Showing papers on "Cooperative binding published in 2014"

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.

Abstract: ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.

Cooperativity and Complexity in the Binding of Anions and Cations to a Tetratopic Ion-Pair Host

Abstract: Cooperative interactions play a very important role in both natural and synthetic supramolecular systems. We report here on the cooperative binding properties of a tetratopic ion-pair host 1. This host combines two isophthalamide anion recognition sites with two unusual “half-crown/two carbonyl” cation recognition sites as revealed by the combination of single-crystal X-ray analysis of the free host and the 1:2 host:calcium cation complex, together with two-dimensional NMR and computational studies. By systematically comparing all of the binding data to several possible binding models and focusing on four different variants of the 1:2 binding model, it was in most cases possible to quantify these complex cooperative interactions. The data showed strong negative cooperativity (α = 0.01–0.05) of 1 toward chloride and acetate anions, while for cations the results were more variable. Interestingly, in the competitive (CDCl3/CD3OD (9:1, v/v)) solvent, the addition of calcium cations to the tetratopic ion-pair ...

Structural and mechanistic basis of zinc regulation across the E. coli Zur regulon.

Abstract: Commensal microbes, whether they are beneficial or pathogenic, are sensitive to host processes that starve or swamp the prokaryote with large fluctuations in local zinc concentration. To understand how microorganisms coordinate a dynamic response to changes in zinc availability at the molecular level, we evaluated the molecular mechanism of the zinc-sensing zinc uptake regulator (Zur) protein at each of the known Zur-regulated genes in Escherichia coli. We solved the structure of zinc-loaded Zur bound to the PznuABC promoter and show that this metalloregulatory protein represses gene expression by a highly cooperative binding of two adjacent dimers to essentially encircle the core element of each of the Zur-regulated promoters. Cooperativity in these protein-DNA interactions requires a pair of asymmetric salt bridges between Arg52 and Asp49′ that connect otherwise independent dimers. Analysis of the protein-DNA interface led to the discovery of a new member of the Zur-regulon: pliG. We demonstrate this gene is directly regulated by Zur in a zinc responsive manner. The pliG promoter forms stable complexes with either one or two Zur dimers with significantly less protein-DNA cooperativity than observed at other Zur regulon promoters. Comparison of the in vitro Zur-DNA binding affinity at each of four Zur-regulon promoters reveals ca. 10,000-fold variation Zur-DNA binding constants. The degree of Zur repression observed in vivo by comparison of transcript copy number in wild-type and Δzur strains parallels this trend spanning a 100-fold difference. We conclude that the number of ferric uptake regulator (Fur)-family dimers that bind within any given promoter varies significantly and that the thermodynamic profile of the Zur-DNA interactions directly correlates with the physiological response at different promoters.

Revised model of calcium and magnesium binding to the bacterial cell wall

Abstract: Metals bind to the bacterial cell wall, yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane as lipoteichoic acid or covalently bound to the cell wall as wall teichoic acid. The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, which contradicts previous reports that calcium binding is 100 % dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger than previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, which means that the binding affinity decreases as more ions become bound to the sample. For Ca2+, Region I has a KA = (1.0 ± 0.2) × 106 M−1 and Region II has a KA = (0.075 ± 0.058) × 106 M−1. For Mg2+, KA1 = (1.5 ± 0.1) × 106 and KA2 = (0.17 ± 0.10) × 106. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70 ± 0.04 and 0.67 ± 0.03 µmol/mg for Ca2+ and Mg2+ respectively. These data contradict the current paradigm of only a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent. This suggests a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations.

Mechanistic insight into ligand binding to G-quadruplex DNA

Abstract: 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.

Allosteric regulation of rhomboid intramembrane proteolysis.

Abstract: Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Forster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases.

A variety of roles for versatile zinc in metallo-β-lactamases.

Abstract: Metallo-β-lactamases are important as a major source of resistance of pathogenic bacteria to the widely used β-lactam antibiotics. They show considerable diversity in terms of sequence and are grouped into three subclasses, B1, B2 and B3, which share a common overall fold. In each case the active enzyme has binding sites for two zinc ions in close proximity, although the amino-acid residues which coordinate the metals vary from one subclass to another. In subclasses B1 and B3, there has been controversy about whether both zinc ions are required for activity, but the most recent evidence indicates that there is positive cooperativity in zinc binding and that the catalytically relevant species is the di-zinc enzyme. Subclass B2 enzymes, on the other hand, are active in the mono-zinc state and are inhibited by the binding of a second zinc ion. Evidence for the importance of the zinc ions in substrate binding has come from structures of product complexes which indicate that the β-lactam core binds to subclass B1 and B3 enzymes in a rather consistent fashion, interactions with the zinc ions being centrally important. The zinc ions play key roles in the catalytic mechanism, including facilitating nucleophilic attack on the amide carbonyl by the zinc-bound hydroxide ion, stabilising the anionic tetrahedral intermediate and coordinating the departing amine nitrogen.

Prepaying the entropic cost for allosteric regulation in KIX

Abstract: The kinase-inducible domain interacting (KIX) domain of the CREB binding protein (CBP) is capable of simultaneously binding two intrinsically disordered transcription factors, such as the mixed-lineage leukemia (MLL) and c-Myb peptides, at isolated interaction sites. In vitro, the affinity for binding c-Myb is approximately doubled when KIX is in complex with MLL, which suggests a positive cooperative binding mechanism, and the affinity for MLL is also slightly increased when KIX is first bound by c-Myb. Expanding the scope of recent NMR and computational studies, we explore the allosteric mechanism at a detailed molecular level that directly connects the microscopic structural dynamics to the macroscopic shift in binding affinities. To this end, we have performed molecular dynamics simulations of free KIX, KIX-c-Myb, MLL-KIX, and MLL-KIX-c-Myb using a topology-based Gō-like model. Our results capture an increase in affinity for the peptide in the allosteric site when KIX is prebound by a complementary effector and both peptides follow an effector-independent folding-and-binding mechanism. More importantly, we discover that MLL binding lowers the entropic cost for c-Myb binding, and vice versa, by stabilizing the L12-G2 loop and the C-terminal region of the α3 helix on KIX. This work demonstrates the importance of entropy in allosteric signaling between promiscuous molecular recognition sites and can inform the rational design of small molecule stabilizers to target important regions of conformationally dynamic proteins.

A study of the molecular mechanism of binding kinetics and long residence times of human CCR5 receptor small molecule allosteric ligands.

Abstract: Background and Purpose The human CCR5 receptor is a co-receptor for HIV-1 infection and a target for anti-viral therapy. A greater understanding of the binding kinetics of small molecule allosteric ligand interactions with CCR5 will lead to a better understanding of the binding process and may help discover new molecules that avoid resistance. Experimental Approach Using [3H] maraviroc as a radioligand, a number of different binding protocols were employed in conjunction with simulations to determine rate constants, kinetic mechanism and mutant kinetic fingerprints for wild-type and mutant human CCR5 with maraviroc, aplaviroc and vicriviroc. Key Results Kinetic characterization of maraviroc binding to the wild-type CCR5 was consistent with a two-step kinetic mechanism that involved an initial receptor–ligand complex (RA), which transitioned to a more stable complex, R'A, with at least a 13-fold increase in affinity. The dissociation rate from R'A, k−2, was 1.2 × 10−3 min−1. The maraviroc time-dependent transition was influenced by F85L, W86A, Y108A, I198A and Y251A mutations of CCR5. Conclusions and Implications The interaction between maraviroc and CCR5 proceeded according to a multi-step kinetic mechanism, whereby initial mass action binding and later reorganizations of the initial maraviroc–receptor complex lead to a complex with longer residence time. Site-directed mutagenesis identified a kinetic fingerprint of residues that affected the binding kinetics, leading to the conclusion that allosteric ligand binding to CCR5 involved the rearrangement of the binding site in a manner specific to each allosteric ligand.

Single-molecule imaging and kinetic analysis of cooperative cofilin–actin filament interactions

Abstract: The actin filament-severing protein actin depolymerizing factor (ADF)/cofilin is ubiquitously distributed among eukaryotes and modulates actin dynamics The cooperative binding of cofilin to actin filaments is crucial for the concentration-dependent unconventional modulation of actin dynamics by cofilin In this study, the kinetic parameters associated with the cooperative binding of cofilin to actin filaments were directly evaluated using a single-molecule imaging technique The on-rate of cofilin binding to the actin filament was estimated to be 006 µM−1⋅s−1 when the cofilin concentration was in the range of 30 nM to 1 µM A dwell time histogram of cofilin bindings decays exponentially to give an off-rate of 06 s−1 During long-term cofilin binding events (>04 s), additional cofilin bindings were observed in the vicinity of the initial binding site The on-rate for these events was 23-fold higher than that for noncontiguous bindings Super-high-resolution image analysis of the cofilin binding location showed that the on-rate enhancement occurred within 65 nm of the original binding event By contrast, the cofilin off-rate was not affected by the presence of prebound cofilin Neither decreasing the temperature nor increasing the viscosity of the test solution altered the on-rates, off-rates, or the cooperative parameter (ω) of the binding These results indicate that cofilin binding enhances additional cofilin binding in the vicinity of the initial binding site (ca 24 subunits), but it does not affect the off-rate, which could be the molecular mechanism of the cooperative binding of cofilin to actin filaments

Cyclization-induced turn-on fluorescence system applicable to dicarboxylate sensing

Abstract: A novel tetraphenylethene-based fluorescence (FL) chemosensor exhibits nonlinear turn-on FL switching though cooperative binding of L-tartarate, where its convergent binding to form cyclic substructures is responsible for the FL increase. This binding scheme achieves selective detection of dicarboxylates over monocarboxylates, thus is potentially applicable to the preliminary screening for metabolic disorders.

Discriminating binding mechanisms of an intrinsically disordered protein via a multi-state coarse-grained model.

Abstract: Many proteins undergo a conformational transition upon binding to their cognate binding partner, with intrinsically disordered proteins (IDPs) providing an extreme example in which a folding transition occurs. However, it is often not clear whether this occurs via an “induced fit” or “conformational selection” mechanism, or via some intermediate scenario. In the first case, transient encounters with the binding partner favour transitions to the bound structure before the two proteins dissociate, while in the second the bound structure must be selected from a subset of unbound structures which are in the correct state for binding, because transient encounters of the incorrect conformation with the binding partner are most likely to result in dissociation. A particularly interesting situation involves those intrinsically disordered proteins which can bind to different binding partners in different conformations. We have devised a multi-state coarse-grained simulation model which is able to capture the binding of IDPs in alternate conformations, and by applying it to the binding of nuclear coactivator binding domain (NCBD) to either ACTR or IRF-3 we are able to determine the binding mechanism. By all measures, the binding of NCBD to either binding partner appears to occur via an induced fit mechanism. Nonetheless, we also show how a scenario closer to conformational selection could arise by choosing an alternative non-binding structure for NCBD.

Determination of protein-ligand binding constants of a cooperatively regulated tetrameric enzyme using electrospray mass spectrometry.

Abstract: This study highlights the benefits of nano electrospray ionization mass spectrometry (nanoESI-MS) as a fast and label-free method not only for determination of dissociation constants (KD) of a cooperatively regulated enzyme but also to better understand the mechanism of enzymatic cooperativity of multimeric proteins. We present an approach to investigate the allosteric mechanism in the binding of inhibitors to the homotetrameric enzyme fructose 1,6-bisphosphatase (FBPase), a potential therapeutic target for glucose control in type 2 diabetes. A series of inhibitors binding at an allosteric site of FBPase were investigated to determine their KDs by nanoESI-MS. The KDs determined by ESI-MS correlate very well with IC50 values in solution. The Hill coefficients derived from nanoESI-MS suggest positive cooperativity. From single-point measurements we could obtain information on relative potency, stoichiometry, conformational changes, and mechanism of cooperativity. A new X-ray crystal structure of FBPase tetr...

The phosphate clamp: sequence selective nucleic acid binding profiles and conformational induction of endonuclease inhibition by cationic Triplatin complexes

Abstract: The substitution-inert polynuclear platinum(II) complex (PPC) series, [{trans-Pt(NH3)2(NH2(CH2)nNH3)}2-μ-(trans-Pt(NH3)2(NH2(CH2)nNH2)2}](NO3)8, where n = 5 (AH78P), 6 (AH78 TriplatinNC) and 7 (AH78H), are potent non-covalent DNA binding agents where nucleic acid recognition is achieved through use of the 'phosphate clamp' where the square-planar tetra-am(m)ine Pt(II) coordination units all form bidentate N-O-N complexes through hydrogen bonding with phosphate oxygens. The modular nature of PPC-DNA interactions results in high affinity for calf thymus DNA (Kapp ∼5 × 10(7) M(-1)). The phosphate clamp-DNA interactions result in condensation of superhelical and B-DNA, displacement of intercalated ethidium bromide and facilitate cooperative binding of Hoechst 33258 at the minor groove. The effect of linker chain length on DNA conformational changes was examined and the pentane-bridged complex, AH78P, was optimal for condensing DNA with results in the nanomolar region. Analysis of binding affinity and conformational changes for sequence-specific oligonucleotides by ITC, dialysis, ICP-MS, CD and 2D-(1)H NMR experiments indicate that two limiting modes of phosphate clamp binding can be distinguished through their conformational changes and strongly suggest that DNA condensation is driven by minor-groove spanning. Triplatin-DNA binding prevents endonuclease activity by type II restriction enzymes BamHI, EcoRI and SalI, and inhibition was confirmed through the development of an on-chip microfluidic protocol.

Membrane interaction of bound ligands contributes to the negative binding cooperativity of the EGF receptor.

Abstract: The epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, migration, and differentiation, and aberrant EGFR signaling is implicated in a variety of cancers. EGFR signaling is triggered by extracellular ligand binding, which promotes EGFR dimerization and activation. Ligand-binding measurements are consistent with a negatively cooperative model in which the ligand-binding affinity at either binding site in an EGFR dimer is weaker when the other site is occupied by a ligand. This cooperativity is widely believed to be central to the effects of ligand concentration on EGFR-mediated intracellular signaling. Although the extracellular portion of the human EGFR dimer has been resolved crystallographically, the crystal structures do not reveal the structural origin of this negative cooperativity, which has remained unclear. Here we report the results of molecular dynamics simulations suggesting that asymmetrical interactions of the two binding sites with the membrane may be responsible (perhaps along with other factors) for this negative cooperativity. In particular, in our simulations the extracellular domains of an EGFR dimer spontaneously lay down on the membrane in an orientation in which favorable membrane contacts were made with one of the bound ligands, but could not be made with the other. Similar interactions were observed when EGFR was glycosylated, as it is in vivo.

Cooperative Binding of Cyclodextrin Dimers to Isoflavone Analogues Elucidated by Free Energy Calculations

Abstract: Dimerization of cyclodextrin (CD) molecules is an elementary step in the construction of CD-based nanostructured materials. Cooperative binding of CD cavities to guest molecules facilitates the dimerization process and, consequently, the overall stability and assembly of CD nanostructures. In the present study, all three dimerization modes (head-to-head, head-to-tail, and tail-to-tail) of β-CD molecules and their binding to three isoflavone drug analogues (puerarin, daidzin, and daidzein) were investigated in explicit water surrounding using molecular dynamics simulations. Total and individual contributions from the binding partners and solvent environment to the thermodynamics of these binding reactions are quantified in detail using free energy calculations. Cooperative drug binding to two CD cavities gives an enhanced binding strength for daidzin and daidzein, whereas for puerarin no obvious enhancement is observed. Head-to-head dimerization yields the most stable complexes for inclusion of the tested ...

The E6AP binding pocket of the HPV16 E6 oncoprotein provides a docking site for a small inhibitory peptide unrelated to E6AP, indicating druggability of E6.

Abstract: The HPV E6 oncoprotein maintains the malignant phenotype of HPV-positive cancer cells and represents an attractive therapeutic target. E6 forms a complex with the cellular E6AP ubiquitin ligase, ultimately leading to p53 degradation. The recently elucidated x-ray structure of a HPV16 E6/E6AP complex showed that HPV16 E6 forms a distinct binding pocket for E6AP. This discovery raises the question whether the E6AP binding pocket is druggable, i. e. whether it provides a docking site for functional E6 inhibitors. To address these issues, we performed a detailed analysis of the HPV16 E6 interactions with two small peptides: (i) E6APpep, corresponding to the E6 binding domain of E6AP, and (ii) pep11**, a peptide that binds to HPV16 E6 and, in contrast to E6APpep, induces apoptosis, specifically in HPV16-positive cancer cells. Surface plasmon resonance, NMR chemical shift perturbation, and mammalian two-hybrid analyses coupled to mutagenesis indicate that E6APpep contacts HPV16 E6 amino acid residues within the E6AP pocket, both in vitro and intracellularly. Many of these amino acids were also important for binding to pep11**, suggesting that the binding sites for the two peptides on HPV16 E6 overlap. Yet, few E6 amino acids were differentially involved which may contribute to the higher binding affinity of pep11**. Data from the HPV16 E6/pep11** interaction allowed the rational design of single amino acid exchanges in HPV18 and HPV31 E6 that enabled their binding to pep11**. Taken together, these results suggest that E6 molecular surfaces mediating E6APpep binding can also accommodate pro-apoptotic peptides that belong to different sequence families. As proof of concept, this study provides the first experimental evidence that the E6AP binding pocket is druggable, opening new possibilities for rational, structure-based drug design.

Discovery and Mapping of an Intracellular Antagonist Binding Site at the Chemokine Receptor CCR2

Abstract: The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence for an intracellular binding site for CCR2-RA-[R], JNJ-27141491, and SD-24. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y(7.53) and phenylalanine F(8.50) of the NPxxYx(5,6)F motif, as well as V(6.36) at the bottom of TM-VI and K(8.49) in helix-VIII. These findings demonstrate for the first time the presence of an allosteric intracellular binding site for CCR2 antagonists. This contributes to an increased understanding of the interactions of diverse ligands at CCR2 and may allow for a more rational design of future allosteric antagonists.

Artificial nucleobase-amino acid conjugates: a new class of TAR RNA binding agents.

Abstract: The human immunodeficiency virus type-1 (HIV-1) Tat protein stimulates transcriptional elongation. Tat is involved in the transcription machinery by binding to the transactivation response region (TAR) RNA stem-loop structure, which is encoded by the 5' leader sequence found in all HIV-1 mRNAs. Herein, we report the rational design, synthesis, and in vitro evaluation of new RNA binding agents that were conceived in order to bind strongly and selectively to the stem-loop structure of TAR RNA and, thus, inhibit the Tat/TAR interaction. We have demonstrated that the conjugation of modified nucleobases, able to interact specifically with an RNA base pair, and various amino acids allows these motifs to bind the target RNA selectively and in a cooperative manner that leads to the inhibition of viral replication in HIV-infected cells.

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

Abstract: 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

NMR characterization of the binding properties and conformation of glycosaminoglycans interacting with interleukin-10

Abstract: The cytokine interleukin-10 (IL-10) is an important regulator of the host immune system with both pro- and anti-inflammatory functions. Glycosaminoglycans (GAGs) play a decisive role in the biology of many growth factors, e.g., for receptor binding or protection from proteolytic degradation. GAGs of the extracellular matrix inhibit IL-10 signaling, however, the molecular mechanism is so far unknown. Here, we studied the interaction between GAGs and IL-10 using a combination of nuclear magnetic resonance (NMR) spectroscopy and computer simulations. The binding region of a set of heparin and chondroitin sulfate GAG disaccharides with varying sulfation pattern were determined by saturation transfer difference (STD) NMR spectroscopy. From the initial growth rate of the STD amplification factor binding affinities were determined and KD values in the low millimolar to micromolar range were obtained. We observed the highest binding affinity to IL-10 with fully sulfated heparin; however, a hyaluronan hexasaccharide did not exhibit binding, which suggests that GAG sulfation is necessary for interaction with IL-10. For octasaccharides or longer GAGs, a cooperative binding behavior was observed, which could indicate simultaneous interaction with both dimer subunits of IL-10. Finally, structural information about the bound GAG was exemplarily obtained for a heparin tetrasaccharide fragment (ΔUA,2S-GlcNS,6S-IdoA,2S-GlcNS,6S) using transferred NOESY experiments, proton-proton scalar couplings and molecular dynamics simulations. The overall backbone conformation is only slightly changed in the presence of IL-10 and the conformational equilibrium between (1)C4 chair and (2)So skew-boat structure of the internal iduronic acid residue is preserved.

Sequence specific binding of beta carboline alkaloid harmalol with deoxyribonucleotides: binding heterogeneity, conformational, thermodynamic and cytotoxic aspects.

Abstract: Background Base dependent binding of the cytotoxic alkaloid harmalol to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by various photophysical and calorimetric studies, and molecular docking. Methodology/principal findings Binding data obtained from absorbance according to neighbor exclusion model indicated that the binding constant decreased in the order poly(dG-dC).poly(dG-dC)>poly(dA-dT).poly(dA-dT)>poly(dA).poly(dT)>poly(dG).poly(dC). The same trend was shown by the competition dialysis, change in fluorescence steady state intensity, stabilization against thermal denaturation, increase in the specific viscosity and perturbations in circular dichroism spectra. Among the polynucleotides, poly(dA).poly(dT) and poly(dG).poly(dC) showed positive cooperativity where as poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) showed non cooperative binding. Isothermal calorimetric data on the other hand showed enthalpy driven exothermic binding with a hydrophobic contribution to the binding Gibbs energy with poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) where as harmalol with poly(dA).poly(dT) showed entropy driven endothermic binding and with poly(dG).poly(dC) it was reported to be entropy driven exothermic binding. The study also tested the in vitro chemotherapeutic potential of harmalol in HeLa, MDA-MB-231, A549, and HepG2 cell line by MTT assay. Conclusions/significance Studies unequivocally established that harmalol binds strongly with hetero GC polymer by mechanism of intercalation where the alkaloid resists complete overlap to the DNA base pairs inside the intercalation cavity and showed maximum cytotoxicity on HepG2 with IC50 value of 14 µM. The results contribute to the understanding of binding, specificity, energetic, cytotoxicity and docking of harmalol-DNA complexation that will guide synthetic efforts of medicinal chemists for developing better therapeutic agents.