the disappearance of the [1H,15N]-TROSY-HSQC cross peaks of IFNα2 caused by the binding of the extracellular domain of IFNAR1 (IFNAR1-EC) to the binary complex of IFNα2 with IFNAR2-EC. The NMR study on the 89 kDa complex was conducted at pH 8 and 308 K using an 800 MHz spectrometer. IFNAR1 binding affected a total of 47 out of 165 IFNα2 residues contained in two large patches on the face of the protein opposing the binding site for IFNAR2 and in a third patch located on the face containing the IFNAR2 binding site. The first two patches form the IFNAR1 binding site and one of these matches the IFNAR1 binding site previously identified by site-directed mutagenesis. The third patch partially matches the IFNα2 binding site for IFNAR2-EC indicating allosteric communication between the binding sites for the two receptor subunits. PMID:20047337

  16. Inhibition of AMP-Activated Protein Kinase at the Allosteric Drug-Binding Site Promotes Islet Insulin Release.

    PubMed

    Scott, John W; Galic, Sandra; Graham, Kate L; Foitzik, Richard; Ling, Naomi X Y; Dite, Toby A; Issa, Samah M A; Langendorf, Chris G; Weng, Qing Ping; Thomas, Helen E; Kay, Thomas W; Birnberg, Neal C; Steinberg, Gregory R; Kemp, Bruce E; Oakhill, Jonathan S

    2015-06-18

    The AMP-activated protein kinase (AMPK) is a metabolic stress-sensing αβγ heterotrimer responsible for energy homeostasis. Pharmacological inhibition of AMPK is regarded as a therapeutic strategy in some disease settings including obesity and cancer; however, the broadly used direct AMPK inhibitor compound C suffers from poor selectivity. We have discovered a dihydroxyquinoline drug (MT47-100) with novel AMPK regulatory properties, being simultaneously a direct activator and inhibitor of AMPK complexes containing the β1 or β2 isoform, respectively. Allosteric inhibition by MT47-100 was dependent on the β2 carbohydrate-binding module (CBM) and determined by three non-conserved CBM residues (Ile81, Phe91, Ile92), but was independent of β2-Ser108 phosphorylation. Whereas MT47-100 regulation of total cellular AMPK activity was determined by β1/β2 expression ratio, MT47-100 augmented glucose-stimulated insulin secretion from isolated mouse pancreatic islets via a β2-dependent mechanism. Our findings highlight the therapeutic potential of isoform-specific AMPK allosteric inhibitors. PMID:26091167

  17. RXR function requires binding to an endogenous terpenoid ligand

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The issue of whether the nuclear receptor RXR must bind to an endogenous, nanomolar affinity ligand in order to perform its natural function is still unsettled (1). On the basis of our previous studies establishing that the Drosophilamelanogaster ortholog of the retinoid X receptor ("ultraspiracle,"...

  18. Enhanced ligand sampling for relative protein-ligand binding free energy calculations.

    PubMed

    Kaus, Joseph W; McCammon, J Andrew

    2015-05-21

    Free energy calculations are used to study how strongly potential drug molecules interact with their target receptors. The accuracy of these calculations depends on the accuracy of the molecular dynamics (MD) force field as well as proper sampling of the major conformations of each molecule. However, proper sampling of ligand conformations can be difficult when there are large barriers separating the major ligand conformations. An example of this is for ligands with an asymmetrically substituted phenyl ring, where the presence of protein loops hinders the proper sampling of the different ring conformations. These ring conformations become more difficult to sample when the size of the functional groups attached to the ring increases. The Adaptive Integration Method (AIM) has been developed, which adaptively changes the alchemical coupling parameter λ during the MD simulation so that conformations sampled at one λ can aid sampling at the other λ values. The Accelerated Adaptive Integration Method (AcclAIM) builds on AIM by lowering potential barriers for specific degrees of freedom at intermediate λ values. However, these methods may not work when there are very large barriers separating the major ligand conformations. In this work, we describe a modification to AIM that improves sampling of the different ring conformations, even when there is a very large barrier between them. This method combines AIM with conformational Monte Carlo sampling, giving improved convergence of ring populations and the resulting free energy. This method, called AIM/MC, is applied to study the relative binding free energy for a pair of ligands that bind to thrombin and a different pair of ligands that bind to aspartyl protease β-APP cleaving enzyme 1 (BACE1). These protein-ligand binding free energy calculations illustrate the improvements in conformational sampling and the convergence of the free energy compared to both AIM and AcclAIM. PMID:25906170

  19. Enhanced Ligand Sampling for Relative Protein–Ligand Binding Free Energy Calculations

    PubMed Central

    2016-01-01

    Free energy calculations are used to study how strongly potential drug molecules interact with their target receptors. The accuracy of these calculations depends on the accuracy of the molecular dynamics (MD) force field as well as proper sampling of the major conformations of each molecule. However, proper sampling of ligand conformations can be difficult when there are large barriers separating the major ligand conformations. An example of this is for ligands with an asymmetrically substituted phenyl ring, where the presence of protein loops hinders the proper sampling of the different ring conformations. These ring conformations become more difficult to sample when the size of the functional groups attached to the ring increases. The Adaptive Integration Method (AIM) has been developed, which adaptively changes the alchemical coupling parameter λ during the MD simulation so that conformations sampled at one λ can aid sampling at the other λ values. The Accelerated Adaptive Integration Method (AcclAIM) builds on AIM by lowering potential barriers for specific degrees of freedom at intermediate λ values. However, these methods may not work when there are very large barriers separating the major ligand conformations. In this work, we describe a modification to AIM that improves sampling of the different ring conformations, even when there is a very large barrier between them. This method combines AIM with conformational Monte Carlo sampling, giving improved convergence of ring populations and the resulting free energy. This method, called AIM/MC, is applied to study the relative binding free energy for a pair of ligands that bind to thrombin and a different pair of ligands that bind to aspartyl protease β-APP cleaving enzyme 1 (BACE1). These protein–ligand binding free energy calculations illustrate the improvements in conformational sampling and the convergence of the free energy compared to both AIM and AcclAIM. PMID:25906170

  20. How Does Confinement Change Ligand-Receptor Binding Equilibrium? Protein Binding in Nanopores and Nanochannels.

    PubMed

    Tagliazucchi, Mario; Szleifer, Igal

    2015-10-01

    We present systematic studies for the binding of small model proteins to ligands attached to the inner walls of long nanochannels and short nanopores by polymeric tethers. Binding of proteins to specific ligands inside nanometric channels and pores leads to changes in their ionic conductance, which have been exploited in sensors that quantify the concentration of the proteins in solution. The theoretical predictions presented in this work are aimed to provide a fundamental understanding of protein binding under geometrically confined environments and to guide the design of this kind of nanochannel-based sensors. The theory predicts that the fraction of the channel volume filled by bound proteins is a nonmonotonic function of the channel radius, the length of the tethers, the surface density of the ligands and the size of the proteins. Notably, increasing the density of ligands, decreasing the size of the channel or increasing the size of the protein may lead to a decrease of the fraction of the channel volume filled by bound proteins. These results are explained from the incomplete binding of proteins to the ligands due to repulsive protein-protein and protein-ligand steric interactions. Our work suggests strategies to optimize the change in conductance due to protein binding, for example: (i) proteins much smaller than the radius of the channel may effectively block the channel if tethers of appropriate length are used, and (ii) a large decrease in conductance upon protein binding can be achieved if the channel and the protein are oppositely charged. PMID:26368839

  1. Biphasic binding kinetics between FepA and its ligands.

    PubMed

    Payne, M A; Igo, J D; Cao, Z; Foster, S B; Newton, S M; Klebba, P E

    1997-08-29

    The Escherichia coli FepA protein is an energy- and TonB-dependent, ligand-binding porin that functions as a receptor for the siderophore ferric enterobactin and colicins B and D. We characterized the kinetic and thermodynamic parameters associated with the initial, energy-independent steps in ligand binding to FepA. In vivo experiments produced Kd values of 24, 185, and 560 nM for ferric enterobactin, colicin B, and colicin D, respectively. The siderophore and colicin B bound to FepA with a 1:1 stoichiometry, but colicin D bound to a maximum level that was 3-fold lower. Preincubation with ferric enterobactin prevented colicin B binding, and preincubation with colicin B prevented ferric enterobactin binding. Colicin B release from FepA was unexpectedly slow in vivo, about 10-fold slower than ferric enterobactin release. This slow dissociation of the colicin B.FepA complex facilitated the affinity purification of FepA and FepA mutants with colicin B-Sepharose. Analysis of a fluorescent FepA derivative showed that ferric enterobactin and colicin B adsorbed with biphasic kinetics, suggesting that both ligands bind in at least two distinct steps, an initial rapid stage and a subsequent slower step, that presumably establishes a transport-competent complex. PMID:9268330

  2. Rationalizing Tight Ligand Binding through Cooperative Interaction Networks

    PubMed Central

    2011-01-01

    Small modifications of the molecular structure of a ligand sometimes cause strong gains in binding affinity to a protein target, rendering a weakly active chemical series suddenly attractive for further optimization. Our goal in this study is to better rationalize and predict the occurrence of such interaction hot-spots in receptor binding sites. To this end, we introduce two new concepts into the computational description of molecular recognition. First, we take a broader view of noncovalent interactions and describe protein–ligand binding with a comprehensive set of favorable and unfavorable contact types, including for example halogen bonding and orthogonal multipolar interactions. Second, we go beyond the commonly used pairwise additive treatment of atomic interactions and use a small world network approach to describe how interactions are modulated by their environment. This approach allows us to capture local cooperativity effects and considerably improves the performance of a newly derived empirical scoring function, ScorpionScore. More importantly, however, we demonstrate how an intuitive visualization of key intermolecular interactions, interaction networks, and binding hot-spots supports the identification and rationalization of tight ligand binding. PMID:22087588

  3. SPACER: server for predicting allosteric communication and effects of regulation

    PubMed Central

    Goncearenco, Alexander; Mitternacht, Simon; Yong, Taipang; Eisenhaber, Birgit; Eisenhaber, Frank; Berezovsky, Igor N.

    2013-01-01

    The SPACER server provides an interactive framework for exploring allosteric communication in proteins with different sizes, degrees of oligomerization and function. SPACER uses recently developed theoretical concepts based on the thermodynamic view of allostery. It proposes easily tractable and meaningful measures that allow users to analyze the effect of ligand binding on the intrinsic protein dynamics. The server shows potential allosteric sites and allows users to explore communication between the regulatory and functional sites. It is possible to explore, for instance, potential effector binding sites in a given structure as targets for allosteric drugs. As input, the server only requires a single structure. The server is freely available at http://allostery.bii.a-star.edu.sg/. PMID:23737445

  4. Dynamics of biomolecules, ligand binding & biological functions

    NASA Astrophysics Data System (ADS)

    Yi, Myunggi

    Proteins are flexible and dynamic. One static structure alone does not often completely explain biological functions of the protein, and some proteins do not even have high resolution structures. In order to provide better understanding to the biological functions of nicotinic acetylcholine receptor, Diphtheria toxin repressor and M2 proton channel, the dynamics of these proteins are investigated using molecular modeling and molecular dynamics (MD) simulations. With absence of high resolution structure of alpha7 receptor, the homology models of apo and cobra toxin bound forms have been built. From the MD simulations of these model structures, we observed one subunit of apo simulation moved away from other four subunits. With local movement of flexible loop regions, the whole subunit tilted clockwise. These conformational changes occurred spontaneously, and were strongly correlated with the conformational change when the channel is activated by agonists. Unlike other computational studies, we directly compared our model of open conformation with the experimental data. However, the subunits of toxin bound form were stable, and conformational change is restricted by the bound cobra toxin. These results provide activation and inhibition mechanisms of alpha7 receptors and a possible explanation for intermediate conductance of the channel. Intramolecular complex of SH3-like domain with a proline-rich (Pr) peptide segment in Diphtheria toxin repressor (DtxR) is stabilized in inactive state. Upon activation of DtxR by transition metal binding, this intramolecular complex should be dissociated. The dynamics of this intramolecular complex is investigated using MD simulations and NMR spectroscopy. We observed spontaneous opening and closing motions of the Pr segment binding pockets in both Pr-SH3 and SH3 simulations. The MD simulation results and NMR relaxation data suggest that the Pr segment exhibits a binding ↔ unbinding equilibrium. Despite a wealth of experimental

  5. Lipid A binding proteins in macrophages detected by ligand blotting

    SciTech Connect

    Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

    1987-05-01

    Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessed using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.

  6. Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

    PubMed Central

    Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

    2013-01-01

    In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

  7. Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin.

    PubMed

    Weber, Roy E; Fago, Angela; Malte, Hans; Storz, Jay F; Gorr, Thomas A

    2013-08-01

    In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3(-)), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3(-) levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3(-) binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

  8. Ligand migration and binding in nonsymbiotic hemoglobins of Arabidopsis thaliana.

    PubMed

    Nienhaus, Karin; Dominici, Paola; Astegno, Alessandra; Abbruzzetti, Stefania; Viappiani, Cristiano; Nienhaus, G Ulrich

    2010-09-01

    We have studied carbon monoxide (CO) migration and binding in the nonsymbiotic hemoglobins AHb1 and AHb2 of Arabidopsis thaliana using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures. Both proteins have similar amino acid sequences but display pronounced differences in ligand binding properties, at both physiological and cryogenic temperatures. Near neutral pH, the distal HisE7 side chain is close to the heme-bound ligand in the majority of AHb1-CO molecules, as indicated by a low CO stretching frequency at 1921 cm(-1). In this fraction, two CO docking sites can be populated, the primary site B and the secondary site C. When the pH is lowered, a high-frequency stretching band at approximately 1964 cm(-1) grows at the expense of the low-frequency band, indicating that HisE7 protonates and, concomitantly, moves away from the bound ligand. Geminate rebinding barriers are markedly different for the two conformations, and docking site C is not accessible in the low-pH conformation. Rebinding of NO ligands was observed only from site B of AHb1, regardless of conformation. In AHb2, the HisE7 side chain is removed from the bound ligand; rebinding barriers are low, and CO molecules can populate only primary docking site B. These results are interpreted in terms of differences in the active site structures and physiological functions. PMID:20666470

  9. Improving Binding Affinity and Selectivity of Computationally Designed Ligand-Binding Proteins Using Experiments.

    PubMed

    Tinberg, Christine E; Khare, Sagar D

    2016-01-01

    The ability to de novo design proteins that can bind small molecules has wide implications for synthetic biology and medicine. Combining computational protein design with the high-throughput screening of mutagenic libraries of computationally designed proteins is emerging as a general approach for creating binding proteins with programmable binding modes, affinities, and selectivities. The computational step enables the creation of a binding site in a protein that otherwise does not (measurably) bind the intended ligand, and targeted mutagenic screening allows for validation and refinement of the computational model as well as provides orders-of-magnitude increases in the binding affinity. Deep sequencing of mutagenic libraries can provide insights into the mutagenic binding landscape and enable further affinity improvements. Moreover, in such a combined computational-experimental approach where the binding mode is preprogrammed and iteratively refined, selectivity can be achieved (and modulated) by the placement of specified amino acid side chain groups around the ligand in defined orientations. Here, we describe the experimental aspects of a combined computational-experimental approach for designing-using the software suite Rosetta-proteins that bind a small molecule of choice and engineering, using fluorescence-activated cell sorting and high-throughput yeast surface display, high affinity and ligand selectivity. We illustrated the utility of this approach by performing the design of a selective digoxigenin (DIG)-binding protein that, after affinity maturation, binds DIG with picomolar affinity and high selectivity over structurally related steroids. PMID:27094290

  10. Discovery of an Allosteric Inhibitor Binding Site in 3-Oxo-acyl-ACP Reductase from Pseudomonas aeruginosa

    PubMed Central

    2013-01-01

    3-Oxo-acyl-acyl carrier protein (ACP) reductase (FabG) plays a key role in the bacterial fatty acid synthesis II system in pathogenic microorganisms, which has been recognized as a potential drug target. FabG catalyzes reduction of a 3-oxo-acyl-ACP intermediate during the elongation cycle of fatty acid biosynthesis. Here, we report gene deletion experiments that support the essentiality of this gene in P. aeruginosa and the identification of a number of small molecule FabG inhibitors with IC50 values in the nanomolar to low micromolar range and good physicochemical properties. Structural characterization of 16 FabG-inhibitor complexes by X-ray crystallography revealed that the compounds bind at a novel allosteric site located at the FabG subunit–subunit interface. Inhibitor binding relies primarily on hydrophobic interactions, but specific hydrogen bonds are also observed. Importantly, the binding cavity is formed upon complex formation and therefore would not be recognized by virtual screening approaches. The structure analysis further reveals that the inhibitors act by inducing conformational changes that propagate to the active site, resulting in a displacement of the catalytic triad and the inability to bind NADPH. PMID:24015914

  11. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate.

    PubMed

    Gelato, Kathy A; Tauber, Maria; Ong, Michelle S; Winter, Stefan; Hiragami-Hamada, Kyoko; Sindlinger, Julia; Lemak, Alexander; Bultsma, Yvette; Houliston, Scott; Schwarzer, Dirk; Divecha, Nullin; Arrowsmith, Cheryl H; Fischle, Wolfgang

    2014-06-19

    UHRF1 is a multidomain protein crucially linking histone H3 modification states and DNA methylation. While the interaction properties of its specific domains are well characterized, little is known about the regulation of these functionalities. We show that UHRF1 exists in distinct active states, binding either unmodified H3 or the H3 lysine 9 trimethylation (H3K9me3) modification. A polybasic region (PBR) in the C terminus blocks interaction of a tandem tudor domain (TTD) with H3K9me3 by occupying an essential peptide-binding groove. In this state the plant homeodomain (PHD) mediates interaction with the extreme N terminus of the unmodified H3 tail. Binding of the phosphatidylinositol phosphate PI5P to the PBR of UHRF1 results in a conformational rearrangement of the domains, allowing the TTD to bind H3K9me3. Our results define an allosteric mechanism controlling heterochromatin association of an essential regulatory protein of epigenetic states and identify a functional role for enigmatic nuclear phosphatidylinositol phosphates. PMID:24813945

  12. Solvent fluctuations in hydrophobic cavity–ligand binding kinetics

    PubMed Central

    Setny, Piotr; Baron, Riccardo; Michael Kekenes-Huskey, Peter; McCammon, J. Andrew; Dzubiella, Joachim

    2013-01-01

    Water plays a crucial part in virtually all protein–ligand binding processes in and out of equilibrium. Here, we investigate the role of water in the binding kinetics of a ligand to a prototypical hydrophobic pocket by explicit-water molecular dynamics (MD) simulations and implicit diffusional approaches. The concave pocket in the unbound state exhibits wet/dry hydration oscillations whose magnitude and time scale are significantly amplified by the approaching ligand. In turn, the ligand’s stochastic motion intimately couples to the slow hydration fluctuations, leading to a sixfold-enhanced friction in the vicinity of the pocket entrance. The increased friction considerably decelerates association in the otherwise barrierless system, indicating the importance of molecular-scale hydrodynamic effects in cavity–ligand binding arising due to capillary fluctuations. We derive and analyze the diffusivity profile and show that the mean first passage time distribution from the MD simulation can be accurately reproduced by a standard Brownian dynamics simulation if the appropriate position-dependent friction profile is included. However, long-time decays in the water–ligand (random) force autocorrelation demonstrate violation of the Markovian assumption, challenging standard diffusive approaches for rate prediction. Remarkably, the static friction profile derived from the force correlations strongly resembles the profile derived on the Markovian assumption apart from a simple shift in space, which can be rationalized by a time–space retardation in the ligand’s downhill dynamics toward the pocket. The observed spatiotemporal hydrodynamic coupling may be of biological importance providing the time needed for conformational receptor–ligand adjustments, typical of the induced-fit paradigm. PMID:23297241

  13. The Allosteric Site for the Nascent Cell Wall in Penicillin-Binding Protein 2a: An Achilles' Heel of Methicillin-Resistant Staphylococcus aureus.

    PubMed

    Acebrón, Iván; Chang, Mayland; Mobashery, Shahriar; Hermoso, Juan A

    2015-01-01

    The ability to resist the effect of a wide range of antibiotics makes methicillin-resistant Staphylococcus aureus (MRSA) a leading global human pathogen. A key determinant of resistance to β-lactam antibiotics in this organism is penicillin-binding protein 2a (PBP2a), an enzyme that catalyzes the crosslinking reaction between two adjacent peptide stems during the peptidoglycan biosynthesis. The recently published crystal structure of the complex of PBP2a with ceftaroline, a cephalosporin antibiotic that shows efficacy against MRSA, has revealed the allosteric site at 60-Å distance from the transpeptidase domain. Binding of ceftaroline to the allosteric site of PBP2a triggers conformational changes that lead to the opening of the active site from a closed conformation, where a second molecule of ceftaroline binds to give inhibition of the enzyme. The discovery of allostery in MRSA remains the only known example of such regulation of cellwall biosynthesis and represents a new paradigm in fighting MRSA. This review summarizes the present knowledge of the allosteric mechanism, the conformational changes allowing PBP2a catalysis and the means by which some clinical strains have acquired resistance to ceftaroline by disrupting the allosteric mechanism. PMID:25760091

  14. Ligand Migration and Binding in Myoglobin Mutant L29W

    NASA Astrophysics Data System (ADS)

    Nienhaus, G. Ulrich; Waschipky, Robert; Nienhaus, Karin; Minkow, Oleksandr; Ostermann, Andreas; Parak, Fritz G.

    2001-09-01

    Myoglobin, a small globular heme protein that binds gaseous ligands such as O2, CO, and NO reversibly at the heme iron, has for many years been a paradigm for studying the effects of structure and dynamics on protein reactions. Time-resolved spectroscopic measurements after photodissociation of the ligand reveal a complex ligand binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein. To observe structural changes induced by ligand dissociation, we have investigated carbonmonoxy myoglobin (MbCO) mutant L29W using time-resolved infrared spectroscopy in combination with x-ray crystallography. The presence of two distinct infrared stretch bands of the bound CO, AI at 1945 cm-1 and AII at 1955 cm-1, implies that L29W MbCO assumes two different conformations at neutral pH. Low-temperature flash photolysis experiments with monitoring of the absorption changes in the individual CO lines reveal markedly different rebinding properties. While recombination in AII is conceptually simple and well described by a two-state transition involving a distribution of enthalpy barriers, recombination in AI is more complicated: Besides a fast kinetic component, a second, slower kinetic component appears; its population grows with increasing temperature. X-ray crystallography of crystals illuminated below 180 K to photodissociate the CO reveals that the slow component arises from ligands that have migrated from their initial docking site to a remote site within the distal heme pocket. This process occurs in an essentially immobilized, frozen protein. Subsequently, ligands rebind by thermal activation over a barrier that is much higher than the barrier for recombination from the initial docking site. Upon photodissociation above 180 K, ligands escape from the distal pocket, aided by protein fluctuations that transiently open exit channels. The x-ray structure shows a large proportion of ligands in a cavity on

  15. Predicting ligand binding affinity with alchemical free energy methods in a polar model binding site

    PubMed Central

    Boyce, Sarah E.; Mobley, David L.; Rocklin, Gabriel; Graves, Alan P.

    2009-01-01

    We present a combined experimental and modeling study of organic ligand molecules binding to a slightly polar engineered cavity site in T4 lysozyme (L99A/M102Q). For modeling, we computed alchemical absolute binding free energies. These were blind tests performed prospectively on 13 diverse, previously untested candidate ligand molecules. We predicted that eight compounds would bind to the cavity and five would not; 11 of 13 predictions were correct at this level. The RMS error to the measurable absolute binding energies was 1.8 kcal/mol. In addition, we computed relative binding free energies for six phenol derivatives starting from two known ligands: phenol and catechol. The average RMS error in the relative free energy prediction was 2.5 (phenol) and 1.1 (catechol) kcal/mol. To understand these results at atomic resolution, we obtained x-ray co-complex structures for nine of the diverse ligands and for all six phenol analogs. The average RMSD of the predicted pose to the experiment was 2.0Å (diverse set), 1.8Å (phenol derived predictions) and 1.2Å (catechol derived predictions). We found that to predict accurate affinities and rank-orderings required near-native starting orientations of the ligand in the binding site. Unanticipated binding modes, multiple ligand binding, and protein conformational change all proved challenging for the free energy methods. We believe these results can help guide future improvements in physics-based absolute binding free energy methods. PMID:19782087

  16. Allosteric Modulators of Class B G-Protein-Coupled Receptors

    PubMed Central

    Hoare, Sam R.J

    2007-01-01

    Class B GPCR’s are activated by peptide ligands, typically 30-40 amino acid residues, that are involved in major physiological functions such as glucose homeostasis (glucagon and glucagon-like peptide 1), calcium homeostasis and bone turnover (parathyroid hormone and calcitonin), and control of the stress axis (corticotropin-releasing factor). Peptide therapeutics have been developed targeting these receptors but development of nonpeptide ligands, enabling oral administration, has proved challenging. Allosteric modulation of these receptors provides a potential route to developing nonpeptide ligands that inhibit, activate, or potentiate activation of these receptors. Here the known mechanisms of allosteric modulators targeting Class B GPCR’s are reviewed, particularly nonpeptide antagonists of the corticotropin-releasing factor 1 receptor and allosteric enhancers of the glucagon-like peptide-1 receptor. Also discussed is the potential for antagonist ligands to operate by competitive inhibition of one of the peptide binding sites, analogous to the Charniere mechanism. These mechanisms are then used to discuss potential strategies and management of pharmacological complexity in the future development of allosteric modulators for Class B GPCR’s. PMID:19305799

  17. The Allosteric Regulatory Mechanism of the Escherichia coli MetNI Methionine ATP Binding Cassette (ABC) Transporter*

    PubMed Central

    Yang, Janet G.; Rees, Douglas C.

    2015-01-01

    The MetNI methionine importer of Escherichia coli, an ATP binding cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of d- and l-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of the MetNI mechanism has thus far been limited to a series of crystal structures in an inward-facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by l-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well. PMID:25678706

  18. Coarse-grained molecular simulations of allosteric cooperativity

    NASA Astrophysics Data System (ADS)

    Nandigrami, Prithviraj; Portman, John J.

    2016-03-01

    Interactions between a protein and a ligand are often accompanied by a redistribution of the population of thermally accessible conformations. This dynamic response of the protein's functional energy landscape enables a protein to modulate binding affinities and control binding sensitivity to ligand concentration. In this paper, we investigate the structural origins of binding affinity and allosteric cooperativity of binding two Ca2+ ions to each domain of Calmodulin (CaM) through simulations of a simple coarse-grained model. In this model, the protein's conformational transitions between open and closed conformational ensembles are simulated explicitly and ligand binding and unbinding are treated implicitly within the grand canonical ensemble. Ligand binding is cooperative because the binding sites are coupled through a shift in the dominant conformational ensemble upon binding. The classic Monod-Wyman-Changeux model of allostery with appropriate binding free energies to the open and closed ensembles accurately describes the simulated binding thermodynamics. The simulations predict that the two domains of CaM have distinct binding affinity and cooperativity. In particular, the C-terminal domain binds Ca2+ with higher affinity and greater cooperativity than the N-terminal domain. From a structural point of view, the affinity of an individual binding loop depends sensitively on the loop's structural compatibility with the ligand in the bound ensemble, as well as the conformational flexibility of the binding site in the unbound ensemble.

  19. Recent improvements to Binding MOAD: a resource for protein-ligand binding affinities and structures.

    PubMed

    Ahmed, Aqeel; Smith, Richard D; Clark, Jordan J; Dunbar, James B; Carlson, Heather A

    2015-01-01

    For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein-ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23,269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. PMID:25378330

  20. Biased signalling and allosteric machines: new vistas and challenges for drug discovery.

    PubMed

    Kenakin, Terry P

    2012-03-01

    Seven transmembrane receptors (7TMRs) are nature's prototype allosteric proteins made to bind molecules at one location to subsequently change their shape to affect the binding of another molecule at another location. This paper attempts to describe the divergent 7TMR behaviours (i.e. third party allostery, receptor oligomerization, biased agonism) observed in pharmacology in terms of a homogeneous group of allosteric behaviours. By considering the bodies involved as a vector defined by a modulator, conduit and guest, these activities can all be described by a simple model of functional allostery made up of the Ehlert allosteric model and the Black/Leff operational model. It will be shown how this model yields parameters that can be used to characterize the activity of any ligand or protein producing effect through allosteric interaction with a 7TMR. PMID:22023017

  1. Coarse-grained molecular dynamics simulations of protein-ligand binding.

    PubMed

    Negami, Tatsuki; Shimizu, Kentaro; Terada, Tohru

    2014-09-30

    Coarse-grained molecular dynamics (CGMD) simulations with the MARTINI force field were performed to reproduce the protein-ligand binding processes. We chose two protein-ligand systems, the levansucrase-sugar (glucose or sucrose), and LinB-1,2-dichloroethane systems, as target systems that differ in terms of the size and shape of the ligand-binding pocket and the physicochemical properties of the pocket and the ligand. Spatial distributions of the Coarse-grained (CG) ligand molecules revealed potential ligand-binding sites on the protein surfaces other than the real ligand-binding sites. The ligands bound most strongly to the real ligand-binding sites. The binding and unbinding rate constants obtained from the CGMD simulation of the levansucrase-sucrose system were approximately 10 times greater than the experimental values; this is mainly due to faster diffusion of the CG ligand in the CG water model. We could obtain dissociation constants close to the experimental values for both systems. Analysis of the ligand fluxes demonstrated that the CG ligand molecules entered the ligand-binding pockets through specific pathways. The ligands tended to move through grooves on the protein surface. Thus, the CGMD simulations produced reasonable results for the two different systems overall and are useful for studying the protein-ligand binding processes. PMID:25043724

  2. An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase

    NASA Astrophysics Data System (ADS)

    Pucheta-Martínez, Encarna; Saladino, Giorgio; Morando, Maria Agnese; Martinez-Torrecuadrada, Jorge; Lelli, Moreno; Sutto, Ludovico; D’Amelio, Nicola; Gervasio, Francesco Luigi

    2016-04-01

    Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe.

  3. An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase

    PubMed Central

    Pucheta-Martínez, Encarna; Saladino, Giorgio; Morando, Maria Agnese; Martinez-Torrecuadrada, Jorge; Lelli, Moreno; Sutto, Ludovico; D’Amelio, Nicola; Gervasio, Francesco Luigi

    2016-01-01

    Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe. PMID:27063862

  4. Controlling allosteric networks in proteins

    NASA Astrophysics Data System (ADS)

    Dokholyan, Nikolay

    2013-03-01

    We present a novel methodology based on graph theory and discrete molecular dynamics simulations for delineating allosteric pathways in proteins. We use this methodology to uncover the structural mechanisms responsible for coupling of distal sites on proteins and utilize it for allosteric modulation of proteins. We will present examples where inference of allosteric networks and its rewiring allows us to ``rescue'' cystic fibrosis transmembrane conductance regulator (CFTR), a protein associated with fatal genetic disease cystic fibrosis. We also use our methodology to control protein function allosterically. We design a novel protein domain that can be inserted into identified allosteric site of target protein. Using a drug that binds to our domain, we alter the function of the target protein. We successfully tested this methodology in vitro, in living cells and in zebrafish. We further demonstrate transferability of our allosteric modulation methodology to other systems and extend it to become ligh-activatable.

  5. An allosteric model of the inositol trisphosphate receptor with nonequilibrium binding

    NASA Astrophysics Data System (ADS)

    Jia, Chen; Jiang, Daquan; Qian, Minping

    2014-10-01

    The inositol trisphosphate receptor (IPR) is a crucial ion channel that regulates the Ca2+ influx from the endoplasmic reticulum (ER) to the cytoplasm. A thorough study of the IPR channel contributes to a better understanding of calcium oscillations and waves. It has long been observed that the IPR channel is a typical biological system which performs adaptation. However, recent advances on the physical essence of adaptation show that adaptation systems with a negative feedback mechanism, such as the IPR channel, must break detailed balance and always operate out of equilibrium with energy dissipation. Almost all previous IPR models are equilibrium models assuming detailed balance and thus violate the dissipative nature of adaptation. In this article, we constructed a nonequilibrium allosteric model of single IPR channels based on the patch-clamp experimental data obtained from the IPR in the outer membranes of isolated nuclei of the Xenopus oocyte. It turns out that our model reproduces the patch-clamp experimental data reasonably well and produces both the correct steady-state and dynamic properties of the channel. Particularly, our model successfully describes the complicated bimodal [Ca2+] dependence of the mean open duration at high [IP3], a steady-state behavior which fails to be correctly described in previous IPR models. Finally, we used the patch-clamp experimental data to validate that the IPR channel indeed breaks detailed balance and thus is a nonequilibrium system which consumes energy.

  6. The Promiscuity of Allosteric Regulation of Nuclear Receptors by Retinoid X Receptor.

    PubMed

    Clark, Alexander K; Wilder, J Heath; Grayson, Aaron W; Johnson, Quentin R; Lindsay, Richard J; Nellas, Ricky B; Fernandez, Elias J; Shen, Tongye

    2016-08-25

    The promiscuous protein retinoid X receptor (RXR) displays essential allosteric regulation of several members in the nuclear hormone receptor superfamily via heterodimerization and (anti)cooperative binding of cognate ligands. Here, the structural basis of the positive allostery of RXR and constitutive androstane receptor (CAR) is revealed. In contrast, a similar computational approach had previously revealed the mechanism for negative allostery in the complex of RXR and thyroid receptor (TR). By comparing the positive and negative allostery of RXR complexed with CAR and TR respectively, we reported the promiscuous allosteric control involving RXR. We characterize the allosteric mechanism by expressing the correlated dynamics of selected residue-residue contacts which was extracted from atomistic molecular dynamics simulation and statistical analysis. While the same set of residues in the binding pocket of RXR may initiate the residue-residue interaction network, RXR uses largely different sets of contacts (only about one-third identical) and allosteric modes to regulate TR and CAR. The promiscuity of RXR control may originate from multiple factors, including (1) the frustrated fit of cognate ligand 9c to the RXR binding pocket and (2) the different ligand-binding features of TR (loose) versus CAR (tight) to their corresponding cognate ligands. PMID:27110634

  7. CO Binding and Ligand Discrimination in Human Myeloperoxidase†

    PubMed Central

    Murphy, Emma J.; Maréchal, Amandine; Segal, Anthony W.; Rich, Peter R.

    2015-01-01

    Despite the fact that ferrous myeloperoxidase (MPO) can bind both O2 and NO, its ability to bind CO has been questioned. UV/visible spectroscopy was used to confirm that CO induces small spectral shifts in ferrous MPO, and Fourier transform infrared difference spectroscopy showed definitively that these arose from formation of a heme ferrous–CO compound. Recombination rates after CO photolysis were monitored at 618 and 645 nm as a function of CO concentration and pH. At pH 6.3, kon and koff were 0.14 mM−1·s−1 and 0.23 s−1, respectively, yielding an unusually high KD of 1.6 mM. This affinity of MPO for CO is 10 times weaker than its affinity for O2. The observed rate constant for CO binding increased with increasing pH and was governed by a single protonatable group with a pKa of 7.8. Fourier transform infrared spectroscopy revealed two different conformations of bound CO with frequencies at 1927 and 1942 cm−1. Their recombination rate constants were identical, indicative of two forms of bound CO that are in rapid thermal equilibrium rather than two distinct protein populations with different binding sites. The ratio of bound states was pH-dependent (pKa ≈ 7.4) with the 1927 cm−1 form favored at high pH. Structural factors that account for the ligand-binding properties of MPO are identified by comparisons with published data on a range of other ligand-binding heme proteins, and support is given to the recent suggestion that the proximal His336 in MPO is in a true imidazolate state. PMID:20146436

  8. Disulfide bonds regulate binding of exogenous ligand to human cytoglobin.

    PubMed

    Tsujino, Hirofumi; Yamashita, Taku; Nose, Azusa; Kukino, Kaori; Sawai, Hitomi; Shiro, Yoshitsugu; Uno, Tadayuki

    2014-06-01

    Cytoglobin (Cgb) was discovered a decade ago and is a fourth member of the group of hexacoordinated globin-folded proteins. Although some crystal structures have been reported and several functions have been proposed for Cgb, its physiological role remains uncertain. In this study, we measured cyanide binding to the ferric state of the wild-type (WT) Cgb, and found that the binding consisted of multiple steps. These results indicated that Cgb may be comprised of several forms, and the presence of monomers, dimers, and tetramers was subsequently confirmed by SDS-PAGE. Remarkably, each species contained two distinguishable forms, and, in the monomer, analyses of alternative cysteine states suggested the presence of an intramolecular disulfide bond (monomer SS form) and a structure with unpaired thiol groups (monomer SH form). These confirmed that forms were separated by gel-exclusion chromatography, and that the cyanide binding of the separated fractions was again measured; they showed different affinities for cyanide, with the monomer fraction showing the highest affinity. In addition, the ferrous state in each fraction showed distinct carbon monoxide (CO)-binding properties, and the affinities for cyanide and CO suggested a linear correlation. Furthermore, we also prepared several variants involving the two cysteine residues. The C38S and C83S variants showed a binding affinity for cyanide similar to the value for the monomer SH form, and hence the fraction with the highest affinity for exogenous ligands was designated as a monomer SS form. We concluded that polymerization could be a mechanism that triggers the exertion of various physiological functions of this protein and that an appropriate disulfide bond between the two cysteine residues was critical for regulating the binding affinity of Cgb, which can act as a ROS scavenger, for exogenous ligands. PMID:24632414

  9. Sequence variation in ligand binding sites in proteins

    PubMed Central

    Magliery, Thomas J; Regan, Lynne

    2005-01-01

    Background The recent explosion in the availability of complete genome sequences has led to the cataloging of tens of thousands of new proteins and putative proteins. Many of these proteins can be structurally or functionally categorized from sequence conservation alone. In contrast, little attention has been given to the meaning of poorly-conserved sites in families of proteins, which are typically assumed to be of little structural or functional importance. Results Recently, using statistical free energy analysis of tetratricopeptide repeat (TPR) domains, we observed that positions in contact with peptide ligands are more variable than surface positions in general. Here we show that statistical analysis of TPRs, ankyrin repeats, Cys2His2 zinc fingers and PDZ domains accurately identifies specificity-determining positions by their sequence variation. Sequence variation is measured as deviation from a neutral reference state, and we present probabilistic and information theory formalisms that improve upon recently suggested methods such as statistical free energies and sequence entropies. Conclusion Sequence variation has been used to identify functionally-important residues in four selected protein families. With TPRs and ankyrin repeats, protein families that bind highly diverse ligands, the effect is so pronounced that sequence "hypervariation" alone can be used to predict ligand binding sites. PMID:16194281

  10. Efficiency parameters in artificial allosteric systems.

    PubMed

    Schneider, Hans-Jörg

    2016-09-14

    It is shown that the until now largely overlooked change of the conformational energy ΔGC is the dominating factor for most synthetic allosteric complexes. Essential is the energy ΔGC required for the formation of a suitable geometry for ligand binding in the absence of an effector molecule E; ΔGC is usually dominated by an increase of strain and/or by high energy solvents in a cavity. The role of the effector molecule E in such systems is to generate a suitable conformation for binding the ligand A and thus to compensate the unfavourable conformational energy ΔGC. Positive cooperativity increases with ΔGC, and decreases with the ΔG0(A) value which reflects the binding energy of A in a strain-free host. As illustrated with a few examples ΔG0(A) cannot be measured directly but can eventually be estimated independently. Many artificial allosteric systems described in the literature, such as those based on ethylene glycol chains or bipyridyl units, lack significant strain differences, and are therefore less efficient. Negative cooperativity is determined only by the difference ΔΔGA,E between the binding energies at the two sites; it can be enhanced or lowered by concomitant changes in ΔGC. PMID:27431438

  11. Proteus and the Design of Ligand Binding Sites.

    PubMed

    Polydorides, Savvas; Michael, Eleni; Mignon, David; Druart, Karen; Archontis, Georgios; Simonson, Thomas

    2016-01-01

    This chapter describes the organization and use of Proteus, a multitool computational suite for the optimization of protein and ligand conformations and sequences, and the calculation of pK α shifts and relative binding affinities. The software offers the use of several molecular mechanics force fields and solvent models, including two generalized Born variants, and a large range of scoring functions, which can combine protein stability, ligand affinity, and ligand specificity terms, for positive and negative design. We present in detail the steps for structure preparation, system setup, construction of the interaction energy matrix, protein sequence and structure optimizations, pK α calculations, and ligand titration calculations. We discuss illustrative examples, including the chemical/structural optimization of a complex between the MHC class II protein HLA-DQ8 and the vinculin epitope, and the chemical optimization of the compstatin analog Ac-Val4Trp/His9Ala, which regulates the function of protein C3 of the complement system. PMID:27094287

  12. A determination of Mg(+)-ligand binding energies

    NASA Technical Reports Server (NTRS)

    Bauschlicher, Charles W., Jr.; Partridge, Harry

    1991-01-01

    Theoretical calculations employing large basis sets and including correlation are carried out for Mg(+) with methanol, water, and formaldehyde. For Mg(+) with ethanol and acetaldehyde, the trends in the binding energies are studied at the self-consistent-field level. The predictions for the binding energy of Mg(+) to methanol and water of 41 + or - 5 and 36 + or - 5 kcal/mol, respectively, are much less than the experimental upper bounds, of 61 + or - 5 and 60 + or - 5 kcal mol, determined by using photodissociation techniques. The theoretical results are inconsistent with the onset of Mg(+) production observed in the photodissociation experiments, as the smallest absorptions are calculated at about 80 kcal/mol for both Mg(+)-CH3OH and Mg(+)-H2O, and these transitions are to bound excited states. The binding energy for Mg(+) with formaldehyde is predicted to be similar to Mg(+)-H2O. The relative binding energies are in reasonable agreement with experiment. The binding energy of a second water molecule to Mg(+) is predicted to be similar to the first. This suggests that the reduced reaction rate observed for the second ligand is not a consequence of a significantly smaller binding energy, at least for the smaller ligards such as those considered in this work.

  13. Sensitive NMR Approach for Determining the Binding Mode of Tightly Binding Ligand Molecules to Protein Targets.

    PubMed

    Chen, Wan-Na; Nitsche, Christoph; Pilla, Kala Bharath; Graham, Bim; Huber, Thomas; Klein, Christian D; Otting, Gottfried

    2016-04-01

    Structure-guided drug design relies on detailed structural knowledge of protein-ligand complexes, but crystallization of cocomplexes is not always possible. Here we present a sensitive nuclear magnetic resonance (NMR) approach to determine the binding mode of tightly binding lead compounds in complex with difficult target proteins. In contrast to established NMR methods, it does not depend on rapid exchange between bound and free ligand or on stable isotope labeling, relying instead on a tert-butyl group as a chemical label. tert-Butyl groups are found in numerous protein ligands and deliver an exceptionally narrow and tall (1)H NMR signal. We show that a tert-butyl group also produces outstandingly intense intra- and intermolecular NOESY cross-peaks. These enable measurements of pseudocontact shifts generated by lanthanide tags attached to the protein, which in turn allows positioning of the ligand on the protein. Once the ligand has been located, assignments of intermolecular NOEs become possible even without prior resonance assignments of protein side chains. The approach is demonstrated with the dengue virus NS2B-NS3 protease in complex with a high-affinity ligand containing a tert-butyl group. PMID:26974502

  14. Exploring Hydrophobic Binding Surfaces Using Comfa and Flexible Hydrophobic Ligands

    NASA Astrophysics Data System (ADS)

    Thakkar, Shraddha; Sanchez, Rosa. I.; Bhuveneswaran, Chidambaram; Compadre, Cesar M.

    2011-06-01

    Cysteine proteinases are a very important group of enzymes involved in a variety of physiological and pathological processes including cancer metastasis and rheumatoid arthritis. In this investigation we used 3D-Quantitative Structure Activity Relationships (3D-QSAR) techniques to model the binding of a variety of substrates to two cysteine proteinases, papain, and cathepsin B. The analysis was performed using Comparative Molecular Field Analysis (CoMFA). The molecules were constructed using standard bond angles and lengths, minimized and aligned. Charges were calculated using the PM3 method in MOPAC. The CoMFA models derived for the binding of the studied substrates to the two proteinases were compared with the expected results from the experimental X-ray crystal structures of the same proteinases. The results showed the value of CoMFA modeling of flexible hydrophobic ligands to analyze ligand binding to protein receptors, and could also serve as the basis to design specific inhibitors of cysteine proteinases with potential therapeutic value.

  15. Observation of Protein Structural Vibrational Mode Sensitivity to Ligand Binding

    NASA Astrophysics Data System (ADS)

    Niessen, Katherine; Xu, Mengyang; Snell, Edward; Markelz, Andrea

    2014-03-01

    We report the first measurements of the dependence of large-scale protein intramolecular vibrational modes on ligand binding. These collective vibrational modes in the terahertz (THz) frequency range (5-100 cm-1) are of great interest due to their predicted relation to protein function. Our technique, Crystals Anisotropy Terahertz Microscopy (CATM), allows for room temperature, table-top measurements of the optically active intramolecular modes. CATM measurements have revealed surprisingly narrowband features. CATM measurements are performed on single crystals of chicken egg-white lysozyme (CEWL) as well as CEWL bound to tri-N-acetylglucosamine (CEWL-3NAG) inhibitor. We find narrow band resonances that dramatically shift with binding. Quasiharmonic calculations are performed on CEWL and CEWL-3NAG proteins with CHARMM using normal mode analysis. The expected CATM response of the crystals is then calculated by summing over all protein orientations within the unit cell. We will compare the CATM measurements with the calculated results and discuss the changes which arise with protein-ligand binding. This work is supported by NSF grant MRI 2 grant DBI2959989.

  16. Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s)

    PubMed Central

    2015-01-01

    Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs. PMID:26529344

  17. Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s).

    PubMed

    Kulkarni, Pushkar M; Kulkarni, Abhijit R; Korde, Anisha; Tichkule, Ritesh B; Laprairie, Robert B; Denovan-Wright, Eileen M; Zhou, Han; Janero, David R; Zvonok, Nikolai; Makriyannis, Alexandros; Cascio, Maria G; Pertwee, Roger G; Thakur, Ganesh A

    2016-01-14

    Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs. PMID:26529344

  18. Allosteric regulation in phosphofructokinase from the extreme thermophile Thermus thermophilus.

    PubMed

    McGresham, Maria S; Lovingshimer, Michelle; Reinhart, Gregory D

    2014-01-14

    An investigation into the kinetics and regulatory properties of the type-1 phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus (TtPFK) reveals an enzyme that is inhibited by PEP and activated by ADP by modifying the affinity exhibited for the substrate fructose 6-phosphate (Fru-6-P) in a manner analogous to other prokaryotic PFKs. However, TtPFK binds both of these allosteric ligands significantly more tightly than other bacterial PFKs while effecting a substantially more modest extent of inhibition or activation at 25 °C, reinforcing the principle that binding affinity and effectiveness can be both independent and uncorrelated to one another. These properties have allowed us to establish rigorously that PEP only inhibits by antagonizing the binding of Fru-6-P and not by influencing turnover, a conclusion that requires kcat to be determined under conditions in which both inhibitor and substrate are saturating simultaneously. In addition, the temperature dependence of the allosteric effects on Fru-6-P binding indicate that the coupling free energies are entropy-dominated, as observed previously for PFK from Bacillus stearothermophilus but not for PFK from Escherichia coli , supporting the hypothesis that entropy-dominated allosteric effects may be a characteristic of enzymes derived from thermostable organisms. For such enzymes, the root cause of the allosteric effect may not be easily discerned from static structural information such as that obtained from X-ray crystallography. PMID:24328040

  19. FOLLITROPIN RECEPTORS CONTAIN CRYPTIC LIGAND BINDING SITES1

    PubMed Central

    Lin, Win; Bernard, Michael P.; Cao, Donghui; Myers, Rebecca V.; Kerrigan, John E.; Moyle, William R.

    2007-01-01

    Human choriogonadotropin (hCG) and follitropin (hFSH) have been shown to contact different regions of the extracellular domains of G-protein coupled lutropin (LHR) and follitropin (FSHR) receptors. We report here that hCG and hFSH analogs interact with an FSHR/LHR chimera having only two unique LHR residues similar to the manners in which they dock with LHR and FSHR, respectively. This shows that although the FSHR does not normally bind hCG, it contains a cryptic lutropin binding site that has the potential to recognize hCG in a manner similar to the LHR. The presence of this cryptic site may explain why equine lutropins bind many mammalian FSHR and why mutations in the transmembrane domain distant from the extracellular domain enable the FSHR to bind hCG. The leucine-rich repeat domain (LRD) of the FSHR also appears to contain a cryptic FSH binding site that is obscured by other parts of the extracellular domain. This will explain why contacts seen in crystals of hFSH complexed with an LRD fragment of the human FSHR are hard to reconcile with the abilities of FSH analogs to interact with membrane G-protein coupled FSHR. We speculate that cryptic lutropin binding sites in the FSHR, which are also likely to be present in thyrotropin receptors (TSHR), permit the physiological regulation of ligand binding specificity. Cryptic FSH binding sites in the LRD may enable alternate spliced forms of the FSHR to interact with FSH. PMID:17059863

  20. Coherent Conformational Degrees of Freedom as a Structural Basis for Allosteric Communication

    PubMed Central

    Mitternacht, Simon; Berezovsky, Igor N.

    2011-01-01

    Conformational changes in allosteric regulation can to a large extent be described as motion along one or a few coherent degrees of freedom. The states involved are inherent to the protein, in the sense that they are visited by the protein also in the absence of effector ligands. Previously, we developed the measure binding leverage to find sites where ligand binding can shift the conformational equilibrium of a protein. Binding leverage is calculated for a set of motion vectors representing independent conformational degrees of freedom. In this paper, to analyze allosteric communication between binding sites, we introduce the concept of leverage coupling, based on the assumption that only pairs of sites that couple to the same conformational degrees of freedom can be allosterically connected. We demonstrate how leverage coupling can be used to analyze allosteric communication in a range of enzymes (regulated by both ligand binding and post-translational modifications) and huge molecular machines such as chaperones. Leverage coupling can be calculated for any protein structure to analyze both biological and latent catalytic and regulatory sites. PMID:22174669

  1. Allosteric transition and binding of small molecule effectors causes curvature change in central β-sheets of selected enzymes

    PubMed Central

    Tolonen, Ellen; Bueno, Brenda; Kulshreshta, Sanjeev; Cieplak, Piotr; Argáez, Miguel; Velázquez, Leticia

    2014-01-01

    A quantitative description of allosteric transition remains a significant science challenge. Many allosteric enzymes contain a central β-sheet in their catalytic domain. When an allosteric protein undergoes the transition between T (tense) and R (relaxed) allosteric states, this central β-sheet undergoes a conformational change. A traditional method of measuring this change, the root mean square deviation (RMSD), appears to be inadequate to describe such changes in meaningful quantitative manner. We designed a novel quantitative method to demonstrate this conformational transition by measuring the change in curvature of the central β-sheet when enzymes transition between allosteric states. The curvature was established by calculating the semiaxes of a 3-D hyperboloid fitted by least squares to the Cα atomic positions of the β-sheet. The two enzymes selected for this study, fructose 1,6-bisphosphatase (FBPase) from pig kidney and aspartate carbamoyltransferase (ATCase) from E. coli, showed while transitioning between the allosteric states (T ⇔ R) a notable change in β-sheet curvature (∼5%) that results in a large lateral shift at the sheet's edge, which is necessary to convey the signal. The results suggest that the β-sheet participates in storing elastic energy associated with the transition. Establishing a tentative link between the energetics of the β-sheet in different allosteric states provides a more objective basis for the naming convention of allosteric states (tense or relaxed), and provides insight into the hysteretic nature of the transition. The approach presented here allows for a better understanding of the internal dynamics of allosteric enzymes by defining the domains that directly participate in the transition. PMID:20602244

  2. Allosteric transition and binding of small molecule effectors causes curvature change in central β-sheets of selected enzymes.

    PubMed

    Tolonen, Ellen; Bueno, Brenda; Kulshreshta, Sanjeev; Cieplak, Piotr; Argáez, Miguel; Velázquez, Leticia; Stec, Boguslaw

    2011-04-01

    A quantitative description of allosteric transition remains a significant science challenge. Many allosteric enzymes contain a central β-sheet in their catalytic domain. When an allosteric protein undergoes the transition between T (tense) and R (relaxed) allosteric states, this central β-sheet undergoes a conformational change. A traditional method of measuring this change, the root mean square deviation (RMSD), appears to be inadequate to describe such changes in meaningful quantitative manner. We designed a novel quantitative method to demonstrate this conformational transition by measuring the change in curvature of the central β-sheet when enzymes transition between allosteric states. The curvature was established by calculating the semiaxes of a 3-D hyperboloid fitted by least squares to the Cα atomic positions of the β-sheet. The two enzymes selected for this study, fructose 1,6-bisphosphatase (FBPase) from pig kidney and aspartate carbamoyltransferase (ATCase) from E. coli, showed while transitioning between the allosteric states (T ⇔ R) a notable change in β-sheet curvature (∼5%) that results in a large lateral shift at the sheet's edge, which is necessary to convey the signal. The results suggest that the β-sheet participates in storing elastic energy associated with the transition. Establishing a tentative link between the energetics of the β-sheet in different allosteric states provides a more objective basis for the naming convention of allosteric states (tense or relaxed), and provides insight into the hysteretic nature of the transition. The approach presented here allows for a better understanding of the internal dynamics of allosteric enzymes by defining the domains that directly participate in the transition. PMID:20602244

  3. The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4.

    PubMed

    Denisov, Ilia G; Mak, Piotr J; Grinkova, Yelena V; Bastien, Dominic; Bérubé, Gervais; Sligar, Stephen G; Kincaid, James R

    2016-05-01

    Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug-drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV-VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis-TST2 and trans-TST2. We directly demonstrate that the binding of two steroid molecules, which can assume multiple possible configurations inside the substrate binding pocket of monomeric CYP3A4, can lead to active site structural changes that affect functional properties. Using resonance Raman spectroscopy, we have documented perturbations in the ferric and Fe-CO states by these substrates, and compared these results with effects caused by binding of monomeric TST. While the binding of trans-TST2 yields results similar to those obtained with monomeric TST, the binding of cis-TST2 is much tighter and results in significantly more pronounced conformational changes of the porphyrin side chains and Fe-CO unit. In addition, binding of an additional monomeric TST molecule in the remote allosteric site significantly improves binding affinity and the overall spin shift for CYP3A4 with trans-TST2 dimer bound inside the substrate binding pocket. This result provides the first direct evidence for an allosteric effect of the peripheral binding site at the protein-membrane interface on the functional properties of CYP3A4. PMID:26774838

  4. Identification of amino acid residues that form part of the ligand-binding pocket of integrin alpha5 beta1.

    PubMed

    Mould, A P; Burrows, L; Humphries, M J

    1998-10-01

    Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel ligand peptide for integrin alpha5 beta1, which blocks alpha5 beta1-mediated cell adhesion to fibronectin (Koivunen, E., Wang, B., and Ruoslahti, E. (1994) J. Cell Biol. 124, 373-380). Here we have localized the binding site for RRETAWA on alpha5 beta1 using inhibitory monoclonal antibodies (mAbs) and site-directed mutagenesis. A cyclic peptide containing this sequence (*CRRETAWAC*) had little effect on the binding of most anti-alpha5 and anti-beta1 mAbs to alpha5 beta1 but completely blocked binding of the anti-alpha5 mAb 16 in a directly competitive manner. Hence, the binding site of RRETAWA appears to closely overlap with the epitope of mAb 16. *CRRETAWAC* also acted as a direct competitive inhibitor of the binding of Arg-Gly-Asp (RGD)-containing fibronectin fragments to alpha5 beta1, suggesting that the binding site for RRETAWA is also closely overlapping with that for RGD. However, differences between the binding sites of RRETAWA and RGD were apparent in that (i) RGD peptides allosterically inhibited the binding of mAb 16 to alpha5 beta1, and (ii) several mAbs that perturbed binding of alpha5 beta1 to RGD had little effect on binding of alpha5 beta1 to RRETAWA. A double mutation in alpha5 (S156G/W157S) blocked the interaction of both RRETAWA and mAb 16 with alpha5 beta1 but had no effect on fibronectin binding or on the binding of other anti-alpha5 mAbs. Ser156-Trp157 is located near the apex of a putative loop region on the upper surface of a predicted beta-propeller structure formed by the NH2-terminal repeats of alpha5. Our findings suggest that this sequence forms part of the ligand-binding pocket of alpha5 beta1. Furthermore, as Ser156-Trp157 is unique to the alpha5 subunit, it may be responsible for the specific recognition of RRETAWA by alpha5 beta1. PMID:9748233

  5. Conformational dynamics and thermodynamics of protein-ligand binding studied by NMR relaxation.

    PubMed

    Akke, Mikael

    2012-04-01

    Protein conformational dynamics can be critical for ligand binding in two ways that relate to kinetics and thermodynamics respectively. First, conformational transitions between different substates can control access to the binding site (kinetics). Secondly, differences between free and ligand-bound states in their conformational fluctuations contribute to the entropy of ligand binding (thermodynamics). In the present paper, I focus on the second topic, summarizing our recent results on the role of conformational entropy in ligand binding to Gal3C (the carbohydrate-recognition domain of galectin-3). NMR relaxation experiments provide a unique probe of conformational entropy by characterizing bond-vector fluctuations at atomic resolution. By monitoring differences between the free and ligand-bound states in their backbone and side chain order parameters, we have estimated the contributions from conformational entropy to the free energy of binding. Overall, the conformational entropy of Gal3C increases upon ligand binding, thereby contributing favourably to the binding affinity. Comparisons with the results from isothermal titration calorimetry indicate that the conformational entropy is comparable in magnitude to the enthalpy of binding. Furthermore, there are significant differences in the dynamic response to binding of different ligands, despite the fact that the protein structure is virtually identical in the different protein-ligand complexes. Thus both affinity and specificity of ligand binding to Gal3C appear to depend in part on subtle differences in the conformational fluctuations that reflect the complex interplay between structure, dynamics and ligand interactions. PMID:22435823

  6. MODELING THE BINDING OF THE METABOLITES OF SOME POLYCYCLIC AROMTIC HYDROCARBONS TO THE LIGAND BINDING DOMAIN OF THE ESTROGEN RECEPTOR

    EPA Science Inventory

    Modeling the binding of the metabolites of some Polycyclic Aromatic Hydrocarbons to the ligand binding domain of the estrogen receptor
    James Rabinowitz, Stephen Little, Katrina Brown, National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC; Un...

  7. Ligand Binding to Chlorite Dismutase from Magnetospirillum sp.

    PubMed

    De Schutter, Amy; Correia, Hugo D; Freire, Diana M; Rivas, María G; Rizzi, Alberto; Santos-Silva, Teresa; González, Pablo J; Van Doorslaer, Sabine

    2015-10-29

    Chlorite dismutase (Cld) catalyzes the reduction of chlorite to chloride and dioxygen. Here, the ligand binding to Cld of Magnetospirillum sp. (MaCld) is investigated with X-ray crystallography and electron paramagnetic resonance (EPR). EPR reveals a large heterogeneity in the structure of wild-type MaCld, showing a variety of low- and high-spin ferric heme forms. Addition of an axial ligand, such as azide or imidazole, removes this heterogeneity almost entirely. This is in line with the two high resolution crystal structures of MaCld obtained in the presence of azide and thiocyanate that show the coordination of the ligands to the heme iron. The crystal structure of the MaCld-azide complex reveals a single well-defined orientation of the azide molecule in the heme pocket. EPR shows, however, a pH-dependent heme structure, probably due to acid-base transitions of the surrounding amino-acid residues stabilizing azide. For the azide and imidazole complex of MaCld, the hyperfine and nuclear quadrupole interactions with the close-by (14)N and (1)H nuclei are determined using pulsed EPR. These values are compared to the corresponding data for the low-spin forms observed in the ferric wild-type MaCld and to existing EPR data on azide and imidazole complexes of other heme proteins. PMID:26287794

  8. Allosteric Regulation of Fibronectin/α5β1 Interaction by Fibronectin-Binding MSCRAMMs

    PubMed Central

    Liang, Xiaowen; Garcia, Brandon L.; Visai, Livia; Prabhakaran, Sabitha; Meenan, Nicola A. G.; Potts, Jennifer R.; Humphries, Martin J.; Höök, Magnus

    2016-01-01

    Adherence of microbes to host tissues is a hallmark of infectious disease and is often mediated by a class of adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Numerous pathogens express MSCRAMMs that specifically bind the heterodimeric human glycoprotein fibronectin (Fn). In addition to roles in adhesion, Fn-binding MSCRAMMs exploit physiological Fn functions. For example, several pathogens can invade host cells by a mechanism whereby MSCRAMM-bound Fn bridges interaction with α5β1 integrin. Here, we investigate two Fn-binding MSCRAMMs, FnBPA (Staphylococcus aureus) and BBK32 (Borrelia burgdorferi) to probe structure-activity relationships of MSCRAMM-induced Fn/α5β1integrin activation. Circular dichroism, fluorescence resonance energy transfer, and dynamic light scattering techniques uncover a conformational rearrangement of Fn involving domains distant from the MSCRAMM binding site. Surface plasmon resonance experiments demonstrate a significant enhancement of Fn/α5β1 integrin affinity in the presence of FnBPA or BBK32. Detailed kinetic analysis of these interactions reveal that this change in affinity can be attributed solely to an increase in the initial Fn/α5β1 on-rate and that this rate-enhancement is dependent on high-affinity Fn-binding by MSCRAMMs. These data implicate MSCRAMM-induced perturbation of specific intramolecular contacts within the Fn heterodimer resulting in activation by exposing previously cryptic α5β1 interaction motifs. By correlating structural changes in Fn to a direct measurement of increased Fn/α5β1 affinity, this work significantly advances our understanding of the structural basis for the modulation of integrin function by Fn-binding MSCRAMMs. PMID:27434228

  9. The Movable Type Method Applied to Protein-Ligand Binding.

    PubMed

    Zheng, Zheng; Ucisik, Melek N; Merz, Kenneth M

    2013-12-10

    Accurately computing the free energy for biological processes like protein folding or protein-ligand association remains a challenging problem. Both describing the complex intermolecular forces involved and sampling the requisite configuration space make understanding these processes innately difficult. Herein, we address the sampling problem using a novel methodology we term "movable type". Conceptually it can be understood by analogy with the evolution of printing and, hence, the name movable type. For example, a common approach to the study of protein-ligand complexation involves taking a database of intact drug-like molecules and exhaustively docking them into a binding pocket. This is reminiscent of early woodblock printing where each page had to be laboriously created prior to printing a book. However, printing evolved to an approach where a database of symbols (letters, numerals, etc.) was created and then assembled using a movable type system, which allowed for the creation of all possible combinations of symbols on a given page, thereby, revolutionizing the dissemination of knowledge. Our movable type (MT) method involves the identification of all atom pairs seen in protein-ligand complexes and then creating two databases: one with their associated pairwise distant dependent energies and another associated with the probability of how these pairs can combine in terms of bonds, angles, dihedrals and non-bonded interactions. Combining these two databases coupled with the principles of statistical mechanics allows us to accurately estimate binding free energies as well as the pose of a ligand in a receptor. This method, by its mathematical construction, samples all of configuration space of a selected region (the protein active site here) in one shot without resorting to brute force sampling schemes involving Monte Carlo, genetic algorithms or molecular dynamics simulations making the methodology extremely efficient. Importantly, this method explores the free

  10. The Movable Type Method Applied to Protein-Ligand Binding

    PubMed Central

    Zheng, Zheng; Ucisik, Melek N.; Merz, Kenneth M.

    2013-01-01

    Accurately computing the free energy for biological processes like protein folding or protein-ligand association remains a challenging problem. Both describing the complex intermolecular forces involved and sampling the requisite configuration space make understanding these processes innately difficult. Herein, we address the sampling problem using a novel methodology we term “movable type”. Conceptually it can be understood by analogy with the evolution of printing and, hence, the name movable type. For example, a common approach to the study of protein-ligand complexation involves taking a database of intact drug-like molecules and exhaustively docking them into a binding pocket. This is reminiscent of early woodblock printing where each page had to be laboriously created prior to printing a book. However, printing evolved to an approach where a database of symbols (letters, numerals, etc.) was created and then assembled using a movable type system, which allowed for the creation of all possible combinations of symbols on a given page, thereby, revolutionizing the dissemination of knowledge. Our movable type (MT) method involves the identification of all atom pairs seen in protein-ligand complexes and then creating two databases: one with their associated pairwise distant dependent energies and another associated with the probability of how these pairs can combine in terms of bonds, angles, dihedrals and non-bonded interactions. Combining these two databases coupled with the principles of statistical mechanics allows us to accurately estimate binding free energies as well as the pose of a ligand in a receptor. This method, by its mathematical construction, samples all of configuration space of a selected region (the protein active site here) in one shot without resorting to brute force sampling schemes involving Monte Carlo, genetic algorithms or molecular dynamics simulations making the methodology extremely efficient. Importantly, this method explores the

  11. Effect of ECM Stiffness on Integrin-Ligand Binding Strength

    NASA Astrophysics Data System (ADS)

    Thomas, Gawain; Wen, Qi

    2014-03-01

    Many studies have shown that cells respond to the stiffness of their extracellular matrix (ECM). However, the mechanism of this stiffness sensing is not fully understood. We believe that cells probe stiffness by applying intracellular force to the ECM via the integrin-mediated adhesions. The linkage of integrins to the cytoskeleton has been modeled as a slip clutch, which has been shown to affect focal adhesion formation and hence force transmission in a stiffness dependent manner. In contrast, the bonds between integrins and ECM have been characterized as ``catch bonds.'' It is unclear how ECM viscoelasticity affects these catch bonds. We report, for the first time, the effects of ECM stiffness on the binding strength of integrins to ECM ligands by measuring the rupture force of individual integrin-ligand bonds of cells on collagen-coated polyacrylamide gels. Results show that the integrin-collagen bonds of 3T3 fibroblasts are nearly four times stronger on a stiff (30 kPa) gel than on a soft (3 kPa) gel. The stronger integrin bonds on stiffer substrates can promote focal adhesion formation. This suggests that the substrate stiffness regulates the cell-ECM adhesions not only by affecting the cytoskeleton-integrin links but also by modulating the binding of integrins to the ECM.

  12. Study on effects of molecular crowding on G-quadruplex-ligand binding and ligand-mediated telomerase inhibition.

    PubMed

    Yaku, Hidenobu; Murashima, Takashi; Tateishi-Karimata, Hisae; Nakano, Shu-ichi; Miyoshi, Daisuke; Sugimoto, Naoki

    2013-11-01

    The telomere G-quadruplex-binding and telomerase-inhibiting capacity of two cationic (TMPyP4 and PIPER) and two anionic (phthalocyanine and Hemin) G-quadruplex-ligands were examined under conditions of molecular crowding (MC). Osmotic experiments showed that binding of the anionic ligands, which bind to G-quadruplex DNA via π-π stacking interactions, caused some water molecules to be released from the G-quadruplex/ligand complex; in contrast, a substantial number of water molecules were taken up upon electrostatic binding of the cationic ligands to G-quadruplex DNA. These behaviors of water molecules maintained or reduced the binding affinity of the anionic and the cationic ligands, respectively, under MC conditions. Consequently, the anionic ligands (phthalocyanine and Hemin) robustly inhibited telomerase activity even with MC; in contrast, the inhibition of telomerase caused by cationic TMPyP4 was drastically reduced by MC. These results allow us to conclude that the binding of G-quadruplex-ligands to G-quadruplex via non-electrostatic interactions is preferable for telomerase inhibition under physiological conditions. PMID:23562626

  13. Protein-Ligand Binding Detected by Terahertz Spectroscopy

    NASA Astrophysics Data System (ADS)

    Knab, J.; Chen, J. Y.; Mader, M.; Markelz, A.

    2004-03-01

    Established measures of protein flexibility through the B-factor use time intensive and facility limited techniques such as X-ray crystallography, NMR structure analysis and inelastic neutron scattering. We demonstrate a novel technique that may be used for determination of ligand binding for proteins as well as a measure of protein flexibility. Using the method of terahertz (THz) time domain spectroscopy, we measured the far infrared dielectric response as a function of the binding of N (1-4)-acetylglucosamine (NAG) to hen egg white lysozyme (HEWL). Vibrational modes associated with tertiary structure conformational motions lay in the THz frequency range. The THz dielectric response reflects the density and amplitude of these normal modes through dipole coupling. Transmission measurements on thin films show that while there is no change in the real part of the refractive index as a function of binding, there is a decrease in the absorbance for the HEWL+NAG thin films relative to HEWL films. This decrease can be attributed to a reduction in the flexibility of the protein with binding. These results are compared to calculated absorbance spectra.

  14. Probing the Sophisticated Synergistic Allosteric Regulation of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis Using ᴅ-Amino Acids

    PubMed Central

    Reichau, Sebastian; Blackmore, Nicola J.; Jiao, Wanting; Parker, Emily J.

    2016-01-01

    Chirality plays a major role in recognition and interaction of biologically important molecules. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of the shikimate pathway, which is responsible for the synthesis of aromatic amino acids in bacteria and plants, and a potential target for the development of antibiotics and herbicides. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) displays an unprecedented complexity of allosteric regulation, with three interdependent allosteric binding sites and a ternary allosteric response to combinations of the aromatic amino acids l-Trp, l-Phe and l-Tyr. In order to further investigate the intricacies of this system and identify key residues in the allosteric network of MtuDAH7PS, we studied the interaction of MtuDAH7PS with aromatic amino acids that bear the non-natural d-configuration, and showed that the d-amino acids do not elicit an allosteric response. We investigated the binding mode of d-amino acids using X-ray crystallography, site directed mutagenesis and isothermal titration calorimetry. Key differences in the binding mode were identified: in the Phe site, a hydrogen bond between the amino group of the allosteric ligands to the side chain of Asn175 is not established due to the inverted configuration of the ligands. In the Trp site, d-Trp forms no interaction with the main chain carbonyl group of Thr240 and less favourable interactions with Asn237 when compared to the l-Trp binding mode. Investigation of the MtuDAH7PSN175A variant further supports the hypothesis that the lack of key interactions in the binding mode of the aromatic d-amino acids are responsible for the absence of an allosteric response, which gives further insight into which residues of MtuDAH7PS play a key role in the transduction of the allosteric signal. PMID:27128682

  15. Probing the Sophisticated Synergistic Allosteric Regulation of Aromatic Amino Acid Biosynthesis in Mycobacterium tuberculosis Using ᴅ-Amino Acids.

    PubMed

    Reichau, Sebastian; Blackmore, Nicola J; Jiao, Wanting; Parker, Emily J

    2016-01-01

    Chirality plays a major role in recognition and interaction of biologically important molecules. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of the shikimate pathway, which is responsible for the synthesis of aromatic amino acids in bacteria and plants, and a potential target for the development of antibiotics and herbicides. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) displays an unprecedented complexity of allosteric regulation, with three interdependent allosteric binding sites and a ternary allosteric response to combinations of the aromatic amino acids l-Trp, l-Phe and l-Tyr. In order to further investigate the intricacies of this system and identify key residues in the allosteric network of MtuDAH7PS, we studied the interaction of MtuDAH7PS with aromatic amino acids that bear the non-natural d-configuration, and showed that the d-amino acids do not elicit an allosteric response. We investigated the binding mode of d-amino acids using X-ray crystallography, site directed mutagenesis and isothermal titration calorimetry. Key differences in the binding mode were identified: in the Phe site, a hydrogen bond between the amino group of the allosteric ligands to the side chain of Asn175 is not established due to the inverted configuration of the ligands. In the Trp site, d-Trp forms no interaction with the main chain carbonyl group of Thr240 and less favourable interactions with Asn237 when compared to the l-Trp binding mode. Investigation of the MtuDAH7PSN175A variant further supports the hypothesis that the lack of key interactions in the binding mode of the aromatic d-amino acids are responsible for the absence of an allosteric response, which gives further insight into which residues of MtuDAH7PS play a key role in the transduction of the allosteric signal. PMID:27128682

  16. Discovery and SAR of a novel series of metabotropic glutamate receptor 5 positive allosteric modulators with high ligand efficiency

    PubMed Central

    Turlington, Mark; Noetzel, Meredith J.; Bridges, Thomas M.; Vinson, Paige N.; Steckler, Thomas; Lavreysen, Hilde; Mackie, Claire; Bartolomé-Nebreda, José M.; Conde-Ceide, Susana; Tong, Han Min; Macdonald, Gregor J.; Daniels, J. Scott; Jones, Carrie K.; Niswender, Colleen M.; Conn, P. Jeffrey; Lindsley, Craig W.; Stauffer, Shaun R.

    2014-01-01

    We report the optimization of a series of novel metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) from a 5,6-bicyclic class of dihydropyrazolo[1,5-a]pyridin-4(5H)-ones containing a phenoxymethyl linker. Studies focused on a survey of nonamide containing hydrogen bond accepting (HBA) pharmacophore replacements. A highly potent and selective PAM, 2-(phenoxymethyl)-6,7-dihydropyrazolo[1,5-a]pyridin-4(5H)-one 11, VU0462054), bearing a simple ketone moiety, was identified (LE = 0.52, LELP = 3.2). In addition, hydroxyl, difluoro, ether, and amino variations were examined. Despite promising lead properties and exploration of alternative core heterocycles, linkers, and ketone replacements, oxidative metabolism and in vivo clearance remained problematic for the series. PMID:24961642

  17. The Dynamic Process of Drug-GPCR Binding at Either Orthosteric or Allosteric Sites Evaluated by Metadynamics

    PubMed Central

    Schneider, Sebastian; Provasi, Davide; Filizola, Marta

    2015-01-01

    Major advances in G Protein-Coupled Receptor (GPCR) structural biology over the past few years have yielded a significant number of high-resolution crystal structures for several different receptor subtypes. This dramatic increase in GPCR structural information has underscored the use of automated docking algorithms for the discovery of novel ligands that can eventually be developed into improved therapeutics. However, these algorithms are often unable to discriminate between different, yet energetically similar, poses because of their relatively simple scoring functions. Here, we describe a metadynamics-based approach to study the dynamic process of ligand binding to/unbinding from GPCRs with a higher level of accuracy and yet satisfying efficiency. PMID:26260607

  18. The first crystal structure of a dTTP-bound deoxycytidylate deaminase validates and details the allosteric-inhibitor binding site.

    PubMed

    Marx, Ailie; Alian, Akram

    2015-01-01

    Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH2 in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed. PMID:25404739

  19. The First Crystal Structure of a dTTP-bound Deoxycytidylate Deaminase Validates and Details the Allosteric-Inhibitor Binding Site*

    PubMed Central

    Marx, Ailie; Alian, Akram

    2015-01-01

    Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH2 in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed. PMID:25404739

  20. Regulation of transcription attenuation and translation initiation by allosteric control of an RNA-binding protein: the Bacillus subtilis TRAP protein.

    PubMed

    Babitzke, Paul

    2004-04-01

    Tryptophan allosterically controls the 11-subunit trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis. When activated by tryptophan, TRAP binds to multiple trinucleotide repeats in target transcripts. TRAP is responsible for the decision to terminate transcription in the leader region of the trpEDCFBA operon or to allow transcription to proceed into the structural genes. TRAP also regulates translation of trpE by promoting formation of an RNA structure that prevents ribosome binding. In addition, bound TRAP regulates translation initiation of pabA, trpP and ycbK by directly blocking ribosome binding. The anti-TRAP protein inhibits TRAP activity by competing with RNA for the RNA binding surface of TRAP. PMID:15063849

  1. Hemoglobin isoform differentiation and allosteric regulation of oxygen binding in the turtle, Trachemys scripta

    PubMed Central

    Damsgaard, Christian; Storz, Jay F.; Hoffmann, Federico G.

    2013-01-01

    When freshwater turtles acclimatize to winter hibernation, there is a gradual transition from aerobic to anaerobic metabolism, which may require adjustments of blood O2 transport before turtles become anoxic. Here, we report the effects of protons, anionic cofactors, and temperature on the O2-binding properties of isolated hemoglobin (Hb) isoforms, HbA and HbD, in the turtle Trachemys scripta. We determined the primary structures of the constituent subunits of the two Hb isoforms, and we related the measured functional properties to differences in O2 affinity between untreated hemolysates from turtles that were acclimated to normoxia and anoxia. Our data show that HbD has a consistently higher O2 affinity compared with HbA, whereas Bohr and temperature effects, as well as thiol reactivity, are similar. Although sequence data show amino acid substitutions at two known β-chain ATP-binding site positions, we find high ATP affinities for both Hb isoforms, suggesting an alternative and stronger binding site for ATP. The high ATP affinities indicate that, although ATP levels decrease in red blood cells of turtles acclimating to anoxia, the O2 affinity would remain largely unchanged, as confirmed by O2-binding measurements of untreated hemolysates from normoxic and anoxic turtles. Thus, the increase in blood-O2 affinity that accompanies winter acclimation is mainly attributable to a decrease in temperature rather than in concentrations of organic phosphates. This is the first extensive study on freshwater turtle Hb isoforms, providing molecular evidence for adaptive changes in O2 transport associated with acclimation to severe hypoxia. PMID:23986362

  2. Modeling spatial correlation of DNA deformations: Allosteric effects of DNA protein binding

    NASA Astrophysics Data System (ADS)

    Xu, Xinliang; Cao, Jianshu; Hao Ge Collaboration; X. Sunney Xie Collaboration

    2013-03-01

    We report a study of DNA deformations by a coarse grained mechanical model. Recent single molecule experimental studies show that when DNA molecule is deformed by its binding to a protein, the binding affinity of a second protein at distance L away from the first binding site is altered. To explain this observation, the relaxation of deformation along the DNA chain is examined. Our method predicts a general exponentially decaying behavior for differenct deformation modes. As an example, inter-helical distance deformation is studied in details, and is found to decay at a previously unknown lengthscale of 10 base pairs as a result of the balance between inter and intra DNA strand energy. This lengthscale is in good agreement with the said single molecule experimental observation. This model of local deformation relaxation helps us better understand many important issues in DNA such as the enhanced flexibility of DNA at short lengthscales and DNA repair mechanism inside cells. Biodynamic Optical Imaging Center, Peking University

  3. Biased Agonism and Biased Allosteric Modulation at the CB1 Cannabinoid Receptor.

    PubMed

    Khajehali, Elham; Malone, Daniel T; Glass, Michelle; Sexton, Patrick M; Christopoulos, Arthur; Leach, Katie

    2015-08-01

    CB1 cannabinoid receptors (CB1Rs) are attractive therapeutic targets for numerous central nervous system disorders. However, clinical application of cannabinoid ligands has been hampered owing to their adverse on-target effects. Ligand-biased signaling from, and allosteric modulation of, CB1Rs offer pharmacological approaches that may enable the development of improved CB1R drugs, through modulation of only therapeutically desirable CB1R signaling pathways. There is growing evidence that CB1Rs are subject to ligand-biased signaling and allosterism. Therefore, in the present study, we quantified ligand-biased signaling and allosteric modulation at CB1Rs. Cannabinoid agonists displayed distinct biased signaling profiles at CB1Rs. For instance, whereas 2-arachidonylglycerol and WIN55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone] showed little preference for inhibition of cAMP and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2), N-arachidonoylethanolamine (anandamide), methanandamide, CP55940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol], and HU-210 [11-hydroxy-Δ(8)-THC-dimethylheptyl] were biased toward cAMP inhibition. The small-molecule allosteric modulator Org27569 [5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide] displayed biased allosteric effects by blocking cAMP inhibition mediated by all cannabinoid ligands tested, at the same time having little or no effect on ERK1/2 phosphorylation mediated by a subset of these ligands. Org27569 also displayed negative binding cooperativity with [(3)H]SR141716A [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide]; however, it had minimal effects on binding of cannabinoid agonists. Furthermore, we highlight the need to validate the reported allosteric effects of the endogenous ligands lipoxin A4 and

  4. Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.

    PubMed

    Kahsai, Alem W; Wisler, James W; Lee, Jungmin; Ahn, Seungkirl; Cahill Iii, Thomas J; Dennison, S Moses; Staus, Dean P; Thomsen, Alex R B; Anasti, Kara M; Pani, Biswaranjan; Wingler, Laura M; Desai, Hemant; Bompiani, Kristin M; Strachan, Ryan T; Qin, Xiaoxia; Alam, S Munir; Sullenger, Bruce A; Lefkowitz, Robert J

    2016-09-01

    G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function through unique, previously unappreciated mechanisms. Here, using a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, the β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs. PMID:27398998

  5. Detection of functional ligand-binding events using synchrotron x-ray scattering.

    SciTech Connect

    Rodi, D. J.; Mandava, S.; Gore, D. B.; Makowski, L.; Fischetti, R. F.; Biosciences Division; IIT

    2007-10-01

    Small-molecule ligands that change the structure of a protein are likely to affect its function, whereas those causing no structural change are less likely to be functional. Wide-angle x-ray scattering (WAXS) can be easily carried out on proteins and small molecules in solution in the absence of chemical tags or derivatives. The authors demonstrate that WAXS is a sensitive probe of ligand binding to proteins in solution and can distinguish between nonfunctional and productive binding. Furthermore, similar ligand-binding modes translate into similar scattering patterns. This approach has high potential as a novel, generic, low-throughput assay for functional ligand binding.

  6. Ligand deconstruction: Why some fragment binding positions are conserved and others are not

    PubMed Central

    Kozakov, Dima; Hall, David R.; Jehle, Stefan; Luo, Lingqi; Ochiana, Stefan O.; Jones, Elizabeth V.; Pollastri, Michael; Allen, Karen N.; Whitty, Adrian; Vajda, Sandor

    2015-01-01

    Fragment-based drug discovery (FBDD) relies on the premise that the fragment binding mode will be conserved on subsequent expansion to a larger ligand. However, no general condition has been established to explain when fragment binding modes will be conserved. We show that a remarkably simple condition can be developed in terms of how fragments coincide with binding energy hot spots—regions of the protein where interactions with a ligand contribute substantial binding free energy—the locations of which can easily be determined computationally. Because a substantial fraction of the free energy of ligand binding comes from interacting with the residues in the energetically most important hot spot, a ligand moiety that sufficiently overlaps with this region will retain its location even when other parts of the ligand are removed. This hypothesis is supported by eight case studies. The condition helps identify whether a protein is suitable for FBDD, predicts the size of fragments required for screening, and determines whether a fragment hit can be extended into a higher affinity ligand. Our results show that ligand binding sites can usefully be thought of in terms of an anchor site, which is the top-ranked hot spot and dominates the free energy of binding, surrounded by a number of weaker satellite sites that confer improved affinity and selectivity for a particular ligand and that it is the intrinsic binding potential of the protein surface that determines whether it can serve as a robust binding site for a suitably optimized ligand. PMID:25918377

  7. Ligand deconstruction: Why some fragment binding positions are conserved and others are not.

    PubMed

    Kozakov, Dima; Hall, David R; Jehle, Stefan; Jehle, Sefan; Luo, Lingqi; Ochiana, Stefan O; Jones, Elizabeth V; Pollastri, Michael; Allen, Karen N; Whitty, Adrian; Vajda, Sandor

    2015-05-19

    Fragment-based drug discovery (FBDD) relies on the premise that the fragment binding mode will be conserved on subsequent expansion to a larger ligand. However, no general condition has been established to explain when fragment binding modes will be conserved. We show that a remarkably simple condition can be developed in terms of how fragments coincide with binding energy hot spots--regions of the protein where interactions with a ligand contribute substantial binding free energy--the locations of which can easily be determined computationally. Because a substantial fraction of the free energy of ligand binding comes from interacting with the residues in the energetically most important hot spot, a ligand moiety that sufficiently overlaps with this region will retain its location even when other parts of the ligand are removed. This hypothesis is supported by eight case studies. The condition helps identify whether a protein is suitable for FBDD, predicts the size of fragments required for screening, and determines whether a fragment hit can be extended into a higher affinity ligand. Our results show that ligand binding sites can usefully be thought of in terms of an anchor site, which is the top-ranked hot spot and dominates the free energy of binding, surrounded by a number of weaker satellite sites that confer improved affinity and selectivity for a particular ligand and that it is the intrinsic binding potential of the protein surface that determines whether it can serve as a robust binding site for a suitably optimized ligand. PMID:25918377

  8. Ligand Binding and Substrate Discrimination by UDP-Galactopyranose Mutase

    SciTech Connect

    Gruber, Todd D.; Borrok, M. Jack; Westler, William M.; Forest, Katrina T.; Kiessling, Laura L.

    2009-07-31

    Galactofuranose (Galf) residues are present in cell wall glycoconjugates of numerous pathogenic microbes. Uridine 5{prime}-diphosphate (UDP) Galf, the biosynthetic precursor of Galf-containing glycoconjugates, is produced from UDP-galactopyranose (UDP-Galp) by the flavoenzyme UDP-galactopyranose mutase (UGM). The gene encoding UGM (glf) is essential for the viability of pathogens, including Mycobacterium tuberculosis, and this finding underscores the need to understand how UGM functions. Considerable effort has been devoted to elucidating the catalytic mechanism of UGM, but progress has been hindered by a lack of structural data for an enzyme-substrate complex. Such data could reveal not only substrate binding interactions but how UGM can act preferentially on two very different substrates, UDP-Galp and UDP-Galf, yet avoid other structurally related UDP sugars present in the cell. Herein, we describe the first structure of a UGM-ligand complex, which provides insight into the catalytic mechanism and molecular basis for substrate selectivity. The structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP-glucose (UDP-Glc) was solved by X-ray crystallographic methods and refined to 2.5 {angstrom} resolution. The ligand is proximal to the cofactor, a finding that is consistent with a proposed mechanism in which the reduced flavin engages in covalent catalysis. Despite this proximity, the glucose ring of the substrate analog is positioned such that it disfavors covalent catalysis. This orientation is consistent with data indicating that UDP-Glc is not a substrate for UGM. The relative binding orientations of UDP-Galp and UDP-Glc were compared using saturation transfer difference NMR. The results indicate that the uridine moiety occupies a similar location in both ligand complexes, and this relevant binding mode is defined by our structural data. In contrast, the orientations of the glucose and galactose sugar moieties differ. To understand the

  9. Effects of ligand binding upon flexibility of proteins.

    PubMed

    Erman, Burak

    2015-05-01

    Binding of a ligand on a protein changes the flexibility of certain parts of the protein, which directly affects its function. These changes are not the same at each point, some parts become more flexible and some others become stiffer. Here, an equation is derived that gives the stiffness map for proteins. The model is based on correlations of fluctuations of pairs of points in proteins, which may be evaluated at different levels of refinement, ranging from all atom molecular dynamics to general elastic network models, including the simplest case of isotropic Gaussian Network Model. The latter is used, as an example, to evaluate the changes of stiffness upon dimerization of ACK1. PMID:25737428

  10. Ligand-binding assays for cyanobacterial neurotoxins targeting cholinergic receptors.

    PubMed

    Aráoz, Rómulo; Vilariño, Natalia; Botana, Luis M; Molgó, Jordi

    2010-07-01

    Toxic cyanobacterial blooms are a threat to public health because of the capacity of some cyanobacterial species to produce potent hepatotoxins and neurotoxins. Cyanobacterial neurotoxins are involved in the rapid death of wild and domestic animals by targeting voltage gated sodium channels and cholinergic synapses, including the neuromuscular junction. Anatoxin-a and its methylene homologue homoanatoxin-a are potent agonists of nicotinic acetylcholine receptors. Since the structural determination of anatoxin-a, several mass spectrometry-based methods have been developed for detection of anatoxin-a and, later, homoanatoxin-a. Mass spectrometry-based techniques provide accuracy, precision, selectivity, sensitivity, reproducibility, adequate limit of detection, and structural and quantitative information for analyses of cyanobacterial anatoxins from cultured and environmental cyanobacterial samples. However, these physicochemical techniques will only detect known toxins for which toxin standards are commercially available, and they require highly specialized laboratory personnel and expensive equipment. Receptor-based assays are functional methods that are based on the mechanism of action of a class of toxins and are thus, suitable tools for survey of freshwater reservoirs for cyanobacterial anatoxins. The competition between cyanobacterial anatoxins and a labelled ligand for binding to nicotinic acetylcholine receptors is measured radioactively or non-radioactively providing high-throughput screening formats for routine detection of this class of neurotoxins. The mouse bioassay is the method of choice for marine toxin monitoring, but has to be replaced by fully validated functional methods. In this paper we review the ligand-binding assays developed for detection of cyanobacterial and algal neurotoxins targeting the nicotinic acetylcholine receptors and for high-throughput screening of novel nicotinic agents. PMID:20238109

  11. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

    PubMed Central

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  12. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins.

    PubMed

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel's ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  13. Heat Capacity Changes and Disorder-to-Order Transitions in Allosteric Activation.

    PubMed

    Cressman, William J; Beckett, Dorothy

    2016-01-19

    Allosteric coupling in proteins is ubiquitous but incompletely understood, particularly in systems characterized by coupling over large distances. Binding of the allosteric effector, bio-5'-AMP, to the Escherichia coli biotin protein ligase, BirA, enhances the protein's dimerization free energy by -4 kcal/mol. Previous studies revealed that disorder-to-order transitions at the effector binding and dimerization sites, which are separated by 33 Å, are integral to functional coupling. Perturbations to the transition at the ligand binding site alter both ligand binding and coupled dimerization. Alanine substitutions in four loops on the dimerization surface yield a range of energetic effects on dimerization. A glycine to alanine substitution at position 142 in one of these loops results in a complete loss of allosteric coupling, disruption of the disorder-to-order transitions at both functional sites, and a decreased affinity for the effector. In this work, allosteric communication between the effector binding and dimerization surfaces in BirA was further investigated by performing isothermal titration calorimetry measurements on nine proteins with alanine substitutions in three dimerization surface loops. In contrast to BirAG142A, at 20 °C all variants bind to bio-5'-AMP with free energies indistinguishable from that measured for wild-type BirA. However, the majority of the variants exhibit altered heat capacity changes for effector binding. Moreover, the ΔCp values correlate with the dimerization free energies of the effector-bound proteins. These thermodynamic results, combined with structural information, indicate that allosteric activation of the BirA monomer involves formation of a network of intramolecular interactions on the dimerization surface in response to bio-5'-AMP binding at the distant effector binding site. PMID:26678378

  14. Dissecting Dynamic Allosteric Pathways Using Chemically Related Small-Molecule Activators.

    PubMed

    Lisi, George P; Manley, Gregory A; Hendrickson, Heidi; Rivalta, Ivan; Batista, Victor S; Loria, J Patrick

    2016-07-01

    The allosteric mechanism of the heterodimeric enzyme imidazole glycerol phosphate synthase was studied in detail with solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. We studied IGPS in complex with a series of allosteric activators corresponding to a large range of catalytic rate enhancements (26- to 4,900-fold), in which ligand binding is entropically driven. Conformational flexibility on the millisecond timescale plays a crucial role in intersubunit communication. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments probing Ile, Leu, and Val methyl groups reveal that the apo- and glutamine-mimicked complexes are static on the millisecond timescale. Domain-wide motions are stimulated in the presence of the allosteric activators. These studies, in conjunction with ligand titrations, demonstrate that the allosteric network is widely dispersed and varies with the identity of the effector. Furthermore, we find that stronger allosteric ligands create more conformational flexibility on the millisecond timescale throughout HisF. This domain-wide loosening leads to maximum catalytic activity. PMID:27238967

  15. The allosteric vestibule of a seven transmembrane helical receptor controls G-protein coupling

    PubMed Central

    Bock, Andreas; Merten, Nicole; Schrage, Ramona; Dallanoce, Clelia; Bätz, Julia; Klöckner, Jessica; Schmitz, Jens; Matera, Carlo; Simon, Katharina; Kebig, Anna; Peters, Lucas; Müller, Anke; Schrobang-Ley, Jasmin; Tränkle, Christian; Hoffmann, Carsten; De Amici, Marco; Holzgrabe, Ulrike; Kostenis, Evi; Mohr, Klaus

    2012-01-01

    Seven transmembrane helical receptors (7TMRs) modulate cell function via different types of G proteins, often in a ligand-specific manner. Class A 7TMRs harbour allosteric vestibules in the entrance of their ligand-binding cavities, which are in the focus of current drug discovery. However, their biological function remains enigmatic. Here we present a new strategy for probing and manipulating conformational transitions in the allosteric vestibule of label-free 7TMRs using the M2 acetylcholine receptor as a paradigm. We designed dualsteric agonists as 'tailor-made' chemical probes to trigger graded receptor activation from the acetylcholine-binding site while simultaneously restricting spatial flexibility of the receptor's allosteric vestibule. Our findings reveal for the first time that a 7TMR's allosteric vestibule controls the extent of receptor movement to govern a hierarchical order of G-protein coupling. This is a new concept assigning a biological role to the allosteric vestibule for controlling fidelity of 7TMR signalling. PMID:22948826

  16. The allosteric communication pathways in KIX domain of CBP

    PubMed Central

    Palazzesi, Ferruccio; Barducci, Alessandro; Tollinger, Martin; Parrinello, Michele

    2013-01-01

    Allosteric regulation plays an important role in a myriad of biomacromolecular processes. Specifically, in a protein, the process of allostery refers to the transmission of a local perturbation, such as ligand binding, to a distant site. Decades after the discovery of this phenomenon, models built on static images of proteins are being reconsidered with the knowledge that protein dynamics plays an important role in its function. Molecular dynamics simulations are a valuable tool for studying complex biomolecular systems, providing an atomistic description of their structure and dynamics. Unfortunately, their predictive power has been limited by the complexity of the biomolecule free-energy surface and by the length of the allosteric timescale (in the order of milliseconds). In this work, we are able to probe the origins of the allosteric changes that transcription factor mixed lineage leukemia (MLL) causes to the interactions of KIX domain of CREB-binding protein (CBP) with phosphorylated kinase inducible domain (pKID), by combing all-atom molecular dynamics with enhanced sampling methods recently developed in our group. We discuss our results in relation to previous NMR studies. We also develop a general simulations protocol to study allosteric phenomena and many other biological processes that occur in the micro/milliseconds timescale. PMID:23940332

  17. Allosteric modulation of caspases.

    PubMed

    Häcker, Hans-Georg; Sisay, Mihiret Tekeste; Gütschow, Michael

    2011-11-01

    Caspases are proteolytic enzymes mainly involved in the induction and execution phases of apoptosis. This type of programmed cell death is an essential regulatory process required to maintain the integrity and homeostasis of multicellular organisms. Inappropriate apoptosis is attributed a key role in many human diseases, including neurodegenerative disorders, ischemic damage, autoimmune diseases and cancer. Allosteric modulation of the function of a protein occurs when the regulatory trigger, such as the binding of a small effector or inhibitor molecule, takes place some distance from the protein's active site. In recent years, several caspases have been identified that possess allosteric sites and binding of small molecule to these sites resulted in the modulation of enzyme activities. Regulation of caspase activity by small molecule allosteric modulators is believed to be of great therapeutic importance. In this review we give brief highlights on recent developments in identifying and characterizing natural and synthetic allosteric inhibitors as well as activators of caspases and discuss their potential in drug discovery and protein engineering. PMID:21807025

  18. Allosteric Pathways in the PPARγ-RXRα nuclear receptor complex

    NASA Astrophysics Data System (ADS)

    Ricci, Clarisse G.; Silveira, Rodrigo L.; Rivalta, Ivan; Batista, Victor S.; Skaf, Munir S.

    2016-01-01

    Understanding the nature of allostery in DNA-nuclear receptor (NR) complexes is of fundamental importance for drug development since NRs regulate the transcription of a myriad of genes in humans and other metazoans. Here, we investigate allostery in the peroxisome proliferator-activated/retinoid X receptor heterodimer. This important NR complex is a target for antidiabetic drugs since it binds to DNA and functions as a transcription factor essential for insulin sensitization and lipid metabolism. We find evidence of interdependent motions of Ω-loops and PPARγ-DNA binding domain with contacts susceptible to conformational changes and mutations, critical for regulating transcriptional functions in response to sequence-dependent DNA dynamics. Statistical network analysis of the correlated motions, observed in molecular dynamics simulations, shows preferential allosteric pathways with convergence centers comprised of polar amino acid residues. These findings are particularly relevant for the design of allosteric modulators of ligand-dependent transcription factors.

  19. Allosteric Pathways in the PPARγ-RXRα nuclear receptor complex

    PubMed Central

    Ricci, Clarisse G.; Silveira, Rodrigo L.; Rivalta, Ivan; Batista, Victor S.; Skaf, Munir S.

    2016-01-01

    Understanding the nature of allostery in DNA-nuclear receptor (NR) complexes is of fundamental importance for drug development since NRs regulate the transcription of a myriad of genes in humans and other metazoans. Here, we investigate allostery in the peroxisome proliferator-activated/retinoid X receptor heterodimer. This important NR complex is a target for antidiabetic drugs since it binds to DNA and functions as a transcription factor essential for insulin sensitization and lipid metabolism. We find evidence of interdependent motions of Ω-loops and PPARγ-DNA binding domain with contacts susceptible to conformational changes and mutations, critical for regulating transcriptional functions in response to sequence-dependent DNA dynamics. Statistical network analysis of the correlated motions, observed in molecular dynamics simulations, shows preferential allosteric pathways with convergence centers comprised of polar amino acid residues. These findings are particularly relevant for the design of allosteric modulators of ligand-dependent transcription factors. PMID:26823026

  20. Muscarinic acetylcholine receptors: location of the ligand binding site

    SciTech Connect

    Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

    1987-05-01

    The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, /sup 3/H-propylbenzilycholine mustard aziridinium ion (/sup 3/H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that /sup 3/H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin.

  1. Crystal structure of the HIV-1 integrase core domain in complex with sucrose reveals details of an allosteric inhibitory binding site

    SciTech Connect

    Wielens, Jerome; Headey, Stephen J.; Jeevarajah, Dharshini; Rhodes, David I.; Deadman, John; Chalmers, David K.; Scanlon, Martin J.; Parker, Michael W.

    2010-04-19

    HIV integrase (IN) is an essential enzyme in HIV replication and an important target for drug design. IN has been shown to interact with a number of cellular and viral proteins during the integration process. Disruption of these important interactions could provide a mechanism for allosteric inhibition of IN. We present the highest resolution crystal structure of the IN core domain to date. We also present a crystal structure of the IN core domain in complex with sucrose which is bound at the dimer interface in a region that has previously been reported to bind integrase inhibitors.

  2. Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain

    SciTech Connect

    Rubio, V.; Cervera, J.; Bendala, E. ); Lusty, C.J. ); Britton, H.G. )

    1991-01-29

    The large subunit of Escherichia coli carbamoyl phosphate synthetase is responsible for carbamoyl phosphate synthesis from NH{sub 3} and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of ({sup 14}C)UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradation with ({sup 14}C)UMP. The ({sup 14}C)UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. Since the binding sites for IMP and UMP overlap, most probably IMP also binds in this domain. The protection from proteolysis by ornithine suggests that ornithine binds in the same domain. To account for the effects of the allosteric effectors on the binding of ATP, the authors propose a scheme where the two halves of the large subunit form a pseudohomodimer by complementary isologous association, thus placing the NH{sub 2} half, which is involved in the binding of the molecule of ATP that yields P{sub i}, close to the regulatory domain.

  3. Origin and evolution of the ligand-binding ability of nuclear receptors.

    PubMed

    Markov, Gabriel V; Laudet, Vincent

    2011-03-01

    The origin of the ligand-binding ability of nuclear receptors is still a matter of discussion. Current opposing models are the early evolution of an ancestral receptor that would bind a specific ligand with high affinity and the early evolution of an ancestral orphan that was a constitutive transcription factor. Here we review the arguments in favour or against these two hypotheses, and we discuss an alternative possibility that the ancestor was a ligand sensor, which would be able to explain the apparently contradictory data generated in previous models for the evolution of ligand binding in nuclear receptors. PMID:21055443

  4. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    ERIC Educational Resources Information Center

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M.

    2016-01-01

    Computational molecular docking is a fast and effective "in silico" method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The…

  5. Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

    PubMed

    Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

    2009-10-01

    Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes. PMID:19754879

  6. Local Unfolding of Fatty Acid Binding Protein to Allow Ligand Entry for Binding.

    PubMed

    Xiao, Tianshu; Fan, Jing-Song; Zhou, Hu; Lin, Qingsong; Yang, Daiwen

    2016-06-01

    Fatty acid binding proteins are responsible for the transportation of fatty acids in biology. Despite intensive studies, the molecular mechanism of fatty acid entry to and exit from the protein cavity is still unclear. Here a cap-closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the helical cap is locked to the β-barrel by a disulfide linkage. Structure determination shows that this variant adopts a closed conformation, but still uptakes fatty acids. Stopped-flow experiments indicate that a rate-limiting step exists before the ligand association and this step corresponds to the conversion of the closed form to the open one. NMR relaxation dispersion and H-D exchange data demonstrate the presence of two excited states: one is native-like, but the other adopts a locally unfolded structure. Local unfolding of helix 2 generates an opening for ligands to enter the protein cavity, and thus controls the ligand association rate. PMID:27105780

  7. Spatial analysis and quantification of the thermodynamic driving forces in protein-ligand binding: binding site variability.

    PubMed

    Raman, E Prabhu; MacKerell, Alexander D

    2015-02-25

    The thermodynamic driving forces behind small molecule-protein binding are still not well-understood, including the variability of those forces associated with different types of ligands in different binding pockets. To better understand these phenomena we calculate spatially resolved thermodynamic contributions of the different molecular degrees of freedom for the binding of propane and methanol to multiple pockets on the proteins Factor Xa and p38 MAP kinase. Binding thermodynamics are computed using a statistical thermodynamics based end-point method applied on a canonical ensemble comprising the protein-ligand complexes and the corresponding free states in an explicit solvent environment. Energetic and entropic contributions of water and ligand degrees of freedom computed from the configurational ensemble provide an unprecedented level of detail into the mechanisms of binding. Direct protein-ligand interaction energies play a significant role in both nonpolar and polar binding, which is comparable to water reorganization energy. Loss of interactions with water upon binding strongly compensates these contributions leading to relatively small binding enthalpies. For both solutes, the entropy of water reorganization is found to favor binding in agreement with the classical view of the "hydrophobic effect". Depending on the specifics of the binding pocket, both energy-entropy compensation and reinforcement mechanisms are observed. It is notable to have the ability to visualize the spatial distribution of the thermodynamic contributions to binding at atomic resolution showing significant differences in the thermodynamic contributions of water to the binding of propane versus methanol. PMID:25625202

  8. Spatial Analysis and Quantification of the Thermodynamic Driving Forces in Protein-Ligand Binding: Binding Site Variability

    PubMed Central

    Raman, E. Prabhu; MacKerell, Alexander D.

    2015-01-01

    The thermodynamic driving forces behind small molecule-protein binding are still not well understood, including the variability of those forces associated with different types of ligands in different binding pockets. To better understand these phenomena we calculate spatially resolved thermodynamic contributions of the different molecular degrees of freedom for the binding of propane and methanol to multiple pockets on the proteins Factor Xa and p38 MAP kinase. Binding thermodynamics are computed using a statistical thermodynamics based end-point method applied on a canonical ensemble comprising the protein-ligand complexes and the corresponding free states in an explicit solvent environment. Energetic and entropic contributions of water and ligand degrees of freedom computed from the configurational ensemble provides an unprecedented level of detail into the mechanisms of binding. Direct protein-ligand interaction energies play a significant role in both non-polar and polar binding, which is comparable to water reorganization energy. Loss of interactions with water upon binding strongly compensates these contributions leading to relatively small binding enthalpies. For both solutes, the entropy of water reorganization is found to favor binding in agreement with the classical view of the “hydrophobic effect”. Depending on the specifics of the binding pocket, both energy-entropy compensation and reinforcement mechanisms are observed. Notable is the ability to visualize the spatial distribution of the thermodynamic contributions to binding at atomic resolution showing significant differences in the thermodynamic contributions of water to the binding of propane versus methanol. PMID:25625202

  9. Evaluation of Metal-Mediated DNA Binding of Benzoazole Ligands by Electrospray Ionization Mass Spectrometry

    PubMed Central

    Mazzitelli, Carolyn L.; Rodriguez, Mireya; Kerwin, Sean; Brodbelt, Jennifer S.

    2008-01-01

    The binding of a series of benzoxazole analogs with different amide- and ester-linked side chains to duplex DNA in the absence and presence of divalent metal cations is examined. All ligands were found to form complexes with Ni2+, Cu2+, and Zn2+, with 2:1 ligand/metal cation binding stoichiometries dominating for ligands containing shorter side chains (2, 6, 7, and 8), while 1:1 complexes were the most abundant for ligands with long side chains (9, 10 and 11). Ligand binding with duplex DNA in the absence of metal cations was assessed, and the long side-chain ligands were found to form low abundance complexes with 1:1 ligand/DNA binding stoichiometries. The ligands with the shorter side chains only formed DNA complexes in the presence of metal cations, most notably for 7 and 8 binding to DNA in the presence of Cu2+. The binding of long side-chain ligands was enhanced by Cu2+ and to a lesser degree by Ni2+ and Zn2+. The cytotoxicities of all of the ligands against the A549 lung cancer and MCF7 breast cancer cell lines were also examined. The ligands exhibiting the most dramatic metal-enhanced DNA binding also demonstrated the greatest cytotoxic activity. Both 7 and 8 were found to be the most cytotoxic against the A549 lung cancer cell line and 8 demonstrated moderate cytotoxicity against MCF7 breast cancer cells. Metal ions also enhanced the DNA binding of the ligands with the long side-chains, especially for 9, which also exhibited the highest level of cytotoxicity of the long side-chain compounds. PMID:17583529

  10. Bioluminescent Ligand-Receptor Binding Assays for Protein or Peptide Hormones.

    PubMed

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    Bioluminescence has been widely used in biomedical research due to its high sensitivity, low background, and broad linear range. In recent studies, we applied bioluminescence to ligand-receptor binding assays for some protein or peptide hormones based on a newly developed small monomeric Nanoluciferase (NanoLuc) reporter that has the so far brightest bioluminescence. The conventional ligand-receptor binding assays rely on radioligands that have drawbacks, such as radioactive hazards and short shelf lives. In contrast, the novel bioluminescent binding assays use the NanoLuc-based protein or peptide tracers that are safe, stable, and ultrasensitive. Thus, the novel bioluminescent ligand-receptor binding assay would be applied to more and more protein or peptide hormones for ligand-receptor interaction studies in future. In the present article, we provided detailed protocols for setting up the novel bioluminescent ligand-receptor binding assays using two representative protein hormones as examples. PMID:27424896

  11. Uncoupling the Structure-Activity Relationships of β2 Adrenergic Receptor Ligands from Membrane Binding.

    PubMed

    Dickson, Callum J; Hornak, Viktor; Velez-Vega, Camilo; McKay, Daniel J J; Reilly, John; Sandham, David A; Shaw, Duncan; Fairhurst, Robin A; Charlton, Steven J; Sykes, David A; Pearlstein, Robert A; Duca, Jose S

    2016-06-23

    Ligand binding to membrane proteins may be significantly influenced by the interaction of ligands with the membrane. In particular, the microscopic ligand concentration within the membrane surface solvation layer may exceed that in bulk solvent, resulting in overestimation of the intrinsic protein-ligand binding contribution to the apparent/measured affinity. Using published binding data for a set of small molecules with the β2 adrenergic receptor, we demonstrate that deconvolution of membrane and protein binding contributions allows for improved structure-activity relationship analysis and structure-based drug design. Molecular dynamics simulations of ligand bound membrane protein complexes were used to validate binding poses, allowing analysis of key interactions and binding site solvation to develop structure-activity relationships of β2 ligand binding. The resulting relationships are consistent with intrinsic binding affinity (corrected for membrane interaction). The successful structure-based design of ligands targeting membrane proteins may require an assessment of membrane affinity to uncouple protein binding from membrane interactions. PMID:27239696

  12. A tandem regression-outlier analysis of a ligand cellular system for key structural modifications around ligand binding

    PubMed Central

    2013-01-01

    Background A tandem technique of hard equipment is often used for the chemical analysis of a single cell to first isolate and then detect the wanted identities. The first part is the separation of wanted chemicals from the bulk of a cell; the second part is the actual detection of the important identities. To identify the key structural modifications around ligand binding, the present study aims to develop a counterpart of tandem technique for cheminformatics. A statistical regression and its outliers act as a computational technique for separation. Results A PPARγ (peroxisome proliferator-activated receptor gamma) agonist cellular system was subjected to such an investigation. Results show that this tandem regression-outlier analysis, or the prioritization of the context equations tagged with features of the outliers, is an effective regression technique of cheminformatics to detect key structural modifications, as well as their tendency of impact to ligand binding. Conclusions The key structural modifications around ligand binding are effectively extracted or characterized out of cellular reactions. This is because molecular binding is the paramount factor in such ligand cellular system and key structural modifications around ligand binding are expected to create outliers. Therefore, such outliers can be captured by this tandem regression-outlier analysis. PMID:23627990

  13. Conformational Transitions upon Ligand Binding: Holo-Structure Prediction from Apo Conformations

    PubMed Central

    Seeliger, Daniel; de Groot, Bert L.

    2010-01-01

    Biological function of proteins is frequently associated with the formation of complexes with small-molecule ligands. Experimental structure determination of such complexes at atomic resolution, however, can be time-consuming and costly. Computational methods for structure prediction of protein/ligand complexes, particularly docking, are as yet restricted by their limited consideration of receptor flexibility, rendering them not applicable for predicting protein/ligand complexes if large conformational changes of the receptor upon ligand binding are involved. Accurate receptor models in the ligand-bound state (holo structures), however, are a prerequisite for successful structure-based drug design. Hence, if only an unbound (apo) structure is available distinct from the ligand-bound conformation, structure-based drug design is severely limited. We present a method to predict the structure of protein/ligand complexes based solely on the apo structure, the ligand and the radius of gyration of the holo structure. The method is applied to ten cases in which proteins undergo structural rearrangements of up to 7.1 Å backbone RMSD upon ligand binding. In all cases, receptor models within 1.6 Å backbone RMSD to the target were predicted and close-to-native ligand binding poses were obtained for 8 of 10 cases in the top-ranked complex models. A protocol is presented that is expected to enable structure modeling of protein/ligand complexes and structure-based drug design for cases where crystal structures of ligand-bound conformations are not available. PMID:20066034

  14. Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities.

    PubMed

    Ribeiro, Teresa; Santos-Silva, Teresa; Alves, Victor D; Dias, Fernando M V; Luís, Ana S; Prates, José A M; Ferreira, Luís M A; Romão, Maria J; Fontes, Carlos M G A

    2010-10-01

    Enzymes that degrade plant cell wall polysaccharides display a modular architecture comprising a catalytic domain bound to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs display considerable variation in primary structure and are grouped into 59 sequence-based families organized in the Carbohydrate-Active enZYme (CAZy) database. Here we report the crystal structure of CtCBM42A together with the biochemical characterization of two other members of family 42 CBMs from Clostridium thermocellum. CtCBM42A, CtCBM42B and CtCBM42C bind specifically to the arabinose side-chains of arabinoxylans and arabinan, suggesting that various cellulosomal components are targeted to these regions of the plant cell wall. The structure of CtCBM42A displays a beta-trefoil fold, which comprises 3 sub-domains designated as alpha, beta and gamma. Each one of the three sub-domains presents a putative carbohydrate-binding pocket where an aspartate residue located in a central position dominates ligand recognition. Intriguingly, the gamma sub-domain of CtCBM42A is pivotal for arabinoxylan binding, while the concerted action of beta and gamma sub-domains of CtCBM42B and CtCBM42C is apparently required for ligand sequestration. Thus, this work reveals that the binding mechanism of CBM42 members is in contrast with that of homologous CBM13s where recognition of complex polysaccharides results from the cooperative action of three protein sub-domains presenting similar affinities. PMID:20637315

  15. Multipurpose ligand, DAKLI (Dynorphin A-analogue Kappa LIgand), with high affinity and selectivity for dynorphin (. kappa. opioid) binding sites

    SciTech Connect

    Goldstein, A.; Nestor, J.J. Jr.; Naidu, A.; Newman, S.R. )

    1988-10-01

    The authors describe a synthetic ligand, DALKI (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as {sup 125}I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin ({kappa} opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.

  16. Pumiliotoxin B binds to a site on the voltage-dependent sodium channel that is allosterically coupled to other binding sites.

    PubMed Central

    Gusovsky, F; Rossignol, D P; McNeal, E T; Daly, J W

    1988-01-01

    Pumiliotoxin B (PTX-B), an alkaloid that has cardiotonic and myotonic activity, increases sodium influx in guinea pig cerebral cortical synaptoneurosomes. In the presence of scorpion venom (Leiurus) or purified alpha-scorpion toxin, the PTX-B-induced sodium influx is enhanced severalfold. PTX-B alone has no effect on sodium flux in N18 neuroblastoma cells but, in the presence of alpha-scorpion toxin, stimulation of sodium influx by PTX-B reaches levels comparable to that attained with the sodium channel activator veratridine. In neuroblastoma LV9 cells, a variant mutant that lacks sodium channels, neither veratridine nor PTX-B induces sodium fluxes in either the presence or absence of alpha-scorpion toxin. In synaptoneurosomes and in N18 cells, the sodium influx induced by the combination of PTX-B and alpha-scorpion toxin is inhibited by tetrodotoxin and local anesthetics. PTX-B does not interact with two of the known toxin sites on the sodium channel, as evidenced by a lack of effect on binding of [3H]saxitoxin or [3H]batrachotoxinin A benzoate to brain synaptoneurosomes. Synergistic effects on sodium influx with alpha-scorpion toxin, beta-scorpion toxin, and brevetoxin indicate that PTX-B does not interact directly with three other toxin sites on the sodium channel. Thus, PTX-B appears to activate sodium influx by interacting with yet another site on the voltage-dependent sodium channel, a site that is coupled allosterically to sites for alpha-scorpion toxin, beta-scorpion toxin, and brevetoxin. PMID:2448797

  17. Ligand preference and orientation in b- and c-type heme-binding proteins

    PubMed Central

    Fufezan, Christian; Zhang, Jun; Gunner, M. R.

    2009-01-01

    Hemes are often incorporated into designed proteins. The importance of the heme ligand type and its orientation is still a matter of debate. Here, heme ligands and ligand orientation were investigated using a nonredundant (87 structures) and a redundant (1503 structures) set of structures to compare and contrast design features of natural b- and c-type heme-binding proteins. Histidine is the most common ligand. Marked differences in ligation motifs between b- and c-type hemes are higher occurrence of His-Met in c-type heme binding motifs (16.4% vs. 1.4%) and higher occurrence of exchangeable, small molecules in b-type heme binding motifs (67.6% vs. 9.9%). Histidine ligands that are part of the c-type CXXCH heme-binding motif show a distinct asymmetric distribution of orientation. They tend to point between either the heme propionates or between the NA and NB heme nitrogens. Molecular mechanics calculations show that this asymmetry is due to the bonded constraints of the covalent attachment between the heme and the protein. In contrast, the orientations of b-type hemes histidine ligands are found evenly distributed with no preference. Observed histidine heme ligand orientations show no dominating influence of electrostatic interactions between the heme propionates and the ligands. Furthermore, ligands in bis-His hemes are found more frequently perpendicular rather than parallel to each other. These correlations support energetic constraints on ligands that can be used in designing proteins. PMID:18491383

  18. DNA-Based Nanostructures: Changes of Mechanical Properties of DNA upon Ligand Binding

    NASA Astrophysics Data System (ADS)

    Nechipurenko, Yury; Grokhovsky, Sergey; Gursky, Georgy; Nechipurenko, Dmitry; Polozov, Robert

    The formation of DNA-based nanostructures involves the binding of different kinds of ligands to DNA as well as the interaction of DNA molecules with each other. Complex formation between ligand and DNA can alter physicochemical properties of the DNA molecule. In the present work, the accessibility of DNA-ligand complexes to cleavage by DNase I are considered, and the exact algorithms for analysis of diagrams of DNase I footprinting for ligand-DNA complexes are obtained. Changes of mechanical properties of the DNA upon ligand binding are also demonstrated by the cleavage patterns generated upon ultrasound irradiation of cis-platin-DNA complexes. Propagation of the mechanical perturbations along DNA in the presence of bound ligands is considered in terms of a string model with a heterogeneity corresponding to the position of a bound ligand on DNA. This model can reproduce qualitatively the cleavage patterns obtained upon ultrasound irradiation of cis-platin-DNA complexes.

  19. Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

    NASA Technical Reports Server (NTRS)

    Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.

    1995-01-01

    A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

  20. Ligand binding cooperativity: Bioisosteric replacement of CO with SO2 among thrombin inhibitors.

    PubMed

    Said, Ahmed M; Hangauer, David G

    2016-08-15

    Ligand-protein binding is a complex process that involves the formation of number of non-covalent interactions, e.g. H-bonds and hydrophobic interactions, between the ligand and the protein host. Upon binding, ligand functional groups can act synergistically (positive cooperativity) to improve the overall ligand binding affinity beyond what would be expected from their individual contributions. In this study, using thrombin as a protein model system, we evaluated the effect of the bioisosteric replacement of a carbonyl functionality with a sulphonyl functionality on positive cooperativity between their H-bonds with thrombin and hydrophobic binding in the adjacent S3 pocket. The positive cooperativity observed was greatly reduced when replacing the carbonyl group with a sulphonyl group. Evaluating how bioisosteric replacements affect cooperativity is important for making better informed ligand optimization SAR decisions. PMID:27445170

  1. Divergent allosteric patterns verify the regulatory paradigm for aspartate transcarbamylase.

    PubMed

    Wales, M E; Madison, L L; Glaser, S S; Wild, J R

    1999-12-17

    The native Escherichia coli aspartate transcarbamoylase (ATCase, E.C. 2.1.3.2) provides a classic allosteric model for the feedback inhibition of a biosynthetic pathway by its end products. Both E. coli and Erwinia herbicola possess ATCase holoenzymes which are dodecameric (2(c3):3(r2)) with 311 amino acid residues per catalytic monomer and 153 and 154 amino acid residues per regulatory (r) monomer, respectively. While the quaternary structures of the two enzymes are identical, the primary amino acid sequences have diverged by 14 % in the catalytic polypeptide and 20 % in the regulatory polypeptide. The amino acids proposed to be directly involved in the active site and nucleotide binding site are strictly conserved between the two enzymes; nonetheless, the two enzymes differ in their catalytic and regulatory characteristics. The E. coli enzyme has sigmoidal substrate binding with activation by ATP, and inhibition by CTP, while the E. herbicola enzyme has apparent first order kinetics at low substrate concentrations in the absence of allosteric ligands, no ATP activation and only slight CTP inhibition. In an apparently important and highly conserved characteristic, CTP and UTP impose strong synergistic inhibition on both enzymes. The co-operative binding of aspartate in the E. coli enzyme is correlated with a T-to-R conformational transition which appears to be greatly reduced in the E. herbicola enzyme, although the addition of inhibitory heterotropic ligands (CTP or CTP+UTP) re-establishes co-operative saturation kinetics. Hybrid holoenzymes assembled in vivo with catalytic subunits from E. herbicola and regulatory subunits from E. coli mimick the allosteric response of the native E. coli holoenzyme and exhibit ATP activation. The reverse hybrid, regulatory subunits from E. herbicola and catalytic subunits from E. coli, exhibited no response to ATP. The conserved structure and diverged functional characteristics of the E. herbicola enzyme provides an opportunity

  2. The Study of the Successive Metal-ligand Binding Energies for Fe(+), Fe(-), V(+) and Co(+)

    NASA Technical Reports Server (NTRS)

    Bauschlicher, Charles W., Jr.; Ricca, Alessandra; Maitre, Philippe; Langhoff, Stephen R. (Technical Monitor)

    1994-01-01

    The successive binding energies of CO and H2O to Fe(+), CO to Fe(-), and H2 to Co(+) and V(+) are presented. Overall the computed results are in good agreement with experiment. The trends in binding energies are analyzed in terms of metal to ligand donation, ligand to metal donation, ligand-ligand repulsion, and changes in the metal atom, such as hybridization, promotion, and spin multiplicity. The geometry and vibrational frequencies are also shown to be directly affected by these effects.

  3. The Study Of The Successive Metal-Ligand Binding Energies For Fe+, Fe-, V+ and Co+

    NASA Technical Reports Server (NTRS)

    Bauschicher, Charles W., Jr.; Ricca, Alessandra; Maitre, Philippe; Langhoff, Stephen R. (Technical Monitor)

    1995-01-01

    The successive binding energies of CO and H2O to Fe(+), CO to Fe(-), and H2 to Co(+) and V(+) are presented. Overall the computed results are in good agreement with experiment. The trends in binding energies are analyzed in terms of metal to ligand donation, ligand to metal donation, ligand-ligand repulsion, and changes in the metal atom, such as hybridization, promotion, and spin multiplicity. The geometry and vibrational frequencies are also shown to be directly affected by these effects.

  4. Detection of persistent organic pollutants binding modes with androgen receptor ligand binding domain by docking and molecular dynamics

    PubMed Central

    2013-01-01

    Background Persistent organic pollutants (POPs) are persistent in the environment after release from industrial compounds, combustion productions or pesticides. The exposure of POPs has been related to various reproductive disturbances, such as reduced semen quality, testicular cancer, and imbalanced sex ratio. Among POPs, dichlorodiphenyldichloroethylene (4,4’-DDE) and polychlorinated biphenyls (PCBs) are the most widespread and well-studied compounds. Recent studies have revealed that 4,4’-DDE is an antagonist of androgen receptor (AR). However, the mechanism of the inhibition remains elusive. CB-153 is the most common congener of PCBs, while the action of CB-153 on AR is still under debate. Results Molecular docking and molecular dynamics (MD) approaches have been employed to study binding modes and inhibition mechanism of 4,4’-DDE and CB-153 against AR ligand binding domain (LBD). Several potential binding sites have been detected and analyzed. One possible binding site is the same binding site of AR natural ligand androgen 5α-dihydrotestosterone (DHT). Another one is on the ligand-dependent transcriptional activation function (AF2) region, which is crucial for the co-activators recruitment. Besides, a novel possible binding site was observed for POPs with low binding free energy with the receptor. Detailed interactions between ligands and the receptor have been represented. The disrupting mechanism of POPs against AR has also been discussed. Conclusions POPs disrupt the function of AR through binding to three possible biding sites on AR/LBD. One of them shares the same binding site of natural ligand of AR. Another one is on AF2 region. The third one is in a cleft near N-terminal of the receptor. Significantly, values of binding free energy of POPs with AR/LBD are comparable to that of natural ligand androgen DHT. PMID:24053684

  5. The multi-drug resistance reversal agent SR33557 and modulation of vinca alkaloid binding to P-glycoprotein by an allosteric interaction

    PubMed Central

    Martin, Catherine; Berridge, Georgina; Higgins, Christopher F; Callaghan, Richard

    1997-01-01

    The interaction of the indolizin sulfone SR33557 with the multidrug resistance P-glycoprotein (P-gp), was used to explore the nature of drug binding site(s) on this transporter. The steady-state accumulation of [3H]-vinblastine in P-gp expressing CHrB30 cells was increased by SR33557 with greater potency than verapamil. Furthermore, SR33557 potentiated the affinity of verapamil to modulate vinblastine transport when added simultaneously. Verapamil elicited a 1.5 to 2.5 fold stimulation of basal ATPase activity in CHrB30 membranes, whereas SR33557 and vinblastine inhibited activity, but only at relatively high concentrations. However, SR33557 and vinblastine decreased the Vmax but not the Km for verapamil stimulation of ATPase activity. This is indicative of a non-competitive interaction, most likely at distinct sites. The specific [3H]-vinblastine binding to P-gp in CHrB30 cell membranes was displaced by SR33557 with an IC50 of 8.3±4.5 nM. Moreover, SR33557 caused a 3 fold increase in the dissociation rate of vinblastine binding to P-gp indicating a negative allosteric effect on the vinca alkaloid acceptor site. These results demonstrate that SR33557 interacts with a site on P-gp which is distinct from, but allosterically linked to the vinca alkaloid site. The apparent broad substrate specificity displayed by P-gp may be explained by a multiple drug binding site model. PMID:9375975

  6. Microassay for measurement of binding of radiolabelled ligands to cell surface molecules.

    PubMed

    Woof, J M; Burton, D R

    1988-07-22

    An improved technique for measuring the binding of radiolabelled ligands to cell surface molecules has been developed by modification of a procedure using centrifugation through a water-immiscible oil to separate free and cell-bound ligand. It maximises the percentage of ligand bound since cell-bound and free ligand can be separated easily and reproducibly even when very small reaction volumes are used. This permits low levels of ligand radiolabelling and relatively low numbers of cells to be used. PMID:2840465

  7. Comparison of ligand migration and binding in heme proteins of the globin family

    NASA Astrophysics Data System (ADS)

    Karin, Nienhaus; Ulrich Nienhaus, G.

    2015-12-01

    The binding of small diatomic ligands such as carbon monoxide or dioxygen to heme proteins is among the simplest biological processes known. Still, it has taken many decades to understand the mechanistic aspects of this process in full detail. Here, we compare ligand binding in three heme proteins of the globin family, myoglobin, a dimeric hemoglobin, and neuroglobin. The combination of structural, spectroscopic, and kinetic experiments over many years by many laboratories has revealed common properties of globins and a clear mechanistic picture of ligand binding at the molecular level. In addition to the ligand binding site at the heme iron, a primary ligand docking site exists that ensures efficient ligand binding to and release from the heme iron. Additional, secondary docking sites can greatly facilitate ligand escape after its dissociation from the heme. Although there is only indirect evidence at present, a preformed histidine gate appears to exist that allows ligand entry to and exit from the active site. The importance of these features can be assessed by studies involving modified proteins (via site-directed mutagenesis) and comparison with heme proteins not belonging to the globin family.

  8. Copper binding ligands: production by marine plankton and characterization by ESI-MS

    NASA Astrophysics Data System (ADS)

    Orians, K.; Ross, A.; Lawrence, M.; Ikonomou, M.

    2003-04-01

    Organic complexation affects the bioavailability and distribution of copper in the surface ocean. The cyanobacterium Synechococcus sp. PCC 7002 was cultured in the lab and subjected to near-toxic Cu concentrations. Strong Cu-binding ligands were produced under these conditions, as found for other species of Synechococcus. The copper-binding ligand produced had a log K'cond. (log conditional stability constant) of 12.2, similar to the natural ligands found in the surface ocean. The amount of ligand produced was proportional to the amount of copper present. Isolation and concentration of these compounds for characterization by electrospray mass spectrometry (ESI-MS) provides information about the structure of the organic ligands and their metal-ion complexes. Using model ligands, we'll show that ligands can be characterized by ESI-MS and that the location of the copper binding site can be determined in complex molecules. We'll also present results of copper-complexing ligands extracted from the coastal waters of British Columbia. Ligand concentrations are higher at low salinity and in surface waters, indicating that these ligands are produced in surface waters and/or delivered to the region via the Fraser River. Analysis of the extracts with highest UV absorbance identified two Cu2+ ligands of molecular weight 259 and 264. The mass and isotopic distributions are consistent with dipeptides and tripeptides containing two metal-binding amino groups. This result is consistent with the findings of other studies attempting to characterize Cu2+ ligands in seawater. The structure of the identified ligand is similar to that of rhodotorulic acid (a microbial siderophore), glutathione, and phytochelatins, indicating that small peptides and related compounds can act as strong, specific metal chelators in natural waters

  9. Theoretical investigation on the diatomic ligand migration process and ligand binding properties in non-O2-binding H-NOX domain.

    PubMed

    Zhang, Yuebin; Liu, Li; Wu, Lei; Li, Shuai; Li, Fei; Li, Zhengqiang

    2013-08-01

    The Nostoc sp (Ns) H-NOX (heme-nitric oxide or OXygen-binding) domain shares 35% sequence identity with soluble guanylate cyclase (sGC) and exhibits similar ligand binding property with the sGC. Previously, our molecular dynamic (MD) simulation work identified that there exists a Y-shaped tunnel system hosted in the Ns H-NOX interior, which servers for ligand migration. The tunnels were then confirmed by Winter et al. [PNAS 2011;108(43):E 881-889] recently using x-ray crystallography with xenon pressured conditions. In this work, to further investigate how the protein matrix of Ns H-NOX modulates the ligand migration process and how the distal residue composition affects the ligand binding prosperities, the free energy profiles for nitric oxide (NO), carbon monooxide (CO), and O2 migration are explored using the steered MDs simulation and the ligand binding energies are calculated using QM/MM schemes. The potential of mean force profiles suggest that the longer branch of the tunnel would be the most favorable route for NO migration and a second NO trapping site other than the distal heme pocket along this route in the Ns H-NOX was identified. On the contrary, CO and O2 would prefer to diffuse via the shorter branch of the tunnel. The QM/MM (quantum mechanics/molecular mechanics) calculations suggest that the hydrophobic distal pocket of Ns H-NOX would provide an approximately vacuum environment and the ligand discrimination would be determined by the intrinsic binding properties of the diatomic gas ligand to the heme group. PMID:23504767

  10. Improved Estimation of Protein-Ligand Binding Free Energy by Using the Ligand-Entropy and Mobility of Water Molecules

    PubMed Central

    Fukunishi, Yoshifumi; Nakamura, Haruki

    2013-01-01

    We previously developed the direct interaction approximation (DIA) method to estimate the protein-ligand binding free energy (ΔG). The DIA method estimates the ΔG value based on the direct van der Waals and electrostatic interaction energies between the protein and the ligand. In the current study, the effect of the entropy of the ligand was introduced with protein dynamic properties by molecular dynamics simulations, and the interaction between each residue of the protein and the ligand was also weighted considering the hydration of each residue. The molecular dynamics simulation of the apo target protein gave the hydration effect of each residue, under the assumption that the residues, which strongly bind the water molecules, are important in the protein-ligand binding. These two effects improved the reliability of the DIA method. In fact, the parameters used in the DIA became independent of the target protein. The averaged error of ΔG estimation was 1.3 kcal/mol and the correlation coefficient between the experimental ΔG value and the calculated ΔG value was 0.75. PMID:24276169

  11. Specific ligand binding attributable to individual epitopes of gonococcal transferrin binding protein A.

    PubMed

    Masri, Heather P; Cornelissen, Cynthia Nau

    2002-02-01

    The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in Escherichia coli, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed. PMID:11796606

  12. Functional anatomy of an allosteric protein

    NASA Astrophysics Data System (ADS)

    Purohit, Prasad; Gupta, Shaweta; Jadey, Snehal; Auerbach, Anthony

    2013-12-01

    Synaptic receptors are allosteric proteins that switch on and off to regulate cell signalling. Here, we use single-channel electrophysiology to measure and map energy changes in the gating conformational change of a nicotinic acetylcholine receptor. Two separated regions in the α-subunits—the transmitter-binding sites and αM2-αM3 linkers in the membrane domain—have the highest ϕ-values (change conformation the earliest), followed by the extracellular domain, most of the membrane domain and the gate. Large gating-energy changes occur at the transmitter-binding sites, α-subunit interfaces, the αM1 helix and the gate. We hypothesize that rearrangements of the linkers trigger the global allosteric transition, and that the hydrophobic gate unlocks in three steps. The mostly local character of side-chain energy changes and the similarly high ϕ-values of separated domains, both with and without ligands, suggest that gating is not strictly a mechanical process initiated by the affinity change for the agonist.

  13. LISE: a server using ligand-interacting and site-enriched protein triangles for prediction of ligand-binding sites.

    PubMed

    Xie, Zhong-Ru; Liu, Chuan-Kun; Hsiao, Fang-Chih; Yao, Adam; Hwang, Ming-Jing

    2013-07-01

    LISE is a web server for a novel method for predicting small molecule binding sites on proteins. It differs from a number of servers currently available for such predictions in two aspects. First, rather than relying on knowledge of similar protein structures, identification of surface cavities or estimation of binding energy, LISE computes a score by counting geometric motifs extracted from sub-structures of interaction networks connecting protein and ligand atoms. These network motifs take into account spatial and physicochemical properties of ligand-interacting protein surface atoms. Second, LISE has now been more thoroughly tested, as, in addition to the evaluation we previously reported using two commonly used small benchmark test sets and targets of two community-based experiments on ligand-binding site predictions, we now report an evaluation using a large non-redundant data set containing >2000 protein-ligand complexes. This unprecedented test, the largest ever reported to our knowledge, demonstrates LISE's overall accuracy and robustness. Furthermore, we have identified some hard to predict protein classes and provided an estimate of the performance that can be expected from a state-of-the-art binding site prediction server, such as LISE, on a proteome scale. The server is freely available at http://lise.ibms.sinica.edu.tw. PMID:23609546

  14. Selective Inhibition of Mutant Isocitrate Dehydrogenase 1 (IDH1) via Disruption of a Metal Binding Network by an Allosteric Small Molecule

    PubMed Central

    Deng, Gejing; Shen, Junqing; Yin, Ming; McManus, Jessica; Mathieu, Magali; Gee, Patricia; He, Timothy; Shi, Chaomei; Bedel, Olivier; McLean, Larry R.; Le-Strat, Frank; Zhang, Ying; Marquette, Jean-Pierre; Gao, Qiang; Zhang, Bailin; Rak, Alexey; Hoffmann, Dietmar; Rooney, Eamonn; Vassort, Aurelie; Englaro, Walter; Li, Yi; Patel, Vinod; Adrian, Francisco; Gross, Stefan; Wiederschain, Dmitri; Cheng, Hong; Licht, Stuart

    2015-01-01

    Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg2+. A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme. PMID:25391653

  15. Computational Prediction of Alanine Scanning and Ligand Binding Energetics in G-Protein Coupled Receptors

    PubMed Central

    Boukharta, Lars; Gutiérrez-de-Terán, Hugo; Åqvist, Johan

    2014-01-01

    Site-directed mutagenesis combined with binding affinity measurements is widely used to probe the nature of ligand interactions with GPCRs. Such experiments, as well as structure-activity relationships for series of ligands, are usually interpreted with computationally derived models of ligand binding modes. However, systematic approaches for accurate calculations of the corresponding binding free energies are still lacking. Here, we report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 receptor and series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. PMID:24743773

  16. Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems.

    PubMed

    Fielding, Lee; Rutherford, Samantha; Fletcher, Dan

    2005-06-01

    The usefulness of bovine serum albumin (BSA) as a model protein for testing NMR methods for the study of protein-ligand interactions is discussed. Isothermal titration calorimetry established the binding affinity and stoichiometry of the specific binding site for L-tryptophan, D-tryptophan, naproxen, ibuprofen, salicylic acid and warfarin. The binding affinities of the same ligands determined by NMR methods are universally weaker (larger KD). This is because the NMR methods are susceptible to interference from additional non-specific binding. The L-tryptophan-BSA and naproxen-BSA systems were the best behaved model systems. PMID:15816062

  17. Molecular decoys: ligand-binding recombinant proteins protect mice from curarimimetic neurotoxins.

    PubMed Central

    Gershoni, J M; Aronheim, A

    1988-01-01

    Mimic ligand-binding sites of the nicotinic acetylcholine receptor bind d-tubocurarine and alpha-bungarotoxin in vitro. Injection of such binding sites into mice could act as molecular decoys in vivo, providing protection against toxic ligands. This hypothesis of molecular "decoyance" has been tested in greater than 250 mice. Bacterially produced cholinergic binding sites provided a 2-fold increase in the survival rate of animals challenged with curarimimetic neurotoxins. Possible considerations for decoy designs and their applications are discussed. Images PMID:3375254

  18. Multifunctionality and mechanism of ligand binding in a mosquito antiinflammatory protein

    SciTech Connect

    Calvo, Eric; Mans, Ben J.; Ribeiro, José M.C.; Andersen, John F.

    2009-04-07

    The mosquito D7 salivary proteins are encoded by a multigene family related to the arthropod odorant-binding protein (OBP) superfamily. Forms having either one or two OBP domains are found in mosquito saliva. Four single-domain and one two-domain D7 proteins from Anopheles gambiae and Aedes aegypti (AeD7), respectively, were shown to bind biogenic amines with high affinity and with a stoichiometry of one ligand per protein molecule. Sequence comparisons indicated that only the C-terminal domain of AeD7 is homologous to the single-domain proteins from A. gambiae, suggesting that the N-terminal domain may bind a different class of ligands. Here, we describe the 3D structure of AeD7 and examine the ligand-binding characteristics of the N- and C-terminal domains. Isothermal titration calorimetry and ligand complex crystal structures show that the N-terminal domain binds cysteinyl leukotrienes (cysLTs) with high affinities (50-60 nM) whereas the C-terminal domain binds biogenic amines. The lipid chain of the cysLT binds in a hydrophobic pocket of the N-terminal domain, whereas binding of norepinephrine leads to an ordering of the C-terminal portion of the C-terminal domain into an alpha-helix that, along with rotations of Arg-176 and Glu-268 side chains, acts to bury the bound ligand.

  19. Cationic Gold Clusters Ligated with Differently Substituted Phosphines: Effect of Substitution on Ligand Reactivity and Binding

    SciTech Connect

    Johnson, Grant E.; Olivares, Astrid M.; Hill, David E.; Laskin, Julia

    2015-01-01

    We present a systematic study of the effect of the number of methyl (Me) and cyclohexyl (Cy) functional groups in monodentate phosphine ligands on the solution-phase synthesis of ligated sub-nanometer gold clusters and their gas-phase fragmentation pathways. Small mixed ligand cationic gold clusters were synthesized using ligand exchange reactions between pre-formed triphenylphosphine ligated (PPh3) gold clusters and monodentate Me- and Cy-substituted ligands in solution and characterized using electrospray ionization mass spectrometry (ESI-MS) and collision-induced dissociation (CID) experiments. Under the same experimental conditions, larger gold-PPh3 clusters undergo efficient exchange of unsubstituted PPh3 ligands for singly Me- and Cy-substituted PPh2Me and PPh2Cy ligands. The efficiency of ligand exchange decreases with an increasing number of Me or Cy groups in the substituted phosphine ligands. CID experiments performed for a series of ligand-exchanged gold clusters indicate that loss of a neutral Me-substituted ligand is preferred over loss of a neutral PPh¬3 ligand while the opposite trend is observed for Cy-substituted ligands. The branching ratio of the competing ligand loss channels is strongly correlated with the electron donating ability of the phosphorous lone pair as determined by the relative proton affinity of the ligand. The results indicate that the relative ligand binding energies increase in the order PMe3 < PPhMe2 < PPh2Me < PPh3< PPh2Cy < PPhCy2< PCy3. Furthermore, the difference in relative ligand binding energies increases with the number of substituted PPh3-mMem or PPh3-mCym ligands (L) exchanged onto each cluster. This study provides the first experimental determination of the relative binding energies of ligated gold clusters containing differently substituted monophosphine ligands, which are important to controlling their synthesis and reactivity in solution. The results also indicate that ligand substitution is an important

  20. Allosteric Modulation of SULT2A1 by Celecoxib and Nimesulide: Computational Analyses

    PubMed Central

    Yalcin, Emine Bihter; Struzik, Scott M.; King, Roberta S.

    2010-01-01

    We used protein-ligand docking and minimization to identify celecoxib as an allosteric modulator of SULT2A1-catalyzed estradiol sulfonation. Subsequent to celecoxib docking and complex minimization, conformational changes in SULT2A1 allowed estradiol docking to an alternative binding region with predicted preference for 17β-OH-E2 sulfonation over 3-OH-E2 sulfonation. PMID:19356094

  1. Antiangiogenic Activity of Alofanib, an Allosteric Inhibitor of Fibroblast Growth Factor Receptor 2.

    PubMed

    Khochenkov, D A; Solomko, E Sch; Peretolchina, N M; Ryabaya, O O; Stepanova, E V

    2015-11-01

    Alofanib is a potential allosteric inhibitor of FGFR2 used in oncology. The inhibitor blocks the extracellular part of the receptor and prevents its binding with the ligand. Alofanib suppressed proliferation of endothelial cells, their migration activity, and ability to form vessellike structures in vitro and significantly decreased the number of microvessels in Matrigel implant and in ovarian cancer (SKOV-3) xenograft in vivo. The results indicate that Alofanib can inhibit angiogenesis. PMID:26597690

  2. Elucidation of Nonadditive Effects in Protein-Ligand Binding Energies: Thrombin as a Case Study.

    PubMed

    Calabrò, Gaetano; Woods, Christopher J; Powlesland, Francis; Mey, Antonia S J S; Mulholland, Adrian J; Michel, Julien

    2016-06-23

    Accurate predictions of free energies of binding of ligands to proteins are challenging partly because of the nonadditivity of protein-ligand interactions; i.e., the free energy of binding is the sum of numerous enthalpic and entropic contributions that cannot be separated into functional group contributions. In principle, molecular simulations methodologies that compute free energies of binding do capture nonadditivity of protein-ligand interactions, but efficient protocols are necessary to compute well-converged free energies of binding that clearly resolve nonadditive effects. To this end, an efficient GPU-accelerated implementation of alchemical free energy calculations has been developed and applied to two congeneric series of ligands of the enzyme thrombin. The results show that accurate binding affinities are computed across the two congeneric series and positive coupling between nonpolar R(1) substituents and a X = NH3(+) substituent is reproduced, albeit with a weaker trend than experimentally observed. By contrast, a docking methodology completely fails to capture nonadditive effects. Further analysis shows that the nonadditive effects are partly due to variations in the strength of a hydrogen-bond between the X = NH3(+) ligands family and thrombin residue Gly216. However, other partially compensating interactions occur across the entire binding site, and no single interaction dictates the magnitude of the nonadditive effects for all the analyzed protein-ligand complexes. PMID:27248478

  3. Trypsin-Ligand Binding Free Energies from Explicit and Implicit Solvent Simulations with Polarizable Potential

    PubMed Central

    Jiao, Dian; Zhang, Jiajing; Duke, Robert E.; Li, Guohui; Ren, Pengyu

    2009-01-01

    We have calculated the binding free energies of a series of benzamidine-like inhibitors to trypsin with a polarizable force field using both explicit and implicit solvent approaches. Free energy perturbation has been performed for the ligands in bulk water and in protein complex with molecular dynamics simulations. The calculated binding free energies are well within the accuracy of experimental measurement and the direction of change is predicted correctly in call cases. We analyzed the molecular dipole moments of the ligands in gas, water and protein environments. Neither binding affinity nor ligand solvation free energy in bulk water shows much dependence on the molecular dipole moments of the ligands. Substitution of the aromatic or the charged group in the ligand results in considerable change in the solvation energy in bulk water and protein whereas the binding affinity varies insignificantly due to cancellation. The effect of chemical modification on ligand charge distribution is mostly local. Replacing benzene with diazine has minimal impact on the atomic multipoles at the amidinium group. We have also utilized an implicit solvent based end-state approach to evaluate the binding free energies of these inhibitors. In this approach, the polarizable multipole model combined with Poisson-Boltzmann/surface area (PMPB/SA) provides the electrostatic interaction energy and the polar solvation free energy. Overall the relative binding free energies obtained from the PMPB/SA model are in good agreement with the experimental data. PMID:19399779

  4. Dynamics and intramolecular ligand binding of DtxR studied by MD simulations and NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Yi, Myunggi; Bhattacharya, Nilakshee; Zhou, Huan-Xiang

    2005-11-01

    Diphtheria toxin repressor (DtxR) regulates the expression of the diphtheria toxin gene through intramolecular ligand binding (Wylie et al., Biochemistry 2005, 44:40-51). Protein dynamics is essential to the binding process of the Pro-rich (Pr) ligand to the C-terminal SH3 domain. We present MD and NMR results on the dynamics and ligand interactions of a Pr-SH3 construct of DtxR. NMR relaxation data (T1, T2, and NOE) showed that the Pr ligand is very flexible, suggesting that it undergoes binding/unbinding transitions. A 50-ns MD trajectory of the protein was used to calculate T1, T2, and NOE, reproducing the NMR results for the SH3 domain but not for the Pr segment. During the MD simulation, the ligand stayed bound to the SH3 domain; thus the simulation represented the bound state. The NMR data for the Pr-segment could be explained by assuming that they represented the average behavior of a fast binding/unbinding exchange. Though unbinding was not observed in the MD simulation, the simulation did show large fluctuations of a loop which forms part of the wall of the binding pocket. The fluctuations led to opening up of the binding pocket, thus weakening the interaction with the Pr segment and perhaps ultimately leading to ligand unbinding.

  5. High affinity and temperature sensitivity of blood oxygen binding in Pangasianodon hypophthalmus due to lack of chloride-hemoglobin allosteric interaction.

    PubMed

    Damsgaard, Christian; Phuong, Le My; Huong, Do Thi Thanh; Jensen, Frank B; Wang, Tobias; Bayley, Mark

    2015-06-01

    Air-breathing fishes represent interesting organisms in terms of understanding the physiological changes associated with the terrestrialization of vertebrates, and, further, are of great socio-economic importance for aquaculture in Southeast Asia. To understand how environmental factors, such as high temperature, affect O2 transport in air-breathing fishes, this study assessed the effects of temperature on O2 binding of blood and Hb in the economically important air-breathing fish Pangasianodon hypophthalmus. To determine blood O2 binding properties, blood was drawn from resting cannulated fishes and O2 binding curves made at 25°C and 35°C. To determine the allosteric regulation and thermodynamics of Hb O2 binding, Hb was purified, and O2 equilibria were recorded at five temperatures in the absence and presence of ATP and Cl(-). Whole blood had a high O2 affinity (O2 tension at half saturation P50 = 4.6 mmHg at extracellular pH 7.6 and 25°C), a high temperature sensitivity of O2 binding (apparent heat of oxygenation ΔH(app) = -28.3 kcal/mol), and lacked a Root effect. Further, the data on Hb revealed weak ATP binding and a complete lack of Cl(-) binding to Hb, which, in part, explains the high O2 affinity and high temperature sensitivity of blood O2 binding. This study demonstrates how a potent mechanism for increasing O2 affinity is linked to increased temperature sensitivity of O2 transport and provides a basic framework for a better understanding of how hypoxia-adapted species will react to increasing temperatures. PMID:25810388

  6. Modulation of global low-frequency motions underlies allosteric regulation: demonstration in CRP/FNR family transcription factors.

    PubMed

    Rodgers, Thomas L; Townsend, Philip D; Burnell, David; Jones, Matthew L; Richards, Shane A; McLeish, Tom C B; Pohl, Ehmke; Wilson, Mark R; Cann, Martin J

    2013-09-01

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distinct site. There is growing evidence that allosteric cooperativity can be communicated by modulation of protein dynamics without conformational change. The mechanisms, however, for communicating dynamic fluctuations between sites are debated. We provide a foundational theory for how allostery can occur as a function of low-frequency dynamics without a change in structure. We have generated coarse-grained models that describe the protein backbone motions of the CRP/FNR family transcription factors, CAP of Escherichia coli and GlxR of Corynebacterium glutamicum. The latter we demonstrate as a new exemplar for allostery without conformation change. We observe that binding the first molecule of cAMP ligand is correlated with modulation of the global normal modes and negative cooperativity for binding the second cAMP ligand without a change in mean structure. The theory makes key experimental predictions that are tested through an analysis of variant proteins by structural biology and isothermal calorimetry. Quantifying allostery as a free energy landscape revealed a protein "design space" that identified the inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, through analyzing CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. This finding provides a link between the position of CRP/FNR transcription factors within the allosteric free energy landscapes and evolutionary selection pressures. Our study therefore reveals significant features of the mechanistic basis for allostery. Changes in low-frequency dynamics correlate with allosteric effects on ligand binding without the requirement for a defined spatial pathway. In addition to evolving suitable three-dimensional structures, CRP/FNR family transcription factors have been selected to

  7. Modulation of Global Low-Frequency Motions Underlies Allosteric Regulation: Demonstration in CRP/FNR Family Transcription Factors

    PubMed Central

    Burnell, David; Jones, Matthew L.; Richards, Shane A.; McLeish, Tom C. B.; Pohl, Ehmke; Wilson, Mark R.; Cann, Martin J.

    2013-01-01

    Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distinct site. There is growing evidence that allosteric cooperativity can be communicated by modulation of protein dynamics without conformational change. The mechanisms, however, for communicating dynamic fluctuations between sites are debated. We provide a foundational theory for how allostery can occur as a function of low-frequency dynamics without a change in structure. We have generated coarse-grained models that describe the protein backbone motions of the CRP/FNR family transcription factors, CAP of Escherichia coli and GlxR of Corynebacterium glutamicum. The latter we demonstrate as a new exemplar for allostery without conformation change. We observe that binding the first molecule of cAMP ligand is correlated with modulation of the global normal modes and negative cooperativity for binding the second cAMP ligand without a change in mean structure. The theory makes key experimental predictions that are tested through an analysis of variant proteins by structural biology and isothermal calorimetry. Quantifying allostery as a free energy landscape revealed a protein “design space” that identified the inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, through analyzing CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. This finding provides a link between the position of CRP/FNR transcription factors within the allosteric free energy landscapes and evolutionary selection pressures. Our study therefore reveals significant features of the mechanistic basis for allostery. Changes in low-frequency dynamics correlate with allosteric effects on ligand binding without the requirement for a defined spatial pathway. In addition to evolving suitable three-dimensional structures, CRP/FNR family transcription factors have been selected to

  8. Two Unique Ligand-Binding Clamps of Rhizopus oryzae Starch Binding Domain for Helical Structure Disruption of Amylose

    PubMed Central

    Jiang, Ting-Ying; Ci, Yuan-Pei; Chou, Wei-I; Lee, Yuan-Chuan; Sun, Yuh-Ju; Chou, Wei-Yao; Li, Kun-Mou; Chang, Margaret Dah-Tsyr

    2012-01-01

    The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21) members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs. PMID:22815939

  9. Ligand-induced conformational changes in a thermophilic ribose-binding protein

    SciTech Connect

    Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.

    2009-05-21

    Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments. Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation (appTm value is 108 C) than the mesophilic Escherichia coli homolog (ecRBP) (appTm value is 56 C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved. Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different

  10. Desformylflustrabromine: A Novel Positive Allosteric Modulator for beta2 Subunit Containing Nicotinic Receptor Sub-Types.

    PubMed

    Pandya, Anshul A

    2016-01-01

    Nicotinic acetylcholine receptors are ligand-gated transmembrane ion channels that are present at the neuromuscular junction and in different locations in the nervous system. The different subtypes of neuronal nicotinic acetylcholine receptors that are found in the brain are thought to be involved in many neurological processes such as pain, cognitive function and depression, as well as in the pathophysiology of numerous neurological diseases and conditions. While the neurotransmitter acetylcholine is an endogenous agonist for all nicotinic receptors subtypes, many drugs that act as agonists and antagonists have also been identified or developed for these receptors. In addition, a novel class of compounds described as allosteric modulators have also been identified or developed for nicotinic acetylcholine receptors. Allosteric modulators are ligands that bind to nicotinic receptors at sites other than the orthosteric site where acetylcholine binds. One such allosteric modulator is desformylflustrabromine. Five chemical analogs along with desformylflustrabromine act as positive allosteric modulator for nAChRs that contain the beta2 subunit in their pentameric structure. Here the discovery and development, medicinal chemistry and pharmacological actions of desformylflustrabromine have been discussed. Desformylflustrabromine and its chemical analogs have the potential to develop into clinically used drugs for neurological diseases and conditions where nicotinic acetylcholine receptors are involved. PMID:26818864

  11. Kuwanon-L as a New Allosteric HIV-1 Integrase Inhibitor: Molecular Modeling and Biological Evaluation.

    PubMed

    Esposito, Francesca; Tintori, Cristina; Martini, Riccardo; Christ, Frauke; Debyser, Zeger; Ferrarese, Roberto; Cabiddu, Gianluigi; Corona, Angela; Ceresola, Elisa Rita; Calcaterra, Andrea; Iovine, Valentina; Botta, Bruno; Clementi, Massimo; Canducci, Filippo; Botta, Maurizio; Tramontano, Enzo

    2015-11-01

    HIV-1 integrase (IN) active site inhibitors are the latest class of drugs approved for HIV treatment. The selection of IN strand-transfer drug-resistant HIV strains in patients supports the development of new agents that are active as allosteric IN inhibitors. Here, a docking-based virtual screening has been applied to a small library of natural ligands to identify new allosteric IN inhibitors that target the sucrose binding pocket. From theoretical studies, kuwanon-L emerged as the most promising binder and was thus selected for biological studies. Biochemical studies showed that kuwanon-L is able to inhibit the HIV-1 IN catalytic activity in the absence and in the presence of LEDGF/p75 protein, the IN dimerization, and the IN/LEDGF binding. Kuwanon-L also inhibited HIV-1 replication in cell cultures. Overall, docking and biochemical results suggest that kuwanon-L binds to an allosteric binding pocket and can be considered an attractive lead for the development of new allosteric IN antiviral agents. PMID:26360521

  12. Binding kinetics of membrane-anchored receptors and ligands: Molecular dynamics simulations and theory

    NASA Astrophysics Data System (ADS)

    Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard; Weikl, Thomas R.

    2015-12-01

    The adhesion of biological membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. Central questions are how the binding kinetics of these proteins is affected by the membranes and by the membrane anchoring of the proteins. In this article, we (i) present detailed data for the binding of membrane-anchored proteins from coarse-grained molecular dynamics simulations and (ii) provide a theory that describes how the binding kinetics depends on the average separation and thermal roughness of the adhering membranes and on the anchoring, lengths, and length variations of the proteins. An important element of our theory is the tilt of bound receptor-ligand complexes and transition-state complexes relative to the membrane normals. This tilt results from an interplay of the anchoring energy and rotational entropy of the complexes and facilitates the formation of receptor-ligand bonds at membrane separations smaller than the preferred separation for binding. In our simulations, we have considered both lipid-anchored and transmembrane receptor and ligand proteins. We find that the binding equilibrium constant and binding on-rate constant of lipid-anchored proteins are considerably smaller than the binding constant and on-rate constant of rigid transmembrane proteins with identical binding domains.

  13. NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

    PubMed

    Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted. PMID:19836400

  14. Development of Photoactivatable Allosteric Modulators for the Chemokine Receptor CXCR3.

    PubMed

    Admas, Tizita Haimanot; Bernat, Viachaslau; Heinrich, Markus R; Tschammer, Nuska

    2016-03-17

    The CXCR3 receptor, a class A G protein-coupled receptor (GPCR), is involved in the regulation and trafficking of various immune cells. CXCR3 antagonists have been proposed to be beneficial for the treatment of a wide range of disorders including but not limited to inflammatory and autoimmune diseases. The structure-based design of CXCR3 ligands remains, however, hampered by a lack of structural information describing in detail the interactions between an allosteric ligand and the receptor. We designed and synthesized photoactivatable probes for the structural and functional characterization, using photoaffinity labeling followed by mass spectrometry, of the CXCR3 allosteric binding pocket of AMG 487 and RAMX3, two potent and selective CXCR3 negative allosteric modulators. Photoaffinity labeling is a common approach to elucidate binding modes of small-molecule ligands of GPCRs through the aid of photoactivatable probes that convert to extremely reactive intermediates upon photolysis. The photolabile probe N-[({1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-{1-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}piperidin-4-yl)methyl]acetamide (10) showed significant labeling of the CXCR3 receptor (80 %) in a [(3) H]RAMX3 radioligand displacement assay. Compound 10 will serve as an important tool compound for the detailed investigation of the binding pocket of CXCR3 by mass spectrometry. PMID:26880380

  15. Thermodynamics of binding of di- and tetrasubstituted naphthalene diimide ligands to DNA G-quadruplex.

    PubMed

    Prato, Gary; Silvent, Samantha; Saka, Sammy; Lamberto, Massimiliano; Kosenkov, Dmytro

    2015-02-26

    Naphthalene diimide ligands have the potential to stabilize human telomeric G-quadruplex DNA via noncovalent interactions. Stabilization of G-quadruplex high order structures has become an important strategy to develop novel anticancer therapeutics. In this study four naphthalene diimide based ligands were analyzed in order to elucidate the principal factors determining contributions to G-quadruplex-ligand binding. Three possible modes of binding and their respective Gibbs free energies for two naphthalene diimide based di-N-alkylpyridinium substituted ligands have been determined using a molecular docking technique and compared to experimental results. The structures obtained from the molecular docking calculations, were analyzed using the ab initio based fragment molecular orbital (FMO) method in order to determine the major enthalpic contributions to the binding and types of interactions between the ligand and specific residues of the G-quadruplex. A computational methodology for the efficient and inexpensive ligand optimization as compared to fully ab initio methods based on the estimation of binding affinities of the naphthalene diimide derived ligands to G-quadruplex is proposed. PMID:25635929

  16. A Sphingosine 1-phosphate receptor 2 selective allosteric agonist

    PubMed Central

    Satsu, Hideo; Schaeffer, Marie-Therese; Guerrero, Miguel; Saldana, Adrian; Eberhart, Christina; Hodder, Peter; Cayanan, Charmagne; Schürer, Stephan; Bhhatarai, Barun; Roberts, Ed; Rosen, Hugh; Brown, Steven J.

    2013-01-01

    Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound. PMID:23849205

  17. The role of hydration on the mechanism of allosteric regulation: in situ measurements of the oxygen-linked kinetics of water binding to hemoglobin.

    PubMed

    Salvay, Andrés G; Grigera, J Raúl; Colombo, Marcio F

    2003-01-01

    We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98% relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo. PMID:12524309

  18. ATP-independent CFTR channel gating and allosteric modulation by phosphorylation

    PubMed Central

    Wang, Wei; Wu, Jianping; Bernard, Karen; Li, Ge; Wang, Guangyu; Bevensee, Mark O.; Kirk, Kevin L.

    2010-01-01

    Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligand-gated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization. PMID:20133716

  19. Imaging G protein-coupled receptors while quantifying their ligand-binding free-energy landscape.

    PubMed

    Alsteens, David; Pfreundschuh, Moritz; Zhang, Cheng; Spoerri, Patrizia M; Coughlin, Shaun R; Kobilka, Brian K; Müller, Daniel J

    2015-09-01

    Imaging native membrane receptors and testing how they interact with ligands is of fundamental interest in the life sciences but has proven remarkably difficult to accomplish. Here, we introduce an approach that uses force-distance curve-based atomic force microscopy to simultaneously image single native G protein-coupled receptors in membranes and quantify their dynamic binding strength to native and synthetic ligands. We measured kinetic and thermodynamic parameters for individual protease-activated receptor-1 (PAR1) molecules in the absence and presence of antagonists, and these measurements enabled us to describe PAR1's ligand-binding free-energy landscape with high accuracy. Our nanoscopic method opens an avenue to directly image and characterize ligand binding of native membrane receptors. PMID:26167642

  20. Large-scale molecular dynamics simulation: Effect of polarization on thrombin-ligand binding energy.

    PubMed

    Duan, Li L; Feng, Guo Q; Zhang, Qing G

    2016-01-01

    Molecular dynamics (MD) simulations lasting 500 ns were performed in explicit water to investigate the effect of polarization on the binding of ligands to human α-thrombin based on the standard nonpolarizable AMBER force field and the quantum-derived polarized protein-specific charge (PPC). The PPC includes the electronic polarization effect of the thrombin-ligand complex, which is absent in the standard force field. A detailed analysis and comparison of the results of the MD simulation with experimental data provided strong evidence that intra-protein, protein-ligand hydrogen bonds and the root-mean-square deviation of backbone atoms were significantly stabilized through electronic polarization. Specifically, two critical hydrogen bonds between thrombin and the ligand were broken at approximately 190 ns when AMBER force field was used and the number of intra-protein backbone hydrogen bonds was higher under PPC than under AMBER. The thrombin-ligand binding energy was computed using the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method, and the results were consistent with the experimental value obtained using PPC. Because hydrogen bonds were unstable, it was failed to predict the binding affinity under the AMBER force field. Furthermore, the results of the present study revealed that differences in the binding free energy between AMBER and PPC almost comes from the electrostatic interaction. Thus, this study provides evidence that protein polarization is critical to accurately describe protein-ligand binding. PMID:27507430

  1. Notch ligand delta-like1: X-ray crystal structure and binding affinity.

    PubMed

    Kershaw, Nadia J; Church, Nicole L; Griffin, Michael D W; Luo, Cindy S; Adams, Timothy E; Burgess, Antony W

    2015-05-15

    The Notch pathway is a fundamental signalling system in most multicellular animals. We have determined the X-ray crystal structure of the extracellular domain of the Notch ligand delta-like ligand-1 (Dll-1). The structure incorporates the N-terminal C2 domain, receptor-binding DSL domain and the first six (of eight) EGF (epidermal growth factor)-like repeats, which form a highly extended conformation, confirmed by analytical ultracentrifugation. Comparison of our structure with a fragment of Jagged1 ligand allows us to dissect the similarities and differences between the ligand families. Differences in the C2 domains of Dll-1 and Jagged1 suggest their lipid-binding properties are likely to differ. A conserved hydrophobic patch on the surface of both Dll-1 and Jagged1 provides a likely receptor-interaction site that is common to both ligands. We also explore the binding affinity of Dll-1 for a fragment of Notch1 using different techniques. Apparent binding affinities vary when different techniques are used, explaining discrepancies in the literature. Using analytical ultracentrifugation, we perform for the first time binding analyses where both receptor and ligand are in solution, which confirms a Kd of 10 μM for this interaction. PMID:25715738

  2. Large-scale molecular dynamics simulation: Effect of polarization on thrombin-ligand binding energy

    PubMed Central

    Duan, Li L.; Feng, Guo Q.; Zhang, Qing G.

    2016-01-01

    Molecular dynamics (MD) simulations lasting 500 ns were performed in explicit water to investigate the effect of polarization on the binding of ligands to human α-thrombin based on the standard nonpolarizable AMBER force field and the quantum-derived polarized protein-specific charge (PPC). The PPC includes the electronic polarization effect of the thrombin-ligand complex, which is absent in the standard force field. A detailed analysis and comparison of the results of the MD simulation with experimental data provided strong evidence that intra-protein, protein-ligand hydrogen bonds and the root-mean-square deviation of backbone atoms were significantly stabilized through electronic polarization. Specifically, two critical hydrogen bonds between thrombin and the ligand were broken at approximately 190 ns when AMBER force field was used and the number of intra-protein backbone hydrogen bonds was higher under PPC than under AMBER. The thrombin-ligand binding energy was computed using the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method, and the results were consistent with the experimental value obtained using PPC. Because hydrogen bonds were unstable, it was failed to predict the binding affinity under the AMBER force field. Furthermore, the results of the present study revealed that differences in the binding free energy between AMBER and PPC almost comes from the electrostatic interaction. Thus, this study provides evidence that protein polarization is critical to accurately describe protein-ligand binding. PMID:27507430

  3. Proton coupling in the ligand-binding reaction of ferric cytochrome P-450 from Pseudomonas putida

    SciTech Connect

    Totani, K.; Iizuka, T.; Shimada, H.; Makino, R.; Ishimura, Y.

    1983-04-01

    Effects of pH on the ligand-binding reactions of ferric heme in cytochrome P-450 from Pseudomonas putida (camphor 5-monooxygenase, EC 1.14.15.1) were studied by using cyanide, N-methylimidazole, pyridine, and ethylisocyanide as ligands. In all cases, affinity of the ferric heme for the ligand was found to increase as pH of the medium was raised from around 6 to 9. Depending on the ligand, the increase was 10- to 1000-fold and the shapes of their pH-affinity curves were remarkably different. Analyses such pH profiles disclosed the presence of a dissociable group in the enzyme with a pK value of approximately 9.5 and that its ionization greatly enhanced the affinity of the heme for ligands. When a dissociable ligand such as hydrogen cyanide and N-methylimidazole was used, the dissociated form of the ligand had a higher affinity toward the heme than the undissociated form. The shapes of the pH-affinity curves were successfully simulated as overlapping curves of ionization reactions of the ligand and the dissociable group. In addition, size of the ligand molecule was shown to be also important in the binding reaction: relatively large molecules such as pyridine, ethylisocyanide, and N-methylimidazole bound to the enzyme in a competitive manner against d-camphor concentration, whereas the binding of a smaller molecule such as cyanide was inhibited by the substrate in a noncompetitive manner. On the basis of these findings, control mechanisms for the ligand-binding reactions of the cytochrome P-450 from P. putida are discussed.

  4. PatchSurfers: Two methods for local molecular property-based binding ligand prediction.

    PubMed

    Shin, Woong-Hee; Bures, Mark Gregory; Kihara, Daisuke

    2016-01-15

    Protein function prediction is an active area of research in computational biology. Function prediction can help biologists make hypotheses for characterization of genes and help interpret biological assays, and thus is a productive area for collaboration between experimental and computational biologists. Among various function prediction methods, predicting binding ligand molecules for a target protein is an important class because ligand binding events for a protein are usually closely intertwined with the proteins' biological function, and also because predicted binding ligands can often be directly tested by biochemical assays. Binding ligand prediction methods can be classified into two types: those which are based on protein-protein (or pocket-pocket) comparison, and those that compare a target pocket directly to ligands. Recently, our group proposed two computational binding ligand prediction methods, Patch-Surfer, which is a pocket-pocket comparison method, and PL-PatchSurfer, which compares a pocket to ligand molecules. The two programs apply surface patch-based descriptions to calculate similarity or complementarity between molecules. A surface patch is characterized by physicochemical properties such as shape, hydrophobicity, and electrostatic potentials. These properties on the surface are represented using three-dimensional Zernike descriptors (3DZD), which are based on a series expansion of a 3 dimensional function. Utilizing 3DZD for describing the physicochemical properties has two main advantages: (1) rotational invariance and (2) fast comparison. Here, we introduce Patch-Surfer and PL-PatchSurfer with an emphasis on PL-PatchSurfer, which is more recently developed. Illustrative examples of PL-PatchSurfer performance on binding ligand prediction as well as virtual drug screening are also provided. PMID:26427548

  5. Evidence of Conformational Selection Driving the Formation of Ligand Binding Sites in Protein-Protein Interfaces

    PubMed Central

    Bohnuud, Tanggis; Kozakov, Dima; Vajda, Sandor

    2014-01-01

    Many protein-protein interactions (PPIs) are compelling targets for drug discovery, and in a number of cases can be disrupted by small molecules. The main goal of this study is to examine the mechanism of binding site formation in the interface region of proteins that are PPI targets by comparing ligand-free and ligand-bound structures. To avoid any potential bias, we focus on ensembles of ligand-free protein conformations obtained by nuclear magnetic resonance (NMR) techniques and deposited in the Protein Data Bank, rather than on ensembles specifically generated for this study. The measures used for structure comparison are based on detecting binding hot spots, i.e., protein regions that are major contributors to the binding free energy. The main tool of the analysis is computational solvent mapping, which explores the surface of proteins by docking a large number of small “probe” molecules. Although we consider conformational ensembles obtained by NMR techniques, the analysis is independent of the method used for generating the structures. Finding the energetically most important regions, mapping can identify binding site residues using ligand-free models based on NMR data. In addition, the method selects conformations that are similar to some peptide-bound or ligand-bound structure in terms of the properties of the binding site. This agrees with the conformational selection model of molecular recognition, which assumes such pre-existing conformations. The analysis also shows the maximum level of similarity between unbound and bound states that is achieved without any influence from a ligand. Further shift toward the bound structure assumes protein-peptide or protein-ligand interactions, either selecting higher energy conformations that are not part of the NMR ensemble, or leading to induced fit. Thus, forming the sites in protein-protein interfaces that bind peptides and can be targeted by small ligands always includes conformational selection, although

  6. Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

    SciTech Connect

    Suino-Powell, Kelly; Xu, Yong; Zhang, Chenghai; Tao, Yong-guang; Tolbert, W. David; Simons, Jr., S. Stoney; Xu, H. Eric

    2010-03-08

    A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 {angstrom}{sup 3}, effectively doubling the size of the GR dexamethasone-binding pocket of 540 {angstrom}{sup 3} and yet leaving the structure of the coactivator binding site intact. DAC occupies only {approx}50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.

  7. Direct Determination of Vibrational Density of States Change on Ligand Binding to a Protein

    NASA Astrophysics Data System (ADS)

    Balog, Erika; Becker, Torsten; Oettl, Martin; Lechner, Ruep; Daniel, Roy; Finney, John; Smith, Jeremy C.

    2004-07-01

    The change in the vibrational density of states of a protein (dihydrofolate reductase) on binding a ligand (methotrexate) is determined using inelastic neutron scattering. The vibrations of the complex soften significantly relative to the unbound protein. The resulting free-energy change, which is directly determined by the density of states change, is found to contribute significantly to the binding equilibrium.

  8. Differential utilization of binding loop flexibility in T cell receptor ligand selection and cross-reactivity.

    PubMed

    Ayres, Cory M; Scott, Daniel R; Corcelli, Steven A; Baker, Brian M

    2016-01-01

    Complementarity determining region (CDR) loop flexibility has been suggested to play an important role in the selection and binding of ligands by T cell receptors (TCRs) of the cellular immune system. However, questions remain regarding the role of loop motion in TCR binding, and crystallographic structures have raised questions about the extent to which generalizations can be made. Here we studied the flexibility of two structurally well characterized αβ TCRs, A6 and DMF5. We found that the two receptors utilize loop motion very differently in ligand binding and cross-reactivity. While the loops of A6 move rapidly in an uncorrelated fashion, those of DMF5 are substantially less mobile. Accordingly, the mechanisms of binding and cross-reactivity are very different between the two TCRs: whereas A6 relies on conformational selection to select and bind different ligands, DMF5 uses a more rigid, permissive architecture with greater reliance on slower motions or induced-fit. In addition to binding site flexibility, we also explored whether ligand-binding resulted in common dynamical changes in A6 and DMF5 that could contribute to TCR triggering. Although binding-linked motional changes propagated throughout both receptors, no common features were observed, suggesting that changes in nanosecond-level TCR structural dynamics do not contribute to T cell signaling. PMID:27118724

  9. VASP: A Volumetric Analysis of Surface Properties Yields Insights into Protein-Ligand Binding Specificity

    PubMed Central

    Chen, Brian Y.; Honig, Barry

    2010-01-01

    Many algorithms that compare protein structures can reveal similarities that suggest related biological functions, even at great evolutionary distances. Proteins with related function often exhibit differences in binding specificity, but few algorithms identify structural variations that effect specificity. To address this problem, we describe the Volumetric Analysis of Surface Properties (VASP), a novel volumetric analysis tool for the comparison of binding sites in aligned protein structures. VASP uses solid volumes to represent protein shape and the shape of surface cavities, clefts and tunnels that are defined with other methods. Our approach, inspired by techniques from constructive solid geometry, enables the isolation of volumetrically conserved and variable regions within three dimensionally superposed volumes. We applied VASP to compute a comparative volumetric analysis of the ligand binding sites formed by members of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains and the serine proteases. Within both families, VASP isolated individual amino acids that create structural differences between ligand binding cavities that are known to influence differences in binding specificity. Also, VASP isolated cavity subregions that differ between ligand binding cavities which are essential for differences in binding specificity. As such, VASP should prove a valuable tool in the study of protein-ligand binding specificity. PMID:20814581

  10. Differential utilization of binding loop flexibility in T cell receptor ligand selection and cross-reactivity

    PubMed Central

    Ayres, Cory M.; Scott, Daniel R.; Corcelli, Steven A.; Baker, Brian M.

    2016-01-01

    Complementarity determining region (CDR) loop flexibility has been suggested to play an important role in the selection and binding of ligands by T cell receptors (TCRs) of the cellular immune system. However, questions remain regarding the role of loop motion in TCR binding, and crystallographic structures have raised questions about the extent to which generalizations can be made. Here we studied the flexibility of two structurally well characterized αβ TCRs, A6 and DMF5. We found that the two receptors utilize loop motion very differently in ligand binding and cross-reactivity. While the loops of A6 move rapidly in an uncorrelated fashion, those of DMF5 are substantially less mobile. Accordingly, the mechanisms of binding and cross-reactivity are very different between the two TCRs: whereas A6 relies on conformational selection to select and bind different ligands, DMF5 uses a more rigid, permissive architecture with greater reliance on slower motions or induced-fit. In addition to binding site flexibility, we also explored whether ligand-binding resulted in common dynamical changes in A6 and DMF5 that could contribute to TCR triggering. Although binding-linked motional changes propagated throughout both receptors, no common features were observed, suggesting that changes in nanosecond-level TCR structural dynamics do not contribute to T cell signaling. PMID:27118724

  11. Differentiation between ligand trapping into intact cells and binding on muscarinic receptors.

    PubMed

    Gossuin, A; Maloteaux, J M; Trouet, A; Laduron, P

    1984-05-22

    Binding properties of [3H] dexetimide , L-quinuclidinyl[phenyl-4-3H] benzilate and [3H]methylscopolamine were compared with intact 108 CC 15 cells and membrane preparations of those. The ability of the three ligands to label specifically muscarinic receptors on membrane fractions was quite similar. By contrast, when performed with intact cells, [3H] dexetimide and L-quinuclidinyl [phenyl-4-3H]benzilate revealed higher nonspecific binding which was prevented by methylamine, suggesting a trapping of the ligands within the cells presumably in the lysosomes. To the contrary, such nonspecific 'binding' or trapping was not detectable when [3H]methylscopolamine was used as ligand, a fact which makes this ligand particularly appropriate for labelling cell surface muscarinic receptors. It is concluded that more caution is needed in binding studies when performed with intact cells; indeed, besides specific binding on receptor sites, [3H]ligand can be entrapped within the cell and can even sometimes give the illusion of specific binding. The use of lysosomal agents which do not interfere with specific receptors on membrane preparations should allow one, in most cases, to discard the possibility of a trapping phenomenon in intact cells. PMID:6722181

  12. Steroid signaling: ligand-binding promiscuity, molecular symmetry, and the need for gating.

    PubMed

    Lathe, Richard; Kotelevtsev, Yuri

    2014-04-01

    Steroid/sterol-binding receptors and enzymes are remarkably promiscuous in the range of ligands they can bind to and, in the case of enzymes, modify - raising the question of how specific receptor activation is achieved in vivo. Estrogen receptors (ER) are modulated by 27-hydroxycholesterol and 5α-androstane-3β,17β-diol (Adiol), in addition to estradiol (E2), and respond to diverse small molecules such as bisphenol A. Steroid-modifying enzymes are also highly promiscuous in ligand binding and metabolism. The specificity problem is compounded by the fact that the steroid core (hydrogenated cyclopentophenanthrene ring system) has several planes of symmetry. Ligand binding can be in symmetrical East-West (rotation) and North-South (inversion) orientations. Hydroxysteroid dehydrogenases (HSDs) can modify symmetrical 7 and 11, also 3 and 17/20, positions, exemplified here by yeast 3α,20β-HSD and mammalian 11β-HSD and 17β-HSD enzymes. Faced with promiscuity and symmetry, other strategies are clearly necessary to promote signaling selectivity in vivo. Gating regulates hormone access via enzymes that preferentially inactivate (or activate) a subclass of ligands, thereby governing which ligands gain receptor access - exemplified by 11β-HSD gating cortisol access to the mineralocorticoid receptor, and P450 CYP7B1 gating Adiol access to ER. Counter-intuitively, the specificity of steroid/sterol action is achieved not by intrinsic binding selectivity but by the combination of local metabolism and binding affinity. PMID:24462647

  13. Coupling of disulfide bond and distal histidine dissociation in human ferrous cytoglobin regulates ligand binding.

    PubMed

    Beckerson, Penny; Reeder, Brandon J; Wilson, Michael T

    2015-02-13

    Earlier kinetics studies on cytoglobin did not assign functional properties to specific structural forms. Here, we used defined monomeric and dimeric forms and cysteine mutants to show that an intramolecular disulfide bond (C38-C83) alters the dissociation rate constant of the intrinsic histidine (H81) (∼1000 fold), thus controlling binding of extrinsic ligands. Through time-resolved spectra we have unequivocally assigned CO binding to hexa- and penta-coordinate forms and have made direct measurement of histidine rebinding following photolysis. We present a model that describes how the cysteine redox state of the monomer controls histidine dissociation rate constants and hence extrinsic ligand binding. PMID:25601563

  14. Effects of the Hydroxyl Group on Phenyl Based Ligand/ERRγ Protein Binding

    PubMed Central

    2015-01-01

    Bisphenol-A (4,4′-dihydroxy-2,2-diphenylpropane, BPA, or BPA-A) and its derivatives, when exposed to humans, may affect functions of multiple organs by specific binding to the human estrogen-related receptor γ (ERRγ). We carried out atomistic molecular dynamics (MD) simulations of three ligand compounds including BPA-A, 4-α-cumylphenol (BPA-C), and 2,2-diphenylpropane (BPA-D) binding to the ligand binding domain (LBD) of a human ERRγ to study the structures and energies associated with the binding. We used the implicit Molecular Mechanics/Poisson–Boltzmann Surface Area (MM/PBSA) method to estimate the free energies of binding for the phenyl based compound/ERRγ systems. The addition of hydroxyl groups to the aromatic ring had only a minor effect on binding structures and a significant effect on ligand/protein binding energy in an aqueous solution. Free binding energies of BPA-D to the ERRγ were found to be considerably less than those of BPA-A and BPA-C to the ERRγ. These results are well correlated with those from experiments where no binding affinities were determined in the BPA-D/ERRγ complex. No conformational change was observed for the helix 12 (H-12) of ERRγ upon binding of these compounds preserving an active transcriptional conformation state. PMID:25098505

  15. Parameterization of an effective potential for protein–ligand binding from host–guest affinity data

    PubMed Central

    Wickstrom, Lauren; Deng, Nanjie; He, Peng; Mentes, Ahmet; Nguyen, Crystal; Gilson, Michael K.; Kurtzman, Tom; Gallicchio, Emilio; Levy, Ronald M.

    2015-01-01

    Force field accuracy is still one of the “stalemates” in biomolecular modeling. Model systems with high quality experimental data are valuable instruments for the validation and improvement of effective potentials. With respect to protein–ligand binding, organic host–guest complexes have long served as models for both experimental and computational studies because of the abundance of binding affinity data available for such systems. Binding affinity data collected for cyclodextrin (CD) inclusion complexes, a popular model for molecular recognition, is potentially a more reliable resource for tuning energy parameters than hydration free energy measurements. Convergence of binding free energy calculations on CD host–guest systems can also be obtained rapidly, thus offering the opportunity to assess the robustness of these parameters. In this work, we demonstrate how implicit solvent parameters can be developed using binding affinity experimental data and the binding energy distribution analysis method (BEDAM) and validated using the Grid Inhomogeneous Solvation Theory analysis. These new solvation parameters were used to study protein–ligand binding in two drug targets against the HIV-1 virus and improved the agreement between the calculated and the experimental binding affinities. This work illustrates how benchmark sets of high quality experimental binding affinity data and physics-based binding free energy models can be used to evaluate and optimize force fields for protein–ligand systems. PMID:26256816

  16. Molecular dynamics studies of Hsp90 with ADP: Protein-ligand binding dynamics

    NASA Astrophysics Data System (ADS)

    Kawaguchi, Kazutomo; Takagi, Hiroyuki; Takasu, Masako; Saito, Hiroaki; Nagao, Hidemi

    2013-02-01

    Ligand binding to a protein molecule plays a key role in the function of many proteins. We performed all-atom model molecular dynamics (MD) simulations of the N-terminal domain of human Hsp90 in complex with ADP, and calculated a free energy profile for ligand binding with thermodynamic integration method. The free energy profile as a function of the distance between the centers of mass of the N-terminal domain of Hsp90 and ADP was calculated using the results of the binding-distance constrained MD simulations. The free energy profile was fit to a harmonic oscillator. We obtained spring constant k = 3.84kJ/molṡnm2 = 9.17×10-1kcal/molṡÅ2. This result indicates that the dynamics of the ligand binding to the protein molecule is about 1,000 times slower than that of the covalent bond.

  17. Allosteric Activation of E2-RING Finger-Mediated Ubiquitylation by a Structurally Defined Specific E2-Binding Region of gp78

    SciTech Connect

    Das, Ranabir; Mariano, Jennifer; Tsai, Yien Che; Kalathur, Ravi C.; Kostova, Zlatka; Li, Jess; Tarasov, Sergey G.; McFeeters, Robert L.; Altieri, Amanda S.; Ji, Xinhua; Byrd, R. Andrew; Weissman, Allan M.

    2010-11-12

    The activity of RING finger ubiquitin ligases (E3) is dependent on their ability to facilitate transfer of ubiquitin from ubiquitin-conjugating enzymes (E2) to substrates. The G2BR domain within the E3 gp78 binds selectively and with high affinity to the E2 Ube2g2. Through structural and functional analyses, we determine that this occurs on a region of Ube2g2 distinct from binding sites for ubiquitin-activating enzyme (E1) and RING fingers. Binding to the G2BR results in conformational changes in Ube2g2 that affect ubiquitin loading. The Ube2g2:G2BR interaction also causes an 50-fold increase in affinity between the E2 and RING finger. This results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger. The significance of this G2BR effect is underscored by enhanced ubiquitylation observed when Ube2g2 is paired with other RING finger E3s. These findings uncover a mechanism whereby allosteric effects on an E2 enhance E2-RING finger interactions and, consequently, ubiquitylation.

  18. Spin-dependent mechanism for diatomic ligand binding to heme

    PubMed Central

    Franzen, Stefan

    2002-01-01

    The nature of diatomic ligand recombination in heme proteins is elucidated by using a Landau–Zener model for the electronic coupling in the recombination rate constant. The model is developed by means of explicit potential energy surfaces calculated by using density functional theory (DFT). The interaction of all possible spin states of the three common diatomic ligands, CO, NO, and O2, and high-spin heme iron is compared. The electronic coupling, rebinding barrier, and Landau–Zener force terms can be obtained and used to demonstrate significant differences among the ligands. In particular the intermediate spin states of NO (S = 3/2) and O2 (S = 1) are shown to be bound states. Rapid recombination occurs from these bound states in agreement with experimental data. The slower phases of O2 recombination can be explained by the presence of two higher spin states, S = 2 and S = 3, which have a small and relatively large barrier to ligand recombination, respectively. By contrast, the intermediate spin state for CO is not a bound state, and the only recombination pathway for CO involves direct recombination from the S = 2 state. This process is significantly slower according to the Landau–Zener model. Quantitative estimates of the parameters used in the rate constants provide a complete description that explains rebinding rates that range from femtoseconds to milliseconds at ambient temperature. PMID:12477933

  19. Dynamics of cellular retinoic acid binding protein I on multiple time scales with implications for ligand binding.

    PubMed

    Krishnan, V V; Sukumar, M; Gierasch, L M; Cosman, M

    2000-08-01

    Cellular retinoic acid binding protein I (CRABPI) belongs to the family of intracellular lipid binding proteins (iLBPs), all of which bind a hydrophobic ligand within an internal cavity. The structures of several iLBPs reveal minimal structural differences between the apo (ligand-free) and holo (ligand-bound) forms, suggesting that dynamics must play an important role in the ligand recognition and binding processes. Here, a variety of nuclear magnetic resonance (NMR) spectroscopy methods were used to systematically study the dynamics of both apo and holo CRABPI at various time scales. Translational and rotational diffusion constant measurements were used to study the overall motions of the proteins. Both apo and holo forms of CRABPI tend to self-associate at high (1.2 mM) concentrations, while at low concentrations (0.2 mM), they are predominantly monomeric. Rapid amide exchange rate and laboratory frame relaxation rate measurements at two spectrometer field strengths (500 and 600 MHz) were used to probe the internal motions of the individual residues. Several residues in the apo form, notably within the ligand recognition region, exhibit millisecond time scale motions that are significantly arrested in the holo form. In contrast, no significant differences in the high-frequency motions were observed between the two forms. These results provide direct experimental evidence for dynamics-induced ligand recognition and binding at a specifically defined time scale. They also exemplify the importance of dynamics in providing a more comprehensive understanding of how a protein functions. PMID:10924105

  20. Crystallographic Study of Novel Transthyretin Ligands Exhibiting Negative-Cooperativity between Two Thyroxine Binding Sites

    PubMed Central

    Singh, Rajiv Ranjan; Mishra, Satyendra; Gupta, Sarika; Surolia, Avadhesha; Salunke, Dinakar M.

    2012-01-01

    Background Transthyretin (TTR) is a homotetrameric serum and cerebrospinal fluid protein that transports thyroxine (T4) and retinol by binding to retinol binding protein. Rate-limiting tetramer dissociation and rapid monomer misfolding and disassembly of TTR lead to amyloid fibril formation in different tissues causing various amyloid diseases. Based on the current understanding of the pathogenesis of TTR amyloidosis, it is considered that the inhibition of amyloid fibril formation by stabilization of TTR in native tetrameric form is a viable approach for the treatment of TTR amyloidosis. Methodology and Principal Findings We have examined interactions of the wtTTR with a series of compounds containing various substitutions at biphenyl ether skeleton and a novel compound, previously evaluated for binding and inhibiting tetramer dissociation, by x-ray crystallographic approach. High resolution crystal structures of five ligands in complex with wtTTR provided snapshots of negatively cooperative binding of ligands in two T4 binding sites besides characterizing their binding orientations, conformations, and interactions with binding site residues. In all complexes, the ligand has better fit and more potent interactions in first T4 site i.e. (AC site) than the second T4 site (BD site). Together, these results suggest that AC site is a preferred ligand binding site and retention of ordered water molecules between the dimer interfaces further stabilizes the tetramer by bridging a hydrogen bond interaction between Ser117 and its symmetric copy. Conclusion Novel biphenyl ether based compounds exhibit negative-cooperativity while binding to two T4 sites which suggests that binding of only single ligand molecule is sufficient to inhibit the TTR tetramer dissociation. PMID:22973437

  1. Automatic generation of bioinformatics tools for predicting protein–ligand binding sites

    PubMed Central

    Banno, Masaki; Ueki, Kokoro; Saad, Gul; Shimizu, Kentaro

    2016-01-01

    Motivation: Predictive tools that model protein–ligand binding on demand are needed to promote ligand research in an innovative drug-design environment. However, it takes considerable time and effort to develop predictive tools that can be applied to individual ligands. An automated production pipeline that can rapidly and efficiently develop user-friendly protein–ligand binding predictive tools would be useful. Results: We developed a system for automatically generating protein–ligand binding predictions. Implementation of this system in a pipeline of Semantic Web technique-based web tools will allow users to specify a ligand and receive the tool within 0.5–1 day. We demonstrated high prediction accuracy for three machine learning algorithms and eight ligands. Availability and implementation: The source code and web application are freely available for download at http://utprot.net. They are implemented in Python and supported on Linux. Contact: shimizu@bi.a.u-tokyo.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26545824

  2. Docking and free energy perturbation studies of ligand binding in the kappa opioid receptor.

    PubMed

    Goldfeld, Dahlia A; Murphy, Robert; Kim, Byungchan; Wang, Lingle; Beuming, Thijs; Abel, Robert; Friesner, Richard A

    2015-01-22

    The kappa opioid receptor (KOR) is an important target for pain and depression therapeutics that lack harmful and addictive qualities of existing medications. We present a model for the binding of morphinan ligands and JDTic to the JDTic/KOR crystal structure based on an atomic level description of the water structure within its active site. The model contains two key interaction motifs that are supported by experimental evidence. The first is the formation of a salt bridge between the ligand and Asp 138(3.32) in transmembrane domain (TM) 3. The second is the stabilization by the ligand of two high energy, isolated, and ice-like waters near TM5 and TM6. This model is incorporated via energetic terms into a new empirical scoring function, WScore, designed to assess interactions between ligands and localized water in a binding site. Pairing WScore with the docking program Glide discriminates known active KOR ligands from large sets of decoy molecules much better than Glide's older generation scoring functions, SP and XP. We also use rigorous free energy perturbation calculations to provide evidence for the proposed mechanism of interaction between ligands and KOR. The molecular description of ligand binding in KOR should provide a good starting point for future drug discovery efforts for this receptor. PMID:25395044

  3. Molecular dynamics simulation of ligand dissociation from liver fatty acid binding protein.

    PubMed

    Long, Dong; Mu, Yuguang; Yang, Daiwen

    2009-01-01

    The mechanisms of how ligands enter and leave the binding cavity of fatty acid binding proteins (FABPs) have been a puzzling question over decades. Liver fatty acid binding protein (LFABP) is a unique family member which accommodates two molecules of fatty acids in its cavity and exhibits the capability of interacting with a variety of ligands with different chemical structures and properties. Investigating the ligand dissociation processes of LFABP is thus a quite interesting topic, which however is rather difficult for both experimental approaches and ordinary simulation strategies. In the current study, random expulsion molecular dynamics simulation, which accelerates ligand motions for rapid dissociation, was used to explore the potential egress routes of ligands from LFABP. The results showed that the previously hypothesized "portal region" could be readily used for the dissociation of ligands at both the low affinity site and the high affinity site. Besides, one alternative portal was shown to be highly favorable for ligand egress from the high affinity site and be related to the unique structural feature of LFABP. This result lends strong support to the hypothesis from the previous NMR exchange studies, which in turn indicates an important role for this alternative portal. Another less favored potential portal located near the N-terminal end was also identified. Identification of the dissociation pathways will allow further mechanistic understanding of fatty acid uptake and release by computational and/or experimental techniques. PMID:19564911

  4. Allosteric mechanisms within the adenosine A2A-dopamine D2 receptor heterotetramer.

    PubMed

    Ferré, Sergi; Bonaventura, Jordi; Tomasi, Dardo; Navarro, Gemma; Moreno, Estefanía; Cortés, Antonio; Lluís, Carme; Casadó, Vicent; Volkow, Nora D

    2016-05-01

    The structure constituted by a G protein coupled receptor (GPCR) homodimer and a G protein provides a main functional unit and oligomeric entities can be viewed as multiples of dimers. For GPCR heteromers, experimental evidence supports a tetrameric structure, comprised of two different homodimers, each able to signal with its preferred G protein. GPCR homomers and heteromers can act as the conduit of allosteric interactions between orthosteric ligands. The well-known agonist/agonist allosteric interaction in the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer, by which A2AR agonists decrease the affinity of D2R agonists, gave the first rationale for the use of A2AR antagonists in Parkinson's disease. We review new pharmacological findings that can be explained in the frame of a tetrameric structure of the A2AR-D2R heteromer: first, ligand-independent allosteric modulations by the D2R that result in changes of the binding properties of A2AR ligands; second, differential modulation of the intrinsic efficacy of D2R ligands for G protein-dependent and independent signaling; third, the canonical antagonistic Gs-Gi interaction within the frame of the heteromer; and fourth, the ability of A2AR antagonists, including caffeine, to also exert the same allosteric modulations of D2R ligands than A2AR agonists, while A2AR agonists and antagonists counteract each other's effects. These findings can have important clinical implications when evaluating the use of A2AR antagonists. They also call for the need of monitoring caffeine intake when evaluating the effect of D2R ligands, when used as therapeutic agents in neuropsychiatric disorders or as probes in imaging studies. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'. PMID:26051403

  5. Conformational selection and adaptation to ligand binding in T4 lysozyme cavity mutants.

    PubMed

    López, Carlos J; Yang, Zhongyu; Altenbach, Christian; Hubbell, Wayne L

    2013-11-12

    The studies presented here explore the relationship between protein packing and molecular flexibility using ligand-binding cavity mutants of T4 lysozyme. Although previously reported crystal structures of the mutants investigated show single conformations that are similar to the WT protein, site-directed spin labeling in solution reveals additional conformational substates in equilibrium exchange with a WT-like population. Remarkably, binding of ligands, including the general anesthetic halothane shifts the population to the WT-like state, consistent with a conformational selection model of ligand binding, but structural adaptation to the ligand is also apparent in one mutant. Distance mapping with double electron-electron resonance spectroscopy and the absence of ligand binding suggest that the new substates induced by the cavity-creating mutations represent alternate packing modes in which the protein fills or partially fills the cavity with side chains, including the spin label in one case; external ligands compete with the side chains for the cavity space, stabilizing the WT conformation. The results have implications for mechanisms of anesthesia, the response of proteins to hydrostatic pressure, and protein engineering. PMID:24167295

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

    PubMed

    Jusuf, Sutjano; Loll, Patrick J; Axelsen, Paul H

    2003-04-01

    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. PMID:12656635

  7. Estimating Protein-Ligand Binding Affinity using High-Throughput Screening by NMR

    PubMed Central

    Shortridge, Matthew D.; Hage, David S.; Harbison, Gerard S.; Powers, Robert

    2009-01-01

    Many of today’s drug discovery programs utilize high-throughput screening methods that rely on quick evaluations of protein activity to rank potential chemical leads. By monitoring biologically relevant protein-ligand interactions, NMR can provide a means to validate these discovery leads and to optimize the drug discovery process. NMR-based screens typically use a change in chemical shift or linewidth to detect a protein-ligand interaction. However, the relatively low throughput of current NMR screens and their high demand on sample requirements generally makes it impractical to collect complete binding curves to measure the affinity for each compound in a large and diverse chemical library. As a result, NMR ligand screens are typically limited to identifying candidates that bind to a protein and do not give any estimate of the binding affinity. To address this issue, a methodology has been developed to rank binding affinities for ligands based on NMR-based screens that use 1D 1H NMR line-broadening experiments. This method was demonstrated by using it to estimate the dissociation equilibrium constants for twelve ligands with the protein human serum albumin (HSA). The results were found to give good agreement with previous affinities that have been reported for these same ligands with HSA. PMID:18831571

  8. The good, the bad and the dubious: VHELIBS, a validation helper for ligands and binding sites

    PubMed Central

    2013-01-01

    Background Many Protein Data Bank (PDB) users assume that the deposited structural models are of high quality but forget that these models are derived from the interpretation of experimental data. The accuracy of atom coordinates is not homogeneous between models or throughout the same model. To avoid basing a research project on a flawed model, we present a tool for assessing the quality of ligands and binding sites in crystallographic models from the PDB. Results The Validation HElper for LIgands and Binding Sites (VHELIBS) is software that aims to ease the validation of binding site and ligand coordinates for non-crystallographers (i.e., users with little or no crystallography knowledge). Using a convenient graphical user interface, it allows one to check how ligand and binding site coordinates fit to the electron density map. VHELIBS can use models from either the PDB or the PDB_REDO databank of re-refined and re-built crystallographic models. The user can specify threshold values for a series of properties related to the fit of coordinates to electron density (Real Space R, Real Space Correlation Coefficient and average occupancy are used by default). VHELIBS will automatically classify residues and ligands as Good, Dubious or Bad based on the specified limits. The user is also able to visually check the quality of the fit of residues and ligands to the electron density map and reclassify them if needed. Conclusions VHELIBS allows inexperienced users to examine the binding site and the ligand coordinates in relation to the experimental data. This is an important step to evaluate models for their fitness for drug discovery purposes such as structure-based pharmacophore development and protein-ligand docking experiments. PMID:23895374

  9. NMR studies of DNA oligomers and their interactions with minor groove binding ligands

    SciTech Connect

    Fagan, P A

    1996-05-01

    The cationic peptide ligands distamycin and netropsin bind noncovalently to the minor groove of DNA. The binding site, orientation, stoichiometry, and qualitative affinity of distamycin binding to several short DNA oligomers were investigated by NMR spectroscopy. The oligomers studied contain A,T-rich or I,C-rich binding sites, where I = 2-desaminodeoxyguanosine. I{center_dot}C base pairs are functional analogs of A{center_dot}T base pairs in the minor groove. The different behaviors exhibited by distamycin and netropsin binding to various DNA sequences suggested that these ligands are sensitive probes of DNA structure. For sites of five or more base pairs, distamycin can form 1:1 or 2:1 ligand:DNA complexes. Cooperativity in distamycin binding is low in sites such as AAAAA which has narrow minor grooves, and is higher in sites with wider minor grooves such as ATATAT. The distamycin binding and base pair opening lifetimes of I,C-containing DNA oligomers suggest that the I,C minor groove is structurally different from the A,T minor groove. Molecules which direct chemistry to a specific DNA sequence could be used as antiviral compounds, diagnostic probes, or molecular biology tools. The author studied two ligands in which reactive groups were tethered to a distamycin to increase the sequence specificity of the reactive agent.

  10. Radioligand binding assays for high affinity binders in the presence of endogenous ligands

    SciTech Connect

    White, H.B. III; McGahan, T.

    1986-05-01

    Endogenous ligands complicate radioligand-binding assays of high-affinity binding proteins by obscuring binding sites or by diluting the labeled ligand. They have developed a mathematical model for such systems where structurally identical radioligand and endogenous ligand can be equilibrated on the binding site and bound radioligand measured. A double-reciprocal plot of bound radioligand, *L/sub B/, versus sample volume, V, yields a straight line. Introduction of scaling factors for sample dilution, F, and total radioligand available, *L/sub T/, produces a plot in which the x-intercept yields the endogenous ligand concentration, (L/sub T/); the slope is the reciprocal of the binding protein concentration, (P/sub T/)/sup -1/; and the y-intercept is the fractional saturation of the high-affinity binder, L/sub T//P/sub T/. This type of analysis has been applied to the assay of high-affinity biotin-binding proteins in egg yolk. Its use led to the detection of a second biotin-binding protein which is heat labile. The conceptual approach can be applied to the assay of other high-affinity binders.

  11. In silico Identification and Characterization of Protein-Ligand Binding Sites.

    PubMed

    Roche, Daniel Barry; McGuffin, Liam James

    2016-01-01

    Protein-ligand binding site prediction methods aim to predict, from amino acid sequence, protein-ligand interactions, putative ligands, and ligand binding site residues using either sequence information, structural information, or a combination of both. In silico characterization of protein-ligand interactions has become extremely important to help determine a protein's functionality, as in vivo-based functional elucidation is unable to keep pace with the current growth of sequence databases. Additionally, in vitro biochemical functional elucidation is time-consuming, costly, and may not be feasible for large-scale analysis, such as drug discovery. Thus, in silico prediction of protein-ligand interactions must be utilized to aid in functional elucidation. Here, we briefly discuss protein function prediction, prediction of protein-ligand interactions, the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated EvaluatiOn (CAMEO) competitions, along with their role in shaping the field. We also discuss, in detail, our cutting-edge web-server method, FunFOLD for the structurally informed prediction of protein-ligand interactions. Furthermore, we provide a step-by-step guide on using the FunFOLD web server and FunFOLD3 downloadable application, along with some real world examples, where the FunFOLD methods have been used to aid functional elucidation. PMID:27094282

  12. Study of Functional and Allosteric Sites in Protein Superfamilies

    PubMed Central

    Suplatov, D.; Švedas, V.

    2015-01-01

    The interaction of proteins (enzymes) with a variety of low-molecular-weight compounds, as well as protein-protein interactions, is the most important factor in the regulation of their functional properties. To date, research effort has routinely focused on studying ligand binding to the functional sites of proteins (active sites of enzymes), whereas the molecular mechanisms of allosteric regulation, as well as binding to other pockets and cavities in protein structures, remained poorly understood. Recent studies have shown that allostery may be an intrinsic property of virtually all proteins. Novel approaches are needed to systematically analyze the architecture and role of various binding sites and establish the relationship between structure, function, and regulation. Computational biology, bioinformatics, and molecular modeling can be used to search for new regulatory centers, characterize their structural peculiarities, as well as compare different pockets in homologous proteins, study the molecular mechanisms of allostery, and understand the communication between topologically independent binding sites in protein structures. The establishment of an evolutionary relationship between different binding centers within protein superfamilies and the discovery of new functional and allosteric (regulatory) sites using computational approaches can improve our understanding of the structure-function relationship in proteins and provide new opportunities for drug design and enzyme engineering. PMID:26798490

  13. Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1

    PubMed Central

    Fay, Jonathan F.; Farrens, David L.

    2015-01-01

    G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1—it simultaneously increases agonist binding, decreases G-protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling. PMID:26100912

  14. Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes.

    PubMed

    Bončina, Matjaž; Podlipnik, Črtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij

    2015-12-01

    Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)3 using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; KTel22 ≈ ligands (Phen-DC3, 360A-Br; KTel22 > KdsDNA). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation. PMID:26546516

  15. Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes

    PubMed Central

    Bončina, Matjaž; Podlipnik, Črtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij

    2015-01-01

    Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)3 using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; KTel22 ≈ ligands (Phen-DC3, 360A-Br; KTel22 > KdsDNA). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation. PMID:26546516

  16. Binding of the sphingolipid S1P to hTERT stabilizes telomerase at the nuclear periphery by allosterically mimicking protein phosphorylation†

    PubMed Central

    Selvam, Shanmugam P.; De Palma, Ryan M.; Oaks, Joshua J.; Oleinik, Natalia; Peterson, Yuri K.; Stahelin, Robert V.; Skordalakes, Emmanuel; Ponnusamy, Suriyan; Garrett-Mayer, Elizabeth; Smith, Charles D.; Ogretmen, Besim

    2015-01-01

    During DNA replication, the enzyme telomerase maintains the ends of chromosomes, called telomeres. Shortened telomeres trigger cell senescence, and cancer cells often have increased telomerase activity to promote their ability to proliferate indefinitely. The catalytic subunit, human telomerase reverse transcriptase (hTERT), is stabilized by phosphorylation. Here, we found that the lysophospholipid sphingosine 1-phosphate (S1P), generated by sphingosine kinase 2 (SK2), bound hTERT at the nuclear periphery in human and mouse fibroblasts. Docking predictions and mutational analyses revealed that binding occurred between a hydroxyl group (C′3-OH) in S1P and Asp684 in hTERT. Inhibiting or depleting SK2 or mutating the S1P binding site decreased the stability of hTERT in cultured cells and promoted senescence and loss of telomere integrity. S1P binding inhibited the interaction of hTERT with MKRN1, an E3 ubiquitin ligase that tags hTERT for degradation. Murine Lewis lung carcinoma (LLC) cells formed smaller tumors in mice lacking SK2 than in wild-type mice, and knocking down SK2 in LLC cells before implantation into mice suppressed their growth. Pharmacologically inhibiting SK2 decreased the growth of subcutaneous A549 lung cancer cell-derived xenografts in mice, and expression of wild-type hTERT, but not an S1P-binding mutant, restored tumor growth. Thus, our data suggest that S1P binding to hTERT allosterically mimicks phosphorylation, promoting telomerase stability and hence telomere maintenance, cell proliferation, and tumor growth PMID:26082434

  17. Analogs of WIN 62,577 define a second allosteric site on muscarinic receptors.

    PubMed

    Lazareno, S; Popham, A; Birdsall, N J M

    2002-12-01

    WIN 51,708 (17-beta-hydroxy-17-alpha-ethynyl-5-alpha-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) and WIN 62,577 (17-beta-hydroxy- 17-alpha-ethynyl-delta(4)-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) are potent and centrally active antagonists at rat, but not human, NK(1) receptors. The interactions of these compounds and some analogs with [(3)H]N-methyl scopolamine ([(3)H]NMS) and unlabeled acetylcholine (ACh) at M(1)-M(4) muscarinic receptors have been studied using equilibrium and nonequilibrium radioligand binding methods. The results are consistent with the predictions of the allosteric ternary complex model. The WIN compounds have log affinities for the unliganded receptor in the range 5 to 6.7, and exhibit positive, negative, or neutral cooperativity with [(3)H]NMS and ACh, depending on the receptor subtype and nature of the interacting ligands. WIN 62,577 is an allosteric enhancer of ACh affinity at M(3) receptors. Although interacting allosterically, WIN 62,577 and WIN 51,708 do not affect [(3)H]NMS dissociation from M(3) receptors. Certain analogs have higher affinities than WIN 62,577, and truncated forms of WIN 62,577, including steroids, also act allosterically. One analog, 17-beta-hydroxy-17-alpha-Delta(4)-androstano[3,2-b]pyrido[2,3-b]indole (PG987), has the unique effect of speeding [(3)H]NMS dissociation; its largest effect, 2.5-fold, is at M(3) receptors. The interaction between PG987 and other allosteric agents on [(3)H]NMS dissociation from M(3) receptors indicate that PG987 binds reversibly to a site distinct from that to which gallamine and strychnine bind: in contrast, PG987 seems to bind to the same site on M(3) receptors as KT5720, staurosporine, and WIN 51,708. Therefore, in addition to the allosteric site that binds strychnine (and probably chloromethyl brucine, another allosteric enhancer) there is a second, nonoverlapping, pharmacologically distinct allosteric site on M(3) receptors that also supports positive cooperativity with

  18. Mass spectrometry-based ligand binding assays on adenosine A1 and A2A receptors.

    PubMed

    Massink, A; Holzheimer, M; Hölscher, A; Louvel, J; Guo, D; Spijksma, G; Hankemeier, T; IJzerman, A P

    2015-12-01

    Conventional methods to measure ligand-receptor binding parameters typically require radiolabeled ligands as probes. Despite the robustness of radioligand binding assays, they carry inherent disadvantages in terms of safety precautions, expensive synthesis, special lab requirements, and waste disposal. Mass spectrometry (MS) is a method that can selectively detect ligands without the need of a label. The sensitivity of MS equipment increases progressively, and currently, it is possible to detect low ligand quantities that are usually found in ligand binding assays. We developed a label-free MS ligand binding (MS binding) assay on the adenosine A(1) and A(2A) receptors (A(1)AR and A(2A)AR), which are well-characterized members of the class A G protein-coupled receptor (GPCR) family. Radioligand binding assays for both receptors are well established, and ample data is available to compare and evaluate the performance of an MS binding assay. 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX) and 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol (ZM-241,385) are high-affinity ligands selective for the A(1)AR and A(2A)AR, respectively. To proof the feasibility of MS binding on the A(1)AR and A(2A)AR, we first developed an MS detection method for unlabeled DPCPX and ZM-241,385. To serve as internal standards, both compounds were also deuterium-labeled. Subsequently, we investigated whether the two unlabeled compounds could substitute for their radiolabeled counterparts as marker ligands in binding experiments, including saturation, displacement, dissociation, and competition association assays. Furthermore, we investigated the accuracy of these assays if the use of internal standards was excluded. The results demonstrate the feasibility of the MS binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other GPCRs. PMID

  19. Discovery of Potential Orthosteric and Allosteric Antagonists of P2Y1R from Chinese Herbs by Molecular Simulation Methods

    PubMed Central

    Lu, Fang; Jiang, Lu-di; Qiao, Lian-sheng; Xiang, Yu-hong

    2016-01-01

    P2Y1 receptor (P2Y1R), which belongs to G protein-coupled receptors (GPCRs), is an important target in ADP-induced platelet aggregation. The crystal structure of P2Y1R has been solved recently, which revealed orthosteric and allosteric ligand-binding sites with the details of ligand-protein binding modes. And it suggests that P2Y1R antagonists, which recognize two distinct sites, could potentially provide an efficacious and safe antithrombotic profile. In present paper, 2D similarity search, pharmacophore based screening, and molecular docking were used to explore the potential natural P2Y1R antagonists. 2D similarity search was used to classify orthosteric and allosteric antagonists of P2Y1R. Based on the result, pharmacophore models were constructed and validated by the test set. Optimal models were selected to discover potential P2Y1R antagonists of orthosteric and allosteric sites from Traditional Chinese Medicine (TCM). And the hits were filtered by Lipinski's rule. Then molecular docking was used to refine the results of pharmacophore based screening and analyze the binding mode of the hits and P2Y1R. Finally, two orthosteric and one allosteric potential compounds were obtained, which might be used in future P2Y1R antagonists design. This work provides a reliable guide for discovering natural P2Y1R antagonists acting on two distinct sites from TCM.

  20. Quantitative Determination of DNA-Ligand Binding Using Fluorescence Spectroscopy

    ERIC Educational Resources Information Center

    Healy, Eamonn F.

    2007-01-01

    The effective use of fluorescence spectroscopy for determining the binding of the intercalcating agent crhidium bromide to DNA is being described. The analysis used simple measurement techniques and hence can be easily adopted by the students for a better understanding.

  1. Metal-coordination-driven mixed ligand binding in supramolecular bisporphyrin tweezers.

    PubMed

    Ikbal, Sk Asif; Dhamija, Avinash; Rath, Sankar Prasad

    2015-09-25

    Mg(II)bisporphyrin has been used as an efficient host for the selective binding of guest ligands. In the presence of heterogeneous guest pairs, 2-aminopyrimidine/pyrazine and 2-aminopyrimidine/1,4-dioxane, 2-aminopyrimidine is bound selectively inside the bisporphyrin cavity whereas pyrazine/1,4-dioxane is bound outside to produce 1D mixed ligand polymers. UV-vis, (1)H NMR spectra and X-ray structure confirm such a selective and orthogonal binding of the guest ligands. The mixed ligand polymer has been synthesized just by mixing the host and guests in one pot and easily isolated as a solid in nearly quantitative yield due to its high stability. PMID:26256242

  2. Carboxylic-Acid-passivated metal oxide nanocrystals: ligand exchange characteristics of a new binding motif.

    PubMed

    De Roo, Jonathan; Justo, Yolanda; De Keukeleere, Katrien; Van den Broeck, Freya; Martins, José C; Van Driessche, Isabel; Hens, Zeger

    2015-05-26

    Ligand exchange is central in the processing of inorganic nanocrystals (NCs) and requires understanding of surface chemistry. Studying sterically stabilized HfO2 and ZrO2 NCs using (1) H solution NMR and IR spectroscopy as well as elemental analysis, this paper demonstrates the reversible exchange of initial oleic acid ligands for octylamine and self-adsorption of oleic acid at NC surfaces. Both processes are incompatible with an X-type binding motif of carboxylic acids as reported for sulfide and selenide NCs. We argue that this behavior stems from the dissociative adsorption of carboxylic acids at the oxide surface. Both proton and carboxylate moieties must be regarded as X-type ligands yielding a combined X2 binding motif that allows for self-adsorption and exchange for L-type ligands. PMID:25866095

  3. Binding of stereognostically designed ligands to trivalent, pentavalent, and hexavalent f-block elements

    SciTech Connect

    Sinkov, Sergey I.; Lumetta, Gregg J.; Warner, Marvin G.; Pittman, Jonathan W.

    2012-03-26

    Stability constants were determined for the complexes formed from two stereognostically designed ligands and the f-block elements Nd(III), Np(V), and Pu(VI). The ligands investigated were tris[3-(2-carboxyphenoxy)propyl]amine (NPB) and tris-N,N',N''-[2-(2-carboxy-4-ethyl-phenoxy)ethyl]-1,4,7-triazacyclononane (EETAC). A stereognostically blind ligand, nitrilotriacetic acid (NTA), was also investigated for comparison. The results suggest that there is no significant stereognostic effect for complexation of NPB or EETAC to Np(V). On the other hand, a modest stereognostic effect is seen for the NPB ligand when complexed to Pu(VI), leading to an approximately 8-fold increase in the binding strength. A more significant effect is observed for the EETAC system in which a 250-fold increase in binding is observed for Pu(VI) versus Nd(III).

  4. The nicotinic acetylcholine receptor and its prokaryotic homologues: Structure, conformational transitions & allosteric modulation.

    PubMed

    Cecchini, Marco; Changeux, Jean-Pierre

    2015-09-01

    Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger - a neurotransmitter - into an ion flux through the postsynaptic membrane. Here, we present an overview of the most recent advances on the signal transduction mechanism boosted by X-ray crystallography of both prokaryotic and eukaryotic homologues of the nicotinic acetylcholine receptor (nAChR) in conjunction with time-resolved analyses based on single-channel electrophysiology and Molecular Dynamics simulations. The available data consistently point to a global mechanism of gating that involves a large reorganization of the receptor mediated by two distinct quaternary transitions: a global twisting and a radial expansion/contraction of the extracellular domain. These transitions profoundly modify the organization of the interface between subunits, which host several sites for orthosteric and allosteric modulatory ligands. The same mechanism may thus mediate both positive and negative allosteric modulations of pLGICs ligand binding at topographically distinct sites. The emerging picture of signal transduction is expected to pave the way to new pharmacological strategies for the development of allosteric modulators of nAChR and pLGICs in general. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. PMID:25529272

  5. Identification of Ligand Binding Sites of Proteins Using the Gaussian Network Model

    PubMed Central

    Tuzmen, Ceren; Erman, Burak

    2011-01-01

    The nonlocal nature of the protein-ligand binding problem is investigated via the Gaussian Network Model with which the residues lying along interaction pathways in a protein and the residues at the binding site are predicted. The predictions of the binding site residues are verified by using several benchmark systems where the topology of the unbound protein and the bound protein-ligand complex are known. Predictions are made on the unbound protein. Agreement of results with the bound complexes indicates that the information for binding resides in the unbound protein. Cliques that consist of three or more residues that are far apart along the primary structure but are in contact in the folded structure are shown to be important determinants of the binding problem. Comparison with known structures shows that the predictive capability of the method is significant. PMID:21283550

  6. "DAKLI": a multipurpose ligand with high affinity and selectivity for dynorphin (kappa opioid) binding sites.

    PubMed Central

    Goldstein, A; Nestor, J J; Naidu, A; Newman, S R

    1988-01-01

    We describe a synthetic ligand, "DAKLI" (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as 125I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin (kappa opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites. PMID:2902630

  7. Classification of Beta-Lactamases and Penicillin Binding Proteins Using Ligand-Centric Network Models

    PubMed Central

    Öztürk, Hakime; Ozkirimli, Elif; Özgür, Arzucan

    2015-01-01

    β-lactamase mediated antibiotic resistance is an important health issue and the discovery of new β-lactam type antibiotics or β-lactamase inhibitors is an area of intense research. Today, there are about a thousand β-lactamases due to the evolutionary pressure exerted by these ligands. While β-lactamases hydrolyse the β-lactam ring of antibiotics, rendering them ineffective, Penicillin-Binding Proteins (PBPs), which share high structural similarity with β-lactamases, also confer antibiotic resistance to their host organism by acquiring mutations that allow them to continue their participation in cell wall biosynthesis. In this paper, we propose a novel approach to include ligand sharing information for classifying and clustering β-lactamases and PBPs in an effort to elucidate the ligand induced evolution of these β-lactam binding proteins. We first present a detailed summary of the β-lactamase and PBP families in the Protein Data Bank, as well as the compounds they bind to. Then, we build two different types of networks in which the proteins are represented as nodes, and two proteins are connected by an edge with a weight that depends on the number of shared identical or similar ligands. These models are analyzed under three different edge weight settings, namely unweighted, weighted, and normalized weighted. A detailed comparison of these six networks showed that the use of ligand sharing information to cluster proteins resulted in modules comprising proteins with not only sequence similarity but also functional similarity. Consideration of ligand similarity highlighted some interactions that were not detected in the identical ligand network. Analysing the β-lactamases and PBPs using ligand-centric network models enabled the identification of novel relationships, suggesting that these models can be used to examine other protein families to obtain information on their ligand induced evolutionary paths. PMID:25689853

  8. Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

    PubMed

    Sato, Shogo; Jung, Hunmin; Nakagawa, Tsutomu; Pawlosky, Robert; Takeshima, Tomomi; Lee, Wan-Ru; Sakiyama, Haruhiko; Laxman, Sunil; Wynn, R Max; Tu, Benjamin P; MacMillan, John B; De Brabander, Jef K; Veech, Richard L; Uyeda, Kosaku

    2016-05-13

    The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis. PMID:26984404

  9. Binding affinity prediction for protein-ligand complexes based on β contacts and B factor.

    PubMed

    Liu, Qian; Kwoh, Chee Keong; Li, Jinyan

    2013-11-25

    Accurate determination of protein-ligand binding affinity is a fundamental problem in biochemistry useful for many applications including drug design and protein-ligand docking. A number of scoring functions have been proposed for the prediction of protein-ligand binding affinity. However, accurate prediction is still a challenging problem because poor performance is often seen in the evaluation under the leave-one-cluster-out cross-validation (LCOCV). We introduce a new scoring function named B2BScore to improve the prediction performance. B2BScore integrates two physicochemical properties for protein-ligand binding affinity prediction. One is the property of β contacts. A β contact between two atoms requires no other atoms to interrupt the atomic contact and assumes that the two atoms should have enough direct contact area. The other is the property of B factor to capture the atomic mobility in the dynamic protein-ligand binding process. Tested on the PDBBind2009 data set, B2BScore shows superior prediction performance to existing methods on independent test data as well as under the LCOCV evaluation framework. In particular, B2BScore achieves a significant LCOCV improvement across 26 protein clusters-a big increase of the averaged Pearson's correlation coefficients from 0.418 to 0.518 and a significant decrease of standard deviation of the coefficients from 0.352 to 0.196. We also identified several important and intuitive contact descriptors of protein-ligand binding through the random forest learning in B2BScore. Some of these descriptors are closely related to contacts between carbon atoms without covalent-bond oxygen/nitrogen, preferred contacts of metal ions, interfacial backbone atoms from proteins, or π rings. Some others are negative descriptors relating to those contacts with nitrogen atoms without covalent-bond hydrogens or nonpreferred contacts of metal ions. These descriptors can be directly used to guide protein-ligand docking. PMID:24191692

  10. Development of a quantitative fluorescence-based ligand-binding assay.

    PubMed

    Breen, Conor J; Raverdeau, Mathilde; Voorheis, H Paul

    2016-01-01

    A major goal of biology is to develop a quantitative ligand-binding assay that does not involve the use of radioactivity. Existing fluorescence-based assays have a serious drawback due to fluorescence quenching that accompanies the binding of fluorescently-labeled ligands to their receptors. This limitation of existing fluorescence-based assays prevents the number of cellular receptors under investigation from being accurately measured. We have developed a method where FITC-labeled proteins bound to a cell surface are proteolyzed extensively to eliminate fluorescence quenching and then the fluorescence of the resulting sample is compared to that of a known concentration of the proteolyzed FITC-protein employed. This step enables the number of cellular receptors to be measured quantitatively. We expect that this method will provide researchers with a viable alternative to the use of radioactivity in ligand binding assays. PMID:27161290

  11. A model for the study of ligand binding to the ribosomal RNA helix h44

    SciTech Connect

    Dibrov, Sergey M.; Parsons, Jerod; Hermann, Thomas

    2010-09-02

    Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand-RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structural model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region.

  12. Development of a quantitative fluorescence-based ligand-binding assay

    PubMed Central

    Breen, Conor J.; Raverdeau, Mathilde; Voorheis, H. Paul

    2016-01-01

    A major goal of biology is to develop a quantitative ligand-binding assay that does not involve the use of radioactivity. Existing fluorescence-based assays have a serious drawback due to fluorescence quenching that accompanies the binding of fluorescently-labeled ligands to their receptors. This limitation of existing fluorescence-based assays prevents the number of cellular receptors under investigation from being accurately measured. We have developed a method where FITC-labeled proteins bound to a cell surface are proteolyzed extensively to eliminate fluorescence quenching and then the fluorescence of the resulting sample is compared to that of a known concentration of the proteolyzed FITC-protein employed. This step enables the number of cellular receptors to be measured quantitatively. We expect that this method will provide researchers with a viable alternative to the use of radioactivity in ligand binding assays. PMID:27161290

  13. Computational exploration of a protein receptor binding space with student proposed peptide ligands.

    PubMed

    King, Matthew D; Phillips, Paul; Turner, Matthew W; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M

    2016-01-01

    Computational molecular docking is a fast and effective in silico method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The DockoMatic tutorial described herein provides a framework by which instructors can guide students through a drug screening exercise. Using receptor models derived from readily available protein crystal structures, docking programs have the ability to predict ligand binding properties, such as preferential binding orientations and binding affinities. The use of computational studies can significantly enhance complimentary wet chemical experimentation by providing insight into the important molecular interactions within the system of interest, as well as guide the design of new candidate ligands based on observed binding motifs and energetics. In this laboratory tutorial, the graphical user interface, DockoMatic, facilitates docking job submissions to the docking engine, AutoDock 4.2. The purpose of this exercise is to successfully dock a 17-amino acid peptide, α-conotoxin TxIA, to the acetylcholine binding protein from Aplysia californica-AChBP to determine the most stable binding configuration. Each student will then propose two specific amino acid substitutions of α-conotoxin TxIA to enhance peptide binding affinity, create the mutant in DockoMatic, and perform docking calculations to compare their results with the class. Students will also compare intermolecular forces, binding energy, and geometric orientation of their prepared analog to their initial α-conotoxin TxIA docking results. PMID:26537635

  14. Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove.

    PubMed

    McMurtrey, Curtis; Trolle, Thomas; Sansom, Tiffany; Remesh, Soumya G; Kaever, Thomas; Bardet, Wilfried; Jackson, Kenneth; McLeod, Rima; Sette, Alessandro; Nielsen, Morten; Zajonc, Dirk M; Blader, Ira J; Peters, Bjoern; Hildebrand, William

    2016-01-01

    HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondii infected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondii encoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1-30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F' pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions. PMID:26824387

  15. Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove

    PubMed Central

    McMurtrey, Curtis; Trolle, Thomas; Sansom, Tiffany; Remesh, Soumya G; Kaever, Thomas; Bardet, Wilfried; Jackson, Kenneth; McLeod, Rima; Sette, Alessandro; Nielsen, Morten; Zajonc, Dirk M; Blader, Ira J; Peters, Bjoern; Hildebrand, William

    2016-01-01

    HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondii infected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondii encoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1–30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F’ pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions. DOI: http://dx.doi.org/10.7554/eLife.12556.001 PMID:26824387

  16. Ligands Binding and Molecular Simulation: the Potential Investigation of a Biosensor Based on an Insect Odorant Binding Protein

    PubMed Central

    Yi, Xin; Zhang, Yanbo; Wang, Peidan; Qi, Jiangwei; Hu, Meiying; Zhong, Guohua

    2015-01-01

    Based on mimicking biological olfaction, biosensors have been applied for the detection of various ligands in complex environment, which could represent one of the most promising research fields. In this study, the basic characters of one insect odorant binding protein (OBP) as a biosensor were explored. To explore the molecular recognition process, the tertiary structure of the protein was modeled and the protein-ligand interactions with 1,536,550 chemicals were investigated by the molecular docking. The availability of large amount of recombinant SlitOBP1 overcame the difficulty to obtain biological sensing material. After obtained the purified recombinant protein, the result of fluorescence binding assays proved the candidate protein has good affinities with the majority of the tested chemicals. With the aid of simulation docking, the key conserved amino acids within the binding site were identified and then mutated to alanine. After mutation, the protein-ligand binding characteristics were recorded, and the competitive binding assays were carried out to provide experimental verification. The detailed information on its structure and affinities investigated in this study could allow the design of specific mutants with desired characteristics, which provides a solid base for tailoring OBP for biosensor and provides a role model for screening the other elements in olfactory system for different applications. PMID:25552932

  17. Ligands binding and molecular simulation: the potential investigation of a biosensor based on an insect odorant binding protein.

    PubMed

    Yi, Xin; Zhang, Yanbo; Wang, Peidan; Qi, Jiangwei; Hu, Meiying; Zhong, Guohua

    2015-01-01

    Based on mimicking biological olfaction, biosensors have been applied for the detection of various ligands in complex environment, which could represent one of the most promising research fields. In this study, the basic characters of one insect odorant binding protein (OBP) as a biosensor were explored. To explore the molecular recognition process, the tertiary structure of the protein was modeled and the protein-ligand interactions with 1,536,550 chemicals were investigated by the molecular docking. The availability of large amount of recombinant SlitOBP1 overcame the difficulty to obtain biological sensing material. After obtained the purified recombinant protein, the result of fluorescence binding assays proved the candidate protein has good affinities with the majority of the tested chemicals. With the aid of simulation docking, the key conserved amino acids within the binding site were identified and then mutated to alanine. After mutation, the protein-ligand binding characteristics were recorded, and the competitive binding assays were carried out to provide experimental verification. The detailed information on its structure and affinities investigated in this study could allow the design of specific mutants with desired characteristics, which provides a solid base for tailoring OBP for biosensor and provides a role model for screening the other elements in olfactory system for different applications. PMID:25552932

  18. Binding affinity prediction of novel estrogen receptor ligands using receptor-based 3-D QSAR methods.

    PubMed

    Sippl, Wolfgang

    2002-12-01

    We have recently reported the development of a 3-D QSAR model for estrogen receptor ligands showing a significant correlation between calculated molecular interaction fields and experimentally measured binding affinity. The ligand alignment obtained from docking simulations was taken as basis for a comparative field analysis applying the GRID/GOLPE program. Using the interaction field derived with a water probe and applying the smart region definition (SRD) variable selection procedure, a significant and robust model was obtained (q(2)(LOO)=0.921, SDEP=0.345). To further analyze the robustness and the predictivity of the established model several recently developed estrogen receptor ligands were selected as external test set. An excellent agreement between predicted and experimental binding data was obtained indicated by an external SDEP of 0.531. Two other traditionally used prediction techniques were applied in order to check the performance of the receptor-based 3-D QSAR procedure. The interaction energies calculated on the basis of receptor-ligand complexes were correlated with experimentally observed affinities. Also ligand-based 3-D QSAR models were generated using program FlexS. The interaction energy-based model, as well as the ligand-based 3-D QSAR models yielded models with lower predictivity. The comparison with the interaction energy-based model and with the ligand-based 3-D QSAR models, respectively, indicates that the combination of receptor-based and 3-D QSAR methods is able to improve the quality of prediction. PMID:12413831

  19. The N-terminal peptide of mammalian GTP cyclohydrolase I is an autoinhibitory control element and contributes to binding the allosteric regulatory protein GFRP.

    PubMed

    Higgins, Christina E; Gross, Steven S

    2011-04-01

    GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may also contribute to autoregulation of GTPCH activity, but mechanisms are undefined. To characterize potential regulatory actions of the N-terminal peptide in rat GTPCH, we expressed, purified, and characterized a truncation mutant, devoid of 45 N-terminal amino acids (Δ45-GTPCH) and contrasted its catalytic and GFRP binding properties to wild type GTPCH (wt-GTPCH). Contrary to prior reports, we show that GFRP binds wt-GTPCH in the absence of any small molecule effector, resulting in allosteric stimulation of GTPCH activity: a 20% increase in Vmax, 50% decrease in KmGTP, and increase in Hill coefficient to 1.6, from 1.0. These features of GFRP-stimulated wt-GTPCH activity were phenocopied by Δ45-GTPCH in the absence of bound GFRP. Addition of GFRP to Δ45-GTPCH failed to elicit complex formation or a substantial further increase in GTPCH catalytic activity. Expression of Δ45-GTPCH in HEK-293 cells elicited 3-fold greater BH4 accumulation than an equivalent of wt-GTPCH. Together, results indicate that the N-terminal peptide exerts autoinhibitory control over rat GTPCH and is required for GFRP binding on its own. Displacement of the autoinhibitory peptide provides a molecular mechanism for physiological up-regulation of GTPCH activity. PMID:21163945

  20. The N-terminal Peptide of Mammalian GTP Cyclohydrolase I Is an Autoinhibitory Control Element and Contributes to Binding the Allosteric Regulatory Protein GFRP*

    PubMed Central

    Higgins, Christina E.; Gross, Steven S.

    2011-01-01

    GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may also contribute to autoregulation of GTPCH activity, but mechanisms are undefined. To characterize potential regulatory actions of the N-terminal peptide in rat GTPCH, we expressed, purified, and characterized a truncation mutant, devoid of 45 N-terminal amino acids (Δ45-GTPCH) and contrasted its catalytic and GFRP binding properties to wild type GTPCH (wt-GTPCH). Contrary to prior reports, we show that GFRP binds wt-GTPCH in the absence of any small molecule effector, resulting in allosteric stimulation of GTPCH activity: a 20% increase in Vmax, 50% decrease in KmGTP, and increase in Hill coefficient to 1.6, from 1.0. These features of GFRP-stimulated wt-GTPCH activity were phenocopied by Δ45-GTPCH in the absence of bound GFRP. Addition of GFRP to Δ45-GTPCH failed to elicit complex formation or a substantial further increase in GTPCH catalytic activity. Expression of Δ45-GTPCH in HEK-293 cells elicited 3-fold greater BH4 accumulation than an equivalent of wt-GTPCH. Together, results indicate that the N-terminal peptide exerts autoinhibitory control over rat GTPCH and is required for GFRP binding on its own. Displacement of the autoinhibitory peptide provides a molecular mechanism for physiological up-regulation of GTPCH activity. PMID:21163945

  1. Identification of a novel family of carbohydrate-binding modules with broad ligand specificity

    PubMed Central

    Duan, Cheng-Jie; Feng, Yu-Liang; Cao, Qi-Long; Huang, Ming-Yue; Feng, Jia-Xun

    2016-01-01

    Most enzymes that act on carbohydrates include non-catalytic carbohydrate-binding modules (CBMs) that recognize and target carbohydrates. CBMs bring their appended catalytic modules into close proximity with the target substrate and increase the hydrolytic rate of enzymes acting on insoluble substrates. We previously identified a novel CBM (CBMC5614-1) at the C-terminus of endoglucanase C5614-1 from an uncultured microorganism present in buffalo rumen. In the present study, that the functional region of CBMC5614-1 involved in ligand binding was localized to 134 amino acids. Two representative homologs of CBMC5614-1, sharing the same ligand binding profile, targeted a range of β-linked polysaccharides that adopt very different conformations. Targeted substrates included soluble and insoluble cellulose, β-1,3/1,4-mixed linked glucans, xylan, and mannan. Mutagenesis revealed that three conserved aromatic residues (Trp-380, Tyr-411, and Trp-423) play an important role in ligand recognition and targeting. These results suggest that CBMC5614-1 and its homologs form a novel CBM family (CBM72) with a broad ligand-binding specificity. CBM72 members can provide new insight into CBM-ligand interactions and may have potential in protein engineering and biocatalysis. PMID:26765840

  2. GATING OF HCN CHANNELS BY CYCLIC NUCLEOTIDES: RESIDUE CONTACTS THAT UNDERLIE LIGAND BINDING, SELECTIVITY AND EFFICACY

    PubMed Central

    Zhou, Lei; Siegelbaum, Steven A.

    2007-01-01

    SUMMARY Cyclic nucleotides regulate the activity of various proteins by interacting with a conserved cyclic nucleotide-binding domain (CNBD). Although X-ray crystallographic studies have revealed the structures of several CNBDs, the residues responsible for generating the high efficacy with which ligand binding leads to protein activation remain unknown. Here we combine molecular dynamics simulations with mutagenesis to identify ligand contacts important for the regulation of the hyperpolarization-activated HCN2 channel by cyclic nucleotides. Surprisingly, out of seven residues that make strong contacts with ligand, only R632 in the C-helix of the CNBD is essential for high ligand efficacy, due to its selective stabilization of cNMP binding to the open state of the channel. Principle component analysis suggests that a local movement of the C-helix upon ligand binding propagates through the CNBD of one subunit to the C-linker of a neighboring subunit to apply force to the gate of the channel. PMID:17562313

  3. Ligand binding sites of Na,K-ATPase.

    PubMed

    Lingrel, J B; Croyle, M L; Woo, A L; Argüello, J M

    1998-08-01

    Our studies have concentrated on two aspects of the Na,K-ATPase, the first relates to the identification of amino acids involved in binding Na+ and K+ during the catalytic cycle and the second involves defining how cardiac glycosides inhibit the enzyme. To date, three amino acids, Ser775, Asp804 and Asp808, all located in transmembrane regions five and six, have been shown to play a major role in K+ binding. These findings are based on site directed mutagenesis and expression studies. In order to understand how cardiac glycosides interact with the Na,K-ATPase, studies again involving mutagenesis coupled with expression have been used. More specifically, amino acid residues have been substituted in an ouabain sensitive alpha subunit using random mutagenesis, and the ability of the resulting enzyme to confer resistance to ouabain sensitive cells was determined. Interestingly, the amino acids of the alpha subunit which alter ouabain sensitivity cluster in two major regions, one comprised of the first and second transmembrane spanning domains and the extracellular loop joining them, and the second formed by the extracellular halves of transmembrane regions four, five, six and seven. As noted above, transmembrane regions five and six also contain the three amino acid residues Ser775, Asp804 and Asp808 which play a key role in cation transport, possibly binding K+. Thus, it is reasonable to propose that cardiac glycosides bind to two sites, the N- terminal region and the central region which contains the cation binding sites. Cardiac glycoside binding to the center region may lock the cation transport region into a configuration such that the enzyme cannot go through the conformational change required for ion transport. PMID:9789548

  4. Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

    NASA Astrophysics Data System (ADS)

    Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju

    2014-06-01

    Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

  5. Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design.

    PubMed Central

    Liang, J.; Edelsbrunner, H.; Woodward, C.

    1998-01-01

    Identification and size characterization of surface pockets and occluded cavities are initial steps in protein structure-based ligand design. A new program, CAST, for automatically locating and measuring protein pockets and cavities, is based on precise computational geometry methods, including alpha shape and discrete flow theory. CAST identifies and measures pockets and pocket mouth openings, as well as cavities. The program specifies the atoms lining pockets, pocket openings, and buried cavities; the volume and area of pockets and cavities; and the area and circumference of mouth openings. CAST analysis of over 100 proteins has been carried out; proteins examined include a set of 51 monomeric enzyme-ligand structures, several elastase-inhibitor complexes, the FK506 binding protein, 30 HIV-1 protease-inhibitor complexes, and a number of small and large protein inhibitors. Medium-sized globular proteins typically have 10-20 pockets/cavities. Most often, binding sites are pockets with 1-2 mouth openings; much less frequently they are cavities. Ligand binding pockets vary widely in size, most within the range 10(2)-10(3)A3. Statistical analysis reveals that the number of pockets and cavities is correlated with protein size, but there is no correlation between the size of the protein and the size of binding sites. Most frequently, the largest pocket/cavity is the active site, but there are a number of instructive exceptions. Ligand volume and binding site volume are somewhat correlated when binding site volume is < or =700 A3, but the ligand seldom occupies the entire site. Auxiliary pockets near the active site have been suggested as additional binding surface for designed ligands (Mattos C et al., 1994, Nat Struct Biol 1:55-58). Analysis of elastase-inhibitor complexes suggests that CAST can identify ancillary pockets suitable for recruitment in ligand design strategies. Analysis of the FK506 binding protein, and of compounds developed in SAR by NMR (Shuker SB et

  6. Does the tissue concentration in receptor binding studies change the affinity of the labelled ligand?

    PubMed

    Ensing, K; De Zeeuw, R A

    1984-12-14

    When the tissue concentration in a radioreceptor assay for anticholinergic drugs was varied in order to obtain optimum conditions, and the receptor concentration Cr and the equilibrium dissociation constant KD were determined by Scatchard analysis, the KD increased with increasing tissue concentrations. This phenomenon was considered as an artefact caused by non-specific binding of the labelled ligand to constituents of the receptor preparation which were not completely retained on the glass-fibre filters used for the separation of bound and free fraction of radio-labelled ligand. The increase in KD in these experiments could be described with a mathematical model of the binding experiments. PMID:6514542

  7. Role of extracellular disulfide-bonded cysteines in the ligand binding function of the. beta. sub 2 -adrenergic receptor

    SciTech Connect

    Dohlman, H.G.; Caron, M.G.; DeBlasi, A.; Frielle, T.; Lefkowitz, R.J. )

    1990-03-06

    Evidence is presented for a role of disulfide bridging in forming the ligand binding site of the {beta}{sub 2}-adrenergic receptor ({beta}AR). The presence of disulfide bonds at the ligand binding site is indicated by competitive inhibition by dithiothreitol (DTT) in radioligand binding assays, by specific protection by {beta}-adrenergic ligands of these effects, and by the requirement of disulfide reduction for limit proteolysis of affinity ligand labeled receptor. The kinetics of binding inhibition by DTT suggest at least two pairs of disulfide-bonded cysteines essential for normal binding. Through site-directed mutagenesis, the authors indeed were able to identify four cysteines which are critical for normal binding affinities and for the proper expression of functional {beta}AR at the cell surface. Unexpectedly, the four cysteines required for normal ligand binding are not those located within the hydrophobic transmembrane domains of the receptor (where ligand binding is presumed to occur) but lie in the extracellular hydrophilic loops connecting these transmembrane segments. These findings indicate that in addition to the well-documented involvement of the membrane-spanning domains of the receptor in ligand binding, there is an important and previously unsuspected role of the hydrophilic extracellular domains in forming the ligand binding site.

  8. CGP7930: a positive allosteric modulator of the GABAB receptor.

    PubMed

    Adams, C L; Lawrence, A J

    2007-01-01

    CGP7930 (3-(3',5'-Di-tert-butyl-4'-hydroxy)phenyl-2,2-dimethylpropanol) is a positive allosteric modulator of the metabotropic GABAB receptor. CGP7930 has been found to modulate the GABAB receptor in the open, or high affinity, state increasing agonist affinity for the receptor and signal transduction efficacy following agonist stimulation. The GABAB heteromeric subunit B2, involved in signal transduction but not ligand binding, seems to be the site of action of CGP7930 and similar allosteric modulators. When administered alone in naïve animals, CGP7930 acts as an anxiolytic in rodents without other overt behavioral effects and has also been demonstrated to reduce self-administration of nicotine, cocaine, or alcohol in rodents, suggesting that "fine tuning" of the GABAB receptor by positive allosteric modulators may be able to regulate abuse of these drugs. Baclofen, the GABAB agonist, is currently finding use in treating addiction and various other disorders, but this can result in off-target effects and tolerance. CGP7930 when co-administered with baclofen enhances its potency, which could in theory minimize deleterious effects. Further study of CGP7930 is required, but this compound, and others like it, holds potential in a clinical setting. PMID:17894647

  9. Identification of common ligand binding determinants of the insulin and insulin-like growth factor 1 receptors. Insights into mechanisms of ligand binding.

    PubMed

    Mynarcik, D C; Williams, P F; Schaffer, L; Yu, G Q; Whittaker, J

    1997-07-25

    Insulin and insulin-like growth factor 1 (IGF-1) are peptides that share nearly 50% sequence homology. However, although their cognate receptors also exhibit significant overall sequence homology, the affinity of each peptide for the non-cognate receptor is 2-3 orders of magnitude lower than for the cognate receptor. The molecular basis for this discrimination is unclear, as are the molecular mechanisms underlying ligand binding. We have recently identified a major ligand binding site of the insulin receptor by alanine scannning mutagenesis. These studies revealed that a number of amino acids critical for insulin binding are conserved in the IGF-1 receptor, suggesting that they may play a role in ligand binding. We therefore performed alanine mutagenesis of these amino acids to determine whether this is the case. cDNAs encoding alanine-substituted secreted recombinant IGF-1 receptors were expressed in 293 EBNA cells, and the ligand binding properties of the expressed proteins were evaluated. Mutation of Phe701 resulted in a receptor with undetectable IGF-1 binding; alanine substitution of the corresponding amino acid of the insulin receptor, Phe714, produces a 140-fold reduction in affinity for insulin. Mutation of Asp8, Asn11, Phe58, Phe692, Glu693, His697, and Asn698 produces a 3.5-6-fold reduction in affinity for IGF-1. In contrast, alanine mutation of the corresponding amino acids of the insulin receptor with the exception of Asp12 produces reductions in affinity that are 50-fold or greater. The affinity of insulin for these mutants relative to wild type receptor was similar to that of their relative affinity for IGF-1 with two exceptions; the IC50 values for insulin binding to the mutants of Arg10, which has normal affinity for IGF-1, and His697, which has a 6-fold reduction in affinity for IGF-1, were both at least 2 orders of magnitude greater than for wild type receptor. The Kd values for insulin of the corresponding alanine mutants of the insulin receptor

  10. Allosteric activation of the Par-6 PDZ via a partial unfolding transition

    PubMed Central

    Whitney, Dustin S.; Peterson, Francis C.; Kovrigin, Evgenii L.; Volkman, Brian F.

    2013-01-01

    Proteins exist in a delicate balance between the native and unfolded states, where thermodynamic stability may be sacrificed to attain the flexibility required for efficient catalysis, binding or allosteric control. Par-6 regulates the Par polarity complex by transmitting a GTPase signal through the CRIB-PDZ module that alters PDZ lig-and binding. Allosteric activation of the PDZ is achieved by local rearrangement of the L164 and K165 side chains to stabilize the interdomain CRIB-PDZ interface and reposition a conserved element of the ligand binding pocket. However, microsecond to millisecond dynamics measurements revealed that L164/K165 exchange requires a larger rearrangement than expected. The margin of thermodynamic stability for the PDZ domain is modest (~3 kcal/mol) and further reduced by transient interactions with the disordered CRIB domain. Measurements of local structural stability revealed that tertiary contacts within the PDZ are disrupted by a partial unfolding transition that enables interconversion of the L/K switch. The unexpected participation of partial PDZ unfolding in the allosteric mechanism of Par-6 suggests that native-state unfolding may be essential for the function of other marginally stable proteins. PMID:23705660

  11. Point mutations of the alpha 1 beta 2 gamma 2 gamma-aminobutyric acid(A) receptor affecting modulation of the channel by ligands of the benzodiazepine binding site.

    PubMed

    Buhr, A; Baur, R; Malherbe, P; Sigel, E

    1996-06-01

    Clinically relevant benzodiazepines allosterically stimulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A(GABAA) receptor. Rat wild-type or mutated alpha 1, beta 2, and gamma 2S subunits were coexpressed in Xenopus oocytes and investigated with electrophysiological techniques. Point mutations in two subunits were identified that affect the response of gamma-aminobutyric acid (GABA)-induced currents by benzodiazepines. Mutation of one of three amino acid residues to alanine (alpha Tyr161 and alpha Thr206) or leucine (gamma Phe77) resulted in a approximately 3-fold increase in potentiation by diazepam. The response to zolpidem was increased in two mutant channels containing the mutated alpha subunit but was nearly absent in channels containing the mutated gamma subunit. In the former cases, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) acted as a negative allosteric modulator of the channel, much stronger than in the wild-type channel, whereas there was no significant difference to the wild-type channel in the latter case. Thus, the mutant gamma subunit has different functional consequences for the various types of ligand of the benzodiazepine binding site. All three amino acid residues, alpha Tyr161, alpha Thr206, and gamma Phe77, are close or identical to homologous residues that are implicated in GABA binding. If the residues binding the channel agonist GABA are located at subunit interfaces, the residues influencing the benzodiazepine effects must also be located at subunit interfaces. PMID:8649346

  12. The FTMap family of web servers for determining and characterizing ligand binding hot spots of proteins

    PubMed Central

    Kozakov, Dima; Grove, Laurie E.; Hall, David R.; Bohnuud, Tanggis; Mottarella, Scott; Luo, Lingqi; Xia, Bing; Beglov, Dmitri; Vajda, Sandor

    2016-01-01

    FTMap is a computational mapping server that identifies binding hot spots of macromolecules, i.e., regions of the surface with major contributions to the ligand binding free energy. To use FTMap, users submit a protein, DNA, or RNA structure in PDB format. FTMap samples billions of positions of small organic molecules used as probes and scores the probe poses using a detailed energy expression. Regions that bind clusters of multiple probe types identify the binding hot spots, in good agreement with experimental data. FTMap serves as basis for other servers, namely FTSite to predict ligand binding sites, FTFlex to account for side chain flexibility, FTMap/param to parameterize additional probes, and FTDyn to map ensembles of protein structures. Applications include determining druggability of proteins, identifying ligand moieties that are most important for binding, finding the most bound-like conformation in ensembles of unliganded protein structures, and providing input for fragment based drug design. FTMap is more accurate than classical mapping methods such as GRID and MCSS, and is much faster than the more recent approaches to protein mapping based on mixed molecular dynamics. Using 16 probe molecules, the FTMap server finds the hot spots of an average size protein in less than an hour. Since FTFlex performs mapping for all low energy conformers of side chains in the binding site, its completion time is proportionately longer. PMID:25855957

  13. Towards a high-affinity allosteric enhancer at muscarinic M1 receptors.

    PubMed

    Lazareno, Sebastian; Popham, Angela; Birdsall, Nigel J M

    2002-01-01

    Loss of forebrain acetylcholine (ACh) is an early neurochemical lesion in Alzheimer's Disease (AD), and muscarinic receptors for ACh are involved in memory and cognition, so a muscarinic agonist could provide 'replacement therapy' in this disease. Muscarinic receptors, which couple to G-proteins, occur throughout the CNS, and in the periphery they mediate the responses of the parasympathetic nervous system, so selectivity is crucial. The five subtypes of muscarinic receptor, M1-M5, have a distinct regional distribution, with M2 and M3 mediating most of the peripheral effects, M2 predominating in hindbrain areas, and M1 predominating in the cortex and hippocampus--the brain regions most associated with memory and cognition, which has lead to a search for a truly M1-selective muscarinic agonist. That search has so far been unsuccessful, but acetylcholinesterase inhibitors such as donepezil (Aricept), which potentiate cholinergic neurotransmission, have a therapeutic role in the management of AD; so the M1 receptor remains a therapeutic target. Our approach is to develop allosteric enhancers--compounds which bind to the receptor at an 'allosteric' site which is distinct from the 'primary' site to which the endogenous ligand binds, and which enhance the affinity (or efficacy) of the endogenous ligand. We have developed radioligand binding assays and analyses for the detection and quantitatitation of allosteric interactions of a test agent with labelled and unlabelled 'primary' ligands, and we report here some results of the initial phase of a chemical synthesis project to develop potent and selective allosteric enhancers at muscarinic M1 receptors. PMID:12212769

  14. Ligand binding to the PDZ domains of postsynaptic density protein 95.

    PubMed

    Toto, Angelo; Pedersen, Søren W; Karlsson, O Andreas; Moran, Griffin E; Andersson, Eva; Chi, Celestine N; Strømgaard, Kristian; Gianni, Stefano; Jemth, Per

    2016-05-01

    Cellular scaffolding and signalling is generally governed by multidomain proteins, where each domain has a particular function. Postsynaptic density protein 95 (PSD-95) is involved in synapse formation and is a typical example of such a multidomain protein. Protein-protein interactions of PSD-95 are well studied and include the following three protein ligands: (i)N-methyl-d-aspartate-type ionotropic glutamate receptor subunit GluN2B, (ii) neuronal nitric oxide synthase and (iii) cysteine-rich protein (CRIPT), all of which bind to one or more of the three PDZ domains in PSD-95. While interactions for individual PDZ domains of PSD-95 have been well studied, less is known about the influence of neighbouring domains on the function of the respective individual domain. We therefore performed a systematic study on the ligand-binding kinetics of PSD-95 using constructs of different size for PSD-95 and its ligands. Regarding the canonical peptide-binding pocket and relatively short peptides (up to 15-mer), the PDZ domains in PSD-95 by and large work as individual binding modules. However, in agreement with previous studies, residues outside of the canonical binding pocket modulate the affinity of the ligands. In particular, the dissociation of the 101 amino acid CRIPT from PSD-95 is slowed down at least 10-fold for full-length PSD-95 when compared with the individual PDZ3 domain. PMID:26941280

  15. Nonlinearly Additive Forces in Multivalent Ligand Binding to a Single Protein Revealed with Force Spectroscopy

    SciTech Connect

    Ratto, T V; Rudd, R E; Langry, K C; Balhorn, R L; McElfresh, M W

    2005-07-15

    We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between ConcanavalinA (ConA) and {alpha}-D-mannose, but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, they do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 66, and 85 pN, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and non-specific binding. We analyze the binding configuration (i.e. serial versus parallel connections) through fitting the polymer stretching data with modified Worm-Like Chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously.

  16. DNA sequence preferences of several AT-selective minor groove binding ligands.

    PubMed Central

    Abu-Daya, A; Brown, P M; Fox, K R

    1995-01-01

    We have examined the interaction of distamycin, netropsin, Hoechst 33258 and berenil, which are AT-selective minor groove-binding ligands, with synthetic DNA fragments containing different arrangements of AT base pairs by DNase I footprinting. For fragments which contain multiple blocks of (A/T)4 quantitative DNase I footprinting reveals that AATT and AAAA are much better binding sites than TTAA and TATA. Hoechst 33258 shows that greatest discrimination between these sites with a 50-fold difference in affinity between AATT and TATA. Alone amongst these ligands, Hoechst 33258 binds to AATT better than AAAA. These differences in binding to the various AT-tracts are interpreted in terms of variations in DNA minor groove width and suggest that TpA steps within an AT-tract decrease the affinity of these ligands. The behaviour of each site also depends on the flanking sequences; adjacent pyrimidine-purine steps cause a decrease in affinity. The precise ranking order for the various binding sites is not the same for each ligand. Images PMID:7567447

  17. Structural Analysis on the Pathologic Mutant Glucocorticoid Receptor Ligand-Binding Domains.

    PubMed

    Hurt, Darrell E; Suzuki, Shigeru; Mayama, Takafumi; Charmandari, Evangelia; Kino, Tomoshige

    2016-02-01

    Glucocorticoid receptor (GR) gene mutations may cause familial or sporadic generalized glucocorticoid resistance syndrome. Most of the missense forms distribute in the ligand-binding domain and impair its ligand-binding activity and formation of the activation function (AF)-2 that binds LXXLL motif-containing coactivators. We performed molecular dynamics simulations to ligand-binding domain of pathologic GR mutants to reveal their structural defects. Several calculated parameters including interaction energy for dexamethasone or the LXXLL peptide indicate that destruction of ligand-binding pocket (LBP) is a primary character. Their LBP defects are driven primarily by loss/reduction of the electrostatic interaction formed by R611 and T739 of the receptor to dexamethasone and a subsequent conformational mismatch, which deacylcortivazol resolves with its large phenylpyrazole moiety and efficiently stimulates transcriptional activity of the mutant receptors with LBP defect. Reduced affinity of the LXXLL peptide to AF-2 is caused mainly by disruption of the electrostatic bonds to the noncore leucine residues of this peptide that determine the peptide's specificity to GR, as well as by reduced noncovalent interaction against core leucines and subsequent exposure of the AF-2 surface to solvent. The results reveal molecular defects of pathologic mutant receptors and provide important insights to the actions of wild-type GR. PMID:26745667

  18. Tension-compression asymmetry in the binding affinity of membrane-anchored receptors and ligands

    NASA Astrophysics Data System (ADS)

    Xu, Guang-Kui; Liu, Zishun; Feng, Xi-Qiao; Gao, Huajian

    2016-03-01

    Cell adhesion plays a crucial role in many biological processes of cells, e.g., immune responses, tissue morphogenesis, and stem cell differentiation. An essential problem in the molecular mechanism of cell adhesion is to characterize the binding affinity of membrane-anchored receptors and ligands under different physiological conditions. In this paper, a theoretical model is presented to study the binding affinity between a large number of anchored receptors and ligands under both tensile and compressive stresses, and corroborated by demonstrating excellent agreement with Monte Carlo simulations. It is shown that the binding affinity becomes lower as the magnitude of the applied stress increases, and drops to zero at a critical tensile or compressive stress. Interestingly, the critical compressive stress is found to be substantially smaller than the critical tensile stress for relatively long and flexible receptor-ligand complexes. This counterintuitive finding is explained by using the Euler instability theory of slender columns under compression. The tension-compression asymmetry in the binding affinity of anchored receptors and ligands depends subtly on the competition between the breaking and instability of their complexes. This study helps in understanding the role of mechanical forces in cell adhesion mediated by specific binding molecules.

  19. A comprehensive ligand based mapping of the σ₂ receptor binding pocket.

    PubMed

    Rhoades, Derek J; Kinder, David H; Mahfouz, Tarek M

    2014-01-01

    The sigma (σ) receptor system consists of at least two major receptor subtypes: σ₁ and σ₂. Several potential therapeutic applications would benefit from structural knowledge of the σ₂ receptor but gaining this knowledge has been hampered by the difficulties associated with its isolation and, thus, characterization. Here, a ligand based approach has been adopted using the program PHASE® and a group of 41 potent and structurally diverse σ₂ ligands to develop several pharmacophore models for different families of σ₂ ligands. These pharmacophores were analyzed to identify the different binding modes to the receptor and were combined together to construct a comprehensive pharmacophore that was used to develop a structural model for the σ₂ binding pocket. A total of six binding modes were identified and could be classified as neutral or charged modes. The results presented here also indicate the significance of hydrophobic interactions to σ₂ binding and the requirement of hydrogen bonding interactions to increase the affinity for this receptor subtype. This work adds breadth to our knowledge of this receptor's binding site, and should contribute significantly to the development of novel selective σ₂ ligands. PMID:23521001

  20. Bringing Clarity to the Prediction of Protein–Ligand Binding Free Energies via “Blurring”

    PubMed Central

    2015-01-01

    We present a method to evaluate the free energies of ligand binding utilizing a Monte Carlo estimation of the configuration integrals concomitant with uncertainty quantification. Ensembles for integration are built through systematically perturbing an initial ligand conformation in a rigid binding pocket, which is optimized separately prior to incorporation of the ligand. We call the procedure producing the ensembles “blurring”, and it is carried out using an in-house developed code. The Boltzmann factor contribution of each pose to the configuration integral is computed and from there the free energy is obtained. Potential function uncertainties are estimated using a fragment-based error propagation method. This method has been applied to a set of small aromatic ligands complexed with T4 Lysozyme L99A mutant. Microstate energies have been determined with the force fields ff99SB and ff94, and the semiempirical method PM6DH2 in conjunction with continuum solvation models including Generalized Born (GB), the Conductor-like Screening Model (COSMO), and SMD. Of the methods studied, PM6DH2-based scoring gave binding free energy estimates, which yielded a good correlation to the experimental binding affinities (R2 = 0.7). All methods overestimated the calculated binding affinities. We trace this to insufficient sampling, the single static protein structure, and inaccuracies in the solvent models we have used in this study. PMID:24803861

  1. A Structural Switch between Agonist and Antagonist Bound Conformations for a Ligand-Optimized Model of the Human Aryl Hydrocarbon Receptor Ligand Binding Domain

    PubMed Central

    Perkins, Arden; Phillips, Jessica L.; Kerkvliet, Nancy I.; Tanguay, Robert L.; Perdew, Gary H.; Kolluri, Siva K.; Bisson, William H.

    2014-01-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates the expression of a diverse group of genes. Exogenous AHR ligands include the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a potent agonist, and the synthetic AHR antagonist N-2-(1H-indol-3yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine (GNF351). As no experimentally determined structure of the ligand binding domain exists, homology models have been utilized for virtual ligand screening (VLS) to search for novel ligands. Here, we have developed an “agonist-optimized” homology model of the human AHR ligand binding domain, and this model aided in the discovery of two human AHR agonists by VLS. In addition, we performed molecular dynamics simulations of an agonist TCDD-bound and antagonist GNF351-bound version of this model in order to gain insights into the mechanics of the AHR ligand-binding pocket. These simulations identified residues 307–329 as a flexible segment of the AHR ligand pocket that adopts discrete conformations upon agonist or antagonist binding. This flexible segment of the AHR may act as a structural switch that determines the agonist or antagonist activity of a given AHR ligand. PMID:25329374

  2. Influence of ligand binding on structure and thermostability of human α1-acid glycoprotein.

    PubMed

    Kopecký, Vladimír; Ettrich, Rüdiger; Pazderka, Tomáš; Hofbauerová, Kateřina; Řeha, David; Baumruk, Vladimír

    2016-02-01

    Ligand binding of neutral progesterone, basic propranolol, and acidic warfarin to human α1-acid glycoprotein (AGP) was investigated by Raman spectroscopy. The binding itself is characterized by a uniform conformational shift in which a tryptophan residue is involved. Slight differences corresponding to different contacts of the individual ligands inside the β-barrel are described. Results are compared with in silico ligand docking into the available crystal structure of deglycosylated AGP using quantum/molecular mechanics. Calculated binding energies are -18.2, -14.5, and -11.5 kcal/mol for warfarin, propranolol, and progesterone, respectively. These calculations are consistent with Raman difference spectroscopy; nevertheless, minor discrepancies in the precise positions of the ligands point to structural differences between deglycosylated and native AGP. Thermal dynamics of AGP with/without bounded warfarin was followed by Raman spectroscopy in a temperature range of 10-95 °C and analyzed by principal component analysis. With increasing temperature, a slight decrease of α-helical content is observed that coincides with an increase in β-sheet content. Above 45 °C, also β-strands tend to unfold, and the observed decrease in β-sheet coincides with an increase of β-turns accompanied by a conformational shift of the nearby disulfide bridge from high-energy trans-gauche-trans to more relaxed gauche-gauche-trans. This major rearrangement in the vicinity of the bridge is not only characterized by unfolding of the β-sheet but also by subsequent ligand release. Hereby, ligand binding alters the protein dynamics, and the more rigid protein-ligand complex shows an improved thermal stability, a finding that contributes to the reported chaperone-like function of AGP. PMID:26400697

  3. Inhibition of RNA Polymerase II Transcription in Human Cells by Synthetic DNA-Binding Ligands

    NASA Astrophysics Data System (ADS)

    Dickinson, Liliane A.; Gulizia, Richard J.; Trauger, John W.; Baird, Eldon E.; Mosier, Donald E.; Gottesfeld, Joel M.; Dervan, Peter B.

    1998-10-01

    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole--imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located with RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

  4. Molecular dynamics simulations of barley and maize lipid transfer proteins show different ligand binding preferences in agreement with experimental data.

    PubMed

    Smith, Lorna J; Roby, Ysobel; Allison, Jane R; van Gunsteren, Wilfred F

    2013-07-30

    Experimental studies of barley and maize lipid transfer proteins (LTPs) show that the two proteins bind the ligand palmitate in opposite orientations in their internal cavities. Moreover, maize LTP is reported to bind the ligand caprate in the internal cavity in a mixture of two orientations with approximately equal occupancy. Six 30 ns molecular dynamics (MD) simulations of maize and barley LTP with ligands bound in two orientations (modes M and B) have been used to understand the different ligand binding preferences. The simulations show that both maize and barley LTP could bind palmitate in the orientation observed experimentally for maize LTP (mode M), with the predominant interaction being a salt bridge between the ligand carboxylate headgroup and a conserved arginine side chain. However, the simulation of barley LTP with palmitate in the mode B orientation shows the most favorable protein-ligand interaction energy. In contrast, the simulations of maize LTP with palmitate and with caprate in the mode B orientation show no persistent ligand binding, the ligands leaving the cavity during the simulations. Sequence differences between maize and barley LTP in the AB loop region, in residues at the base of the hydrophobic cavity, and in the helix A region are identified as contributing to the different behavior. The simulations reproduce well the experimentally observed binding preferences for palmitate and suggest that the experimental data for maize LTP with caprate reflect ligand mobility in binding mode M rather than the population of binding modes M and B. PMID:23834513

  5. Graphlet signature-based scoring method to estimate protein–ligand binding affinity

    PubMed Central

    Singh, Omkar; Sawariya, Kunal; Aparoy, Polamarasetty

    2014-01-01

    Over the years, various computational methodologies have been developed to understand and quantify receptor–ligand interactions. Protein–ligand interactions can also be explained in the form of a network and its properties. The ligand binding at the protein-active site is stabilized by formation of new interactions like hydrogen bond, hydrophobic and ionic. These non-covalent interactions when considered as links cause non-isomorphic sub-graphs in the residue interaction network. This study aims to investigate the relationship between these induced sub-graphs and ligand activity. Graphlet signature-based analysis of networks has been applied in various biological problems; the focus of this work is to analyse protein–ligand interactions in terms of neighbourhood connectivity and to develop a method in which the information from residue interaction networks, i.e. graphlet signatures, can be applied to quantify ligand affinity. A scoring method was developed, which depicts the variability in signatures adopted by different amino acids during inhibitor binding, and was termed as GSUS (graphlet signature uniqueness score). The score is specific for every individual inhibitor. Two well-known drug targets, COX-2 and CA-II and their inhibitors, were considered to assess the method. Residue interaction networks of COX-2 and CA-II with their respective inhibitors were used. Only hydrogen bond network was considered to calculate GSUS and quantify protein–ligand interaction in terms of graphlet signatures. The correlation of the GSUS with pIC50 was consistent in both proteins and better in comparison to the Autodock results. The GSUS scoring method was better in activity prediction of molecules with similar structure and diverse activity and vice versa. This study can be a major platform in developing approaches that can be used alone or together with existing methods to predict ligand affinity from protein–ligand complexes. PMID:26064572

  6. Spectrochemical evidence for the presence of a tyrosine residue in the allosteric site of glucosamine-6-phosphate deaminase from Escherichia coli.

    PubMed

    Altamirano, M M; Hernandez-Arana, A; Tello-Solis, S; Calcagno, M L

    1994-03-01

    The interaction of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli with its allosteric activator, N-acetyl-D-glucosamine 6-phosphate, was studied by different spectrophotometric methods. Analysis of the circular-dichroism differential spectra produced by the binding of the allosteric activator or the competitive inhibitor 2-amino-2-deoxy-D-glucitol 6-phosphate (a homotropic ligand displacing the allosteric equilibrium to the R conformer), strongly suggests the presence of tyrosine residues at or near the allosteric site, although a conformational effect cannot be ruled out. The involvement of a single tyrosine residue in the N-acetyl-D-glucosamine-6-phosphate binding site of glucosamine-6-phosphate deaminase was supported by spectrophotometric pH titrations performed in the presence or absence of the homotropic and heterotropic ligand. In these experiments, a single titrated tyrosine residue is completely protected by saturation with the allosteric activator; this group is considerably acidic (pK 8.75). The analysis of the amino acid sequence of the deaminase using a set of indices for the prediction of surface accessibility of amino acid residues, suggests that the involved residue may be Tyr121 or Tyr254. PMID:8125098

  7. Structure and ligand-binding mechanism of a cysteinyl leukotriene-binding protein from a blood-feeding disease vector

    PubMed Central

    Jablonka, Willy; Pham, Van; Nardone, Glenn; Gittis, Apostolos; Silva-Cardoso, Lívia; Atella, Georgia C.; Ribeiro, José M.C.; Andersen, John F.

    2016-01-01

    Blood-feeding disease vectors mitigate the negative effects of hemostasis and inflammation through the binding of small-molecule agonists of these processes by salivary proteins. In this study, a lipocalin protein family member (LTBP1) from the saliva of Rhodnius prolixus, a vector of the pathogen Trypanosoma cruzi, is shown to sequester cysteinyl leukotrienes during feeding to inhibit immediate inflammatory responses. Calorimetric binding experiments showed that LTBP1 binds leukotrienes C4 (LTC4) and D4 (LTD4) and E4 (LTE4) but not biogenic amines, adenosine diphosphate or other eicosanoid compounds. Crystal structures of ligand-free LTBP1 and its complexes with LTC4 and LTD4 reveal a conformational change during binding that brings Tyr 114 into close contact with the ligand. LTC4 is cleaved in the complex leaving free glutathione, and a C20 fatty acid. Chromatographic analysis of bound ligands showed only intact LTC4, suggesting that cleavage could be radiation-mediated. PMID:27124118

  8. Structure and Ligand-Binding Mechanism of a Cysteinyl Leukotriene-Binding Protein from a Blood-Feeding Disease Vector.

    PubMed

    Jablonka, Willy; Pham, Van; Nardone, Glenn; Gittis, Apostolos; Silva-Cardoso, Lívia; Atella, Georgia C; Ribeiro, José M C; Andersen, John F

    2016-07-15

    Blood-feeding disease vectors mitigate the negative effects of hemostasis and inflammation through the binding of small-molecule agonists of these processes by salivary proteins. In this study, a lipocalin protein family member (LTBP1) from the saliva of Rhodnius prolixus, a vector of the pathogen Trypanosoma cruzi, is shown to sequester cysteinyl leukotrienes during feeding to inhibit immediate inflammatory responses. Calorimetric binding experiments showed that LTBP1 binds leukotrienes C4 (LTC4), D4 (LTD4), and E4 (LTE4) but not biogenic amines, adenosine diphosphate, or other eicosanoid compounds. Crystal structures of ligand-free LTBP1 and its complexes with LTC4 and LTD4 reveal a conformational change during binding that brings Tyr114 into close contact with the ligand. LTC4 is cleaved in the complex, leaving free glutathione and a C20 fatty acid. Chromatographic analysis of bound ligands showed only intact LTC4, suggesting that cleavage could be radiation-mediated. PMID:27124118

  9. Global Low Frequency Protein Motions in Long-Range Allosteric Signaling

    NASA Astrophysics Data System (ADS)

    McLeish, Tom; Rogers, Thomas; Townsend, Philip; Burnell, David; Pohl, Ehmke; Wilson, Mark; Cann, Martin; Richards, Shane; Jones, Matthew

    2015-03-01

    We present a foundational theory for how allostery can occur as a function of low frequency dynamics without a change in protein structure. Elastic inhomogeneities allow entropic ``signalling at a distance.'' Remarkably, many globular proteins display just this class of elastic structure, in particular those that support allosteric binding of substrates (long-range co-operative effects between the binding sites of small molecules). Through multi-scale modelling of global normal modes we demonstrate negative co-operativity between the two cAMP ligands without change to the mean structure. Crucially, the value of the co-operativity is itself controlled by the interactions around a set of third allosteric ``control sites.'' The theory makes key experimental predictions, validated by analysis of variant proteins by a combination of structural biology and isothermal calorimetry. A quantitative description of allostery as a free energy landscape revealed a protein ``design space'' that identified the key inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, by analyzing naturally occurring CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. The methodology establishes the means to engineer allosteric mechanisms that are driven by low frequency dynamics.

  10. Characterization of Aluminum-Binding Ligands in Pisolithus tinctorius

    NASA Astrophysics Data System (ADS)

    McCauley, R. L.; Cumming, J.

    2009-12-01

    Highly abundant in soil, Al is found in non-toxic forms under neutral pH conditions. However, when the pH of the soil decreases, the presence of cationic Al increases, creating a toxic environment for plants and fungi. Certain plants and their ectomycorrhizal symbiotic fungi have higher tolerance for Al in the soil and surrounding media. A particular fungus, Pisolithus tinctorius, has been found to produce Al-binding pigments which chelate and detoxify cationic Al in the environment. The objectives of this study are to 1) determine the resistance of different ectomycorrhizal fungi species to Al, 2) characterize the production of Al binding compounds by fungi, and 3) quantify Al partitioning between free and bound forms in the environment. Pisolithus tinctorius, Amanita muscaria, Lacaria bicolor, and Rhizopogon rubescens were grown under varying Al concentration in vitro (0 and 200 µM for all species; 0, 100, 200, and 400 µM for P. tinctorius). Biomass was measured and media was analyzed for Al speciation and organic acid profiles post experiment. The Al-binding exudates of P. tinctorius were isolated using immobilized metal affinity chromatography (IMAC) and further separated with reverse phase HPLC (UV). All fungi were resistant to Al at the concentrations tested. Pisolithus was found to have a significantly higher mass than other ectomycorrhizae studied. Organic Al levels were found to increase with an increase in Al treatment for P. tinctorius. These techniques revealed at least eleven compounds active in the Al-binding IMAC fraction with seven peaks having brown pigmentation. These compounds may assist in Al detoxification by P. tinctorius.

  11. Broadly Neutralizing Antibody PGT121 Allosterically Modulates CD4 Binding via Recognition of the HIV-1 gp120 V3 Base and Multiple Surrounding Glycans

    PubMed Central

    Julien, Jean-Philippe; Sok, Devin; Khayat, Reza; Lee, Jeong Hyun; Doores, Katie J.; Walker, Laura M.; Ramos, Alejandra; Diwanji, Devan C.; Pejchal, Robert; Cupo, Albert; Katpally, Umesh; Depetris, Rafael S.; Stanfield, Robyn L.; McBride, Ryan; Marozsan, Andre J.; Paulson, James C.; Sanders, Rogier W.; Moore, John P.; Burton, Dennis R.; Poignard, Pascal; Ward, Andrew B.; Wilson, Ian A.

    2013-01-01

    New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml−1. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained. PMID:23658524

  12. Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans.

    PubMed

    Julien, Jean-Philippe; Sok, Devin; Khayat, Reza; Lee, Jeong Hyun; Doores, Katie J; Walker, Laura M; Ramos, Alejandra; Diwanji, Devan C; Pejchal, Robert; Cupo, Albert; Katpally, Umesh; Depetris, Rafael S; Stanfield, Robyn L; McBride, Ryan; Marozsan, Andre J; Paulson, James C; Sanders, Rogier W; Moore, John P; Burton, Dennis R; Poignard, Pascal; Ward, Andrew B; Wilson, Ian A

    2013-01-01

    New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml(-1). Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained. PMID:23658524

  13. Allosteric interactions at adenosine A1 and A3 receptors: new insights into the role of small molecules and receptor dimerization

    PubMed Central

    Hill, Stephen J; May, Lauren T; Kellam, Barrie; Woolard, Jeanette

    2014-01-01

    The purine nucleoside adenosine is present in all cells in tightly regulated concentrations. It is released under a variety of physiological and pathophysiological conditions to facilitate protection and regeneration of tissues. Adenosine acts via specific GPCRs to either stimulate cyclic AMP formation, as exemplified by Gs-protein-coupled adenosine receptors (A2A and A2B), or inhibit AC activity, in the case of Gi/o-coupled adenosine receptors (A1 and A3). Recent advances in our understanding of GPCR structure have provided insights into the conformational changes that occur during receptor activation following binding of agonists to orthosteric (i.e. at the same binding site as an endogenous modulator) and allosteric regulators to allosteric sites (i.e. at a site that is topographically distinct from the endogenous modulator). Binding of drugs to allosteric sites may lead to changes in affinity or efficacy, and affords considerable potential for increased selectivity in new drug development. Herein, we provide an overview of the properties of selective allosteric regulators of the adenosine A1 and A3 receptors, focusing on the impact of receptor dimerization, mechanistic approaches to single-cell ligand-binding kinetics and the effects of A1- and A3-receptor allosteric modulators on in vivo pharmacology. Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5 PMID:24024783

  14. Nucleotide binding and allosteric modulation of the second AAA+ domain of ClpB probed by transient kinetic studies.

    PubMed

    Werbeck, Nicolas D; Kellner, Julian N; Barends, Thomas R M; Reinstein, Jochen

    2009-08-01

    The bacterial AAA+ chaperone ClpB provides thermotolerance by disaggregating aggregated proteins in collaboration with the DnaK chaperone system. Like many other AAA+ proteins, ClpB is believed to act as a biological motor converting the chemical energy of ATP into molecular motion. ClpB has two ATPase domains, NBD1 and NBD2, on one polypeptide chain. The functional unit of ClpB is a homohexameric ring, with a total of 12 potential nucleotide binding sites. Previously, two separate constructs, one each containing NBD1 or NBD2, have been shown to form a functional complex with chaperone activity when mixed. Here we aimed to elucidate the nucleotide binding properties of the ClpB complex using pre-steady state kinetics and fluorescent nucleotides. For this purpose, we first disassembled the complex and characterized in detail the binding kinetics of a construct comprising NBD2 and the C-terminal domain of ClpB. The monomeric construct bound nucleotides very tightly. ADP bound 2 orders of magnitude more tightly than ATP; this difference in binding affinity resulted almost exclusively from different dissociation rate constants. The nucleotide binding properties of NBD2 changed when this construct was complemented with a construct comprising NBD1 and the middle domain. Our approach shows how complex formation can influence the binding properties of the individual domains and allows us to assign nucleotide binding features of this highly complex, multimeric enzyme to specific domains. PMID:19594134

  15. Effects of co-operative ligand binding on protein amide NH hydrogen exchange.

    PubMed

    Polshakov, Vladimir I; Birdsall, Berry; Feeney, James

    2006-03-01

    Amide protection factors have been determined from NMR measurements of hydrogen/deuterium amide NH exchange rates measured on assigned signals from Lactobacillus casei apo-DHFR and its binary and ternary complexes with trimethoprim (TMP), folinic acid and coenzymes (NADPH/NADP(+)). The substantial sizes of the residue-specific DeltaH and TDeltaS values for the opening/closing events in NH exchange for most of the measurable residues in apo-DHFR indicate that sub-global or global rather than local exchange mechanisms are usually involved. The amide groups of residues in helices and sheets are those most protected in apo-DHFR and its complexes, and the protection factors are generally related to the tightness of ligand binding. The effects of ligand binding that lead to changes in amide protection are not localised to specific binding sites but are spread throughout the structure via a network of intramolecular interactions. Although the increase in protein stability in the DHFR.TMP.NADPH complex involves increased ordering in the protein structure (requiring TDeltaS energy) this is recovered, to a large extent, by the stronger binding (enthalpic DeltaH) interactions made possible by the reduced motion in the protein. The ligand-induced protection effects in the ternary complexes DHFR.TMP.NADPH (large positive binding co-operativity) and DHFR.folinic acid.NADPH (large negative binding co-operativity) mirror the co-operative effects seen in the ligand binding. For the DHFR.TMP.NADPH complex, the ligand-induced protection factors result in DeltaDeltaG(o) values for many residues being larger than the DeltaDeltaG(o) values in the corresponding binary complexes. In contrast, for DHFR.folinic acid.NADPH, the DeltaDeltaG(o) values are generally smaller than many of those in the corresponding binary complexes. The results indicate that changes in protein conformational flexibility on formation of the ligand complex play an important role in determining the co-operativity in

  16. YC-1 BINDING TO THE BETA SUBUNIT OF SOLUBLE GUANYLYL CYCLASE OVERCOMES ALLOSTERIC INHIBITION BY THE ALPHA SUBUNIT

    PubMed Central

    Purohit, Rahul; Fritz, Bradley G.; The, Juliana; Issaian, Aaron; Weichsel, Andrzej; David, Cynthia L.; Campbell, Eric; Hausrath, Andrew C.; Rassouli-Taylor, Leida; Garcin, Elsa D.; Gage, Matthew J.; Montfort, William R.

    2014-01-01

    Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remains unknown. Using linked-equilibria binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the beta subunit. In the absence of CO, YC-1 binds with Kd = 9–21 μM, depending on construct. In the presence of CO, these values decrease to 0.6–1.1 μM. Pfizer compound 25 bound ~10-fold weaker than YC-1 in the absence of CO whereas compound BAY 41–2272 bound particularly tightly in the presence of CO (Kd = 30–90 nM). Additionally, we found that CO binding is much weaker to heterodimeric sGC proteins (Kd = 50–100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca beta H-NOX/PAS). YC-1 greatly enhanced CO binding to heterodimeric sGC, as expected (Kd = ~1 μM). These data indicate the alpha subunit induces a heme pocket conformation with lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the alpha subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity. PMID:24328155

  17. Theory and Normal Mode Analysis of Change in Protein Vibrational Dynamics on Ligand Binding

    SciTech Connect

    Mortisugu, Kei; Njunda, Brigitte; Smith, Jeremy C

    2009-12-01

    The change of protein vibrations on ligand binding is of functional and thermodynamic importance. Here, this process is characterized using a simple analytical 'ball-and-spring' model and all-atom normal-mode analysis (NMA) of the binding of the cancer drug, methotrexate (MTX) to its target, dihydrofolate reductase (DHFR). The analytical model predicts that the coupling between protein vibrations and ligand external motion generates entropy-rich, low-frequency vibrations in the complex. This is consistent with the atomistic NMA which reveals vibrational softening in forming the DHFR-MTX complex, a result also in qualitative agreement with neutron-scattering experiments. Energy minimization of the atomistic bound-state (B) structure while gradually decreasing the ligand interaction to zero allows the generation of a hypothetical 'intermediate' (I) state, without the ligand force field but with a structure similar to that of B. In going from I to B, it is found that the vibrational entropies of both the protein and MTX decrease while the complex structure becomes enthalpically stabilized. However, the relatively weak DHFR:MTX interaction energy results in the net entropy gain arising from coupling between the protein and MTX external motion being larger than the loss of vibrational entropy on complex formation. This, together with the I structure being more flexible than the unbound structure, results in the observed vibrational softening on ligand binding.

  18. Protein:Ligand binding free energies: A stringent test for computational protein design.

    PubMed

    Druart, Karen; Palmai, Zoltan; Omarjee, Eyaz; Simonson, Thomas

    2016-02-01

    A computational protein design method is extended to allow Monte Carlo simulations where two ligands are titrated into a protein binding pocket, yielding binding free energy differences. These provide a stringent test of the physical model, including the energy surface and sidechain rotamer definition. As a test, we consider tyrosyl-tRNA synthetase (TyrRS), which has been extensively redesigned experimentally. We consider its specificity for its substrate l-tyrosine (l-Tyr), compared to the analogs d-Tyr, p-acetyl-, and p-azido-phenylalanine (ac-Phe, az-Phe). We simulate l- and d-Tyr binding to TyrRS and six mutants, and compare the structures and binding free energies to a more rigorous "MD/GBSA" procedure: molecular dynamics with explicit solvent for structures and a Generalized Born + Surface Area model for binding free energies. Next, we consider l-Tyr, ac- and az-Phe binding to six other TyrRS variants. The titration results are sensitive to the precise rotamer definition, which involves a short energy minimization for each sidechain pair to help relax bad contacts induced by the discrete rotamer set. However, when designed mutant structures are rescored with a standard GBSA energy model, results agree well with the more rigorous MD/GBSA. As a third test, we redesign three amino acid positions in the substrate coordination sphere, with either l-Tyr or d-Tyr as the ligand. For two, we obtain good agreement with experiment, recovering the wildtype residue when l-Tyr is the ligand and a d-Tyr specific mutant when d-Tyr is the ligand. For the third, we recover His with either ligand, instead of wildtype Gln. PMID:26503829

  19. Dopaminergic receptor-ligand binding assays based on molecularly imprinted polymers on quartz crystal microbalance sensors.

    PubMed

    Naklua, Wanpen; Suedee, Roongnapa; Lieberzeit, Peter A

    2016-07-15

    Molecularly imprinted polymers (MIPs) have been successfully applied as selective materials for assessing the binding activity of agonist and antagonist of dopamine D1 receptor (D1R) by using quartz crystal microbalance (QCM). In this study, D1R derived from rat hypothalamus was used as a template and thus self-organized on stamps. Those were pressed into an oligomer film consisting of acrylic acid: N-vinylpyrrolidone: N,N'-(1,2-dihydroxyethylene) bis-acrylamide in a ratio of 2:3:12 spin coated onto a dual electrode QCM. Such we obtained one D1R-MIP-QCM electrode, whereas the other electrode carried the non-imprinted control polymer (NIP) that had remained untreated. Successful imprinting of D1R was confirmed by AFM. The polymer can re-incorporate D1R leading to frequency responses of 100-1200Hz in a concentration range of 5.9-47.2µM. In a further step such frequency changes proved inherently useful for examining the binding properties of test ligands to D1R. The resulting mass-sensitive measurements revealed Kd of dopamine∙HCl, haloperidol, and (+)-SCH23390 at 0.874, 25.6, and 0.004nM, respectively. These results correlate well with the values determined in radio ligand binding assays. Our experiments revealed that D1R-MIP sensors are useful for estimating the strength of ligand binding to the active single site. Therefore, we have developed a biomimetic surface imprinting strategy for QCM studies of D1R-ligand binding and presented a new method to ligand binding assay for D1R. PMID:26926593

  20. Charge is Major Determinant of Activation of the Ligand-Responsive Multidrug Resistance Gene Regulator, BmrR.

    PubMed

    Bachas, Sharrol; Kohrs, Bryan; Wade, Herschel

    2016-05-19

    A medium-throughput approach (80+ compounds) to investigate allosteric transcriptional control in the multidrug resistance gene regulator BmrR, with cations, zwitterions, uncharged compounds and anions, is described. Even at the allosteric level, BmrR is quite promiscuous with regard to molecular shape and structure, but it is sensitive to molecular charge. A role for charge is further supported by differences in the activation properties of structurally similar ligands displaying variable charge properties as well as differences in activation by zwitterions and uncharged ligands, which show similar binding affinities. A comparison of allosteric selectivity with the distribution of differently charged ligands in bacterial cellular environments suggests that the selectivity of charge is a major factor in discrimination of xenobiotics, and native biological compounds and metabolites. Interestingly, in eukaryotic cells, the selectivity of cationic ligands might be a protective mechanism against chemical agents that act in a promiscuous fashion. PMID:27010425

  1. Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer

    PubMed Central

    Duesterberg, Van K; Fischer-Hwang, Irena T; Perez, Christian F; Hogan, Daniel W; Block, Steven M

    2015-01-01

    The thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in mRNA that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and Förster resonance energy transfer (smFRET), using an optical trap equipped for simultaneous smFRET. The ‘Force-FRET’ approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in smFRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity. DOI: http://dx.doi.org/10.7554/eLife.12362.001 PMID:26709838

  2. In silico identification of anthropogenic chemicals as ligands of zebrafish sex hormone binding globulin

    SciTech Connect

    Thorsteinson, Nels; Ban, Fuqiang; Santos-Filho, Osvaldo; Tabaei, Seyed M.H.; Miguel-Queralt, Solange; Underhill, Caroline; Cherkasov, Artem Hammond, Geoffrey L.

    2009-01-01

    Anthropogenic compounds with the capacity to interact with the steroid-binding site of sex hormone binding globulin (SHBG) pose health risks to humans and other vertebrates including fish. Building on studies of human SHBG, we have applied in silico drug discovery methods to identify potential binders for SHBG in zebrafish (Danio rerio) as a model aquatic organism. Computational methods, including; homology modeling, molecular dynamics simulations, virtual screening, and 3D QSAR analysis, successfully identified 6 non-steroidal substances from the ZINC chemical database that bind to zebrafish SHBG (zfSHBG) with low-micromolar to nanomolar affinities, as determined by a competitive ligand-binding assay. We also screened 80,000 commercial substances listed by the European Chemicals Bureau and Environment Canada, and 6 non-steroidal hits from this in silico screen were tested experimentally for zfSHBG binding. All 6 of these compounds displaced the [{sup 3}H]5{alpha}-dihydrotestosterone used as labeled ligand in the zfSHBG screening assay when tested at a 33 {mu}M concentration, and 3 of them (hexestrol, 4-tert-octylcatechol, and dihydrobenzo(a)pyren-7(8H)-one) bind to zfSHBG in the micromolar range. The study demonstrates the feasibility of large-scale in silico screening of anthropogenic compounds that may disrupt or highjack functionally important protein:ligand interactions. Such studies could increase the awareness of hazards posed by existing commercial chemicals at relatively low cost.

  3. A simple model-free method for direct assessment of fluorescent ligand binding by linear spectral summation.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2014-01-01

    Fluorescent tagged ligands are commonly used to determine binding to proteins. However, bound and free ligand concentrations are not directly determined. Instead the response in a fluorescent ligand titration experiment is considered to be proportional to the extent of binding and, therefore, the maximum value of binding is scaled to the total protein concentration. Here, a simple model-free method is presented to be performed in two steps. In the first step, normalized bound and free spectra of the ligand are determined. In the second step, these spectra are used to fit composite spectra as the sum of individual components or linear spectral summation. Using linear spectral summation, free and bound 1-Anilinonaphthalene-8-Sulfonic Acid (ANS) fluorescent ligand concentrations are directly calculated to determine ANS binding to tear lipocalin (TL), an archetypical ligand binding protein. Error analysis shows that the parameters that determine bound and free ligand concentrations were recovered with high certainty. The linear spectral summation method is feasible when fluorescence intensity is accompanied by a spectral shift upon protein binding. Computer simulations of the experiments of ANS binding to TL indicate that the method is feasible when the fluorescence spectral shift between bound and free forms of the ligand is just 8 nm. Ligands tagged with environmentally sensitive fluorescent dyes, e.g., dansyl chromophore, are particularly suitable for this method. PMID:24043458

  4. A Simple Model-Free Method for Direct Assessment of Fluorescent Ligand Binding by Linear Spectral Summation

    PubMed Central

    Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

    2013-01-01

    Fluorescent tagged ligands are commonly used to determine binding to proteins. However, bound and free ligand concentrations are not directly determined. Instead the response in a fluorescent ligand titration experiment is considered to be proportional to the extent of binding and, therefore, the maximum value of binding is scaled to the total protein concentration. Here, a simple model-free method is presented to be performed in two steps. In the first step, normalized bound and free spectra of the ligand are determined. In the second step, these spectra are used to fit composite spectra as the sum of individual components or linear spectral summation. Using linear spectral summation, free and bound 1-Anilinonaphthalene-8-Sulfonic Acid (ANS) fluorescent ligand concentrations are directly calculated to determine ANS binding to tear lipocalin (TL), an archetypical ligand binding protein. Error analysis shows that the parameters that determine bound and free ligand concentrations were recovered with high certainty. The linear spectral summation method is feasible when fluorescence intensity is accompanied by a spectral shift upon protein binding. Computer simulations of the experiments of ANS binding to TL indicate that the method is feasible when the fluorescence spectral shift between bound and free forms of the ligand is just 8 nm. Ligands tagged with environmentally sensitive fluorescent dyes, e.g., dansyl chromophore, are particularly suitable for this method. PMID:24043458

  5. Calculations of distance distributions and probabilities of binding by ligands between parallel plane membranes comprising receptors

    NASA Astrophysics Data System (ADS)

    Plante, Ianik; Devroye, Luc; Cucinotta, Francis A.

    2014-03-01

    Cell communication through biochemical signaling pathways is a key determinant of tissue responses to radiation. Several molecules, such as the transforming growth factor β (TGFβ), are implicated in radiation-induced signaling between cells. Brownian Dynamics (BD) algorithms have recently been used to simulate the interaction of ligands with receptors and to elucidate signal transduction and autocrine loops in ligand-receptors systems. In this paper, we discuss the simulation of particle diffusion and binding kinetics in a space bounded by two parallel plane membranes, using an exact algorithm to sample the propagator (Green’s function) of a particle located between 2 membranes. We also show that the simulation results are independent of the number of time steps used, in accordance with time discretization equations. These simulations could be used to simulate the motion and binding of ligand molecules in a cell culture, and possibly in neuronal synapses.

  6. Adaptive ligand binding by the purine riboswitch in the recognition of guanine and adenine analogs

    PubMed Central

    Gilbert, Sunny D.; Reyes, Francis E.; Edwards, Andrea L.; Batey, Robert T.

    2009-01-01

    SUMMARY Purine riboswitches discriminate between guanine and adenine by at least 10,000-fold based on the identity of a single pyrimidine (Y74) that forms a Watson-Crick base pair with the ligand. To understand how this high degree of specificity for closely related compounds is achieved through simple pairing, we investigated their interaction with purine analogs with varying functional groups at the 2- and 6-positions that have the potential to alter interactions with Y74. Using a combination of crystallographic and calorimetric approaches, we find that binding these purines is often facilitated by either small structural changes in the RNA or tautomeric changes in the ligand. This work also reveals that, along with base pairing, conformational restriction of Y74 significantly contributes to nucleobase selectivity. These results reveal that compounds that exploit the inherent local flexibility within riboswitch binding pockets can alter their ligand specificity. PMID:19523903

  7. Vibrational Softening of a Protein on Ligand Binding

    SciTech Connect

    Balog, Erica; Perahia, David; Smith, Jeremy C; Merzel, Franci

    2011-01-01

    Neutron scattering experiments have demonstrated that binding of the cancer drug methotrexate softens the low-frequency vibrations of its target protein, dihydrofolate reductase (DHFR). Here, this softening is fully reproduced using atomic detail normal-mode analysis. Decomposition of the vibrational density of states demonstrates that the largest contributions arise from structural elements of DHFR critical to stability and function. Mode-projection analysis reveals an increase of the breathing-like character of the affected vibrational modes consistent with the experimentally observed increased adiabatic compressibility of the protein on complexation.

  8. Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM

    NASA Astrophysics Data System (ADS)

    Pfreundschuh, Moritz; Alsteens, David; Wieneke, Ralph;