Cooperative Binding Model Research Articles

Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Reveals Buffer-Modulated Cooperativity of Metal-Templated Protein Dimerization.

Self-assembly of protein monomers directed by metal ion coordination constitutes a promising strategy for designing supramolecular architectures complicated by the noncovalent interaction between monomers. Herein, two pulse dipolar electron paramagnetic resonance spectroscopy (PDS) techniques, pulse electron–electron double resonance and relaxation-induced dipolar modulation enhancement, were simultaneously employed to study the CuII-templated dimerization behavior of a model protein (Streptococcus sp. group G, protein G B1 domain) in both phosphate and Tris-HCl buffers. A cooperative binding model could simultaneously fit all data and demonstrate that the cooperativity of protein dimerization across α-helical double-histidine motifs in the presence of CuII is strongly modulated by the buffer, representing a platform for highly tunable buffer-switchable templated dimerization. Hence, PDS enriches the family of techniques for monitoring binding processes, supporting the development of novel strategies for bioengineering structures and stable architectures assembled by an initial metal-templated dimerization.

Selectivity via Cooperativity: Preferential Stabilization of the p65/14-3-3 Interaction with Semisynthetic Natural Products.

Natural compounds are an important class of potent drug molecules including some retrospectively found to act as stabilizers of protein–protein interactions (PPIs). However, the design of synthetic PPI stabilizers remains an understudied approach. To date, there are limited examples where cooperativity has been utilized to guide the optimization of a PPI stabilizer. The 14-3-3 scaffold proteins provide an excellent platform to explore PPI stabilization because these proteins mediate several hundred PPIs, and a class of natural compounds, the fusicoccanes, are known to stabilize a subset of 14-3-3 protein interactions. 14-3-3 has been reported to negatively regulate the p65 subunit of the NF-κB transcription factor, which qualifies this protein complex as a potential target for drug discovery to control cell proliferation. Here, we report the high-resolution crystal structures of two 14-3-3 binding motifs of p65 in complex with 14-3-3. A semisynthetic natural product derivative, DP-005, binds to an interface pocket of the p65/14-3-3 complex and concomitantly stabilizes it. Cooperativity analyses of this interaction, and other disease relevant 14-3-3-PPIs, demonstrated selectivity of DP-005 for the p65/14-3-3 complex. The adaptation of a cooperative binding model provided a general approach to characterize stabilization and to assay for selectivity of PPI stabilizers.

Quantitative Cooperative Binding Model for Intrinsically Disordered Proteins Interacting with Nanomaterials.

Intrinsically disordered proteins (IDPs) can display a broad spectrum of binding modes and highly variable binding affinities when interacting with both biological and nonbiological materials. A quantitative model of such behavior is important for the better understanding of the function of IDPs when encountering inorganic nanomaterials with the potential to control their behavior in vivo and in vitro. Depending on their amino acid composition and chain length, binding properties can vary strongly between different IDPs. Moreover, due to differences in the physical chemical properties of clusters of amino acid residues along the IDP primary sequence, individual residues can adopt a wide range of bound state populations. Quantitative experimental binding affinities with synthetic silica nanoparticles (SNPs) at residue-level resolution, which were obtained for a set of IDPs by solution NMR relaxation experiments, are explained here by a first-principle analytical statistical mechanical model termed SILC. SILC quantitatively predicts residue-specific binding affinities to nanoparticles and it expresses binding cooperativity as the cumulative result of pairwise residue effects. The model, which was parametrized for anionic SNPs and applied to experimental data of four IDP systems with distinctive binding behavior, successfully predicts differences in overall binding affinities, fine details of IDP-SNP affinity profiles, and site-directed mutagenesis effects with a spatial resolution at the individual residue level. The SILC model provides an analytical description of such types of fuzzy IDP-SNP complexes and may help advance understanding nanotoxicity and in vivo targeting of IDPs by specifically designed nanomaterials.

Evaluation of the binding properties of drugs to albumin from DSC thermograms.

There is a demand in rapid and robust methods to determine the affinity of drugs to receptors, enzymes, and transport proteins. Differential scanning calorimetry (DSC) is a common method to prove the existence of ligand-protein binding from the shift of denaturation peak, but it is rarely used to obtain the binding constant values. The work is aimed to prove that the DSC experiments can be a source of reliable values of the binding constants and information on the stoichiometry of drug-albumin binding. DSC thermograms of bovine serum albumin denaturation in the presence of several drugs with different affinity and stoichiometry of binding to albumin: naproxen, warfarin, ibuprofen, and isoniazid were recorded. The dependences of the denaturation peak maximum temperature and area on the molar drug/protein ratio, which varied from 0 to 100, were considered. With the help of numerical modeling of the DSC curves, these dependences were predicted using the binding parameters determined in independent experiments and a simple two-state model of denaturation. The DSC data at relatively small concentrations of ligands are in good agreement with the calculation results. The deviations from the model predictions at high ligand concentrations in the cases of naproxen and ibuprofen indicate that albumin is able to bind several additional molecules of these drugs with its low-affinity sites. The fit was improved by using a sequential binding model with two binding constants K1=1.0×107 and K2=1.0×104 for naproxen and a cooperative binding model for ibuprofen. The stoichiometry of drug-albumin complexes fully saturated with drug ligand was calculated from the dependence of the denaturation temperature on the drug concentration. In the case of isoniazid, DSC thermograms indicated very weak binding to albumin.

Mobility of Lewis acids within the secondary coordination sphere: toward a model for cooperative substrate binding.

Distance dependence of appended Lewis acids in N2H4 binding and deprotonation was evaluated within a series of zinc complexes. Variation of spacer-length to a tethered trialkylborane Lewis acid revealed distinct preferences for binding and stabilization of the resulting deprotonated N2H3- unit.

Mechanism of allosteric activation of human mRNA cap methyltransferase (RNMT) by RAM: insights from accelerated molecular dynamics simulations.

The RNA guanine-N7 methyltransferase (RNMT) in complex with RNMT-activating miniprotein (RAM) catalyses the formation of a N7-methylated guanosine cap structure on the 5′ end of nascent RNA polymerase II transcripts. The mRNA cap protects the primary transcript from exonucleases and recruits cap-binding complexes that mediate RNA processing, export and translation. By using microsecond standard and accelerated molecular dynamics simulations, we provide for the first time a detailed molecular mechanism of allosteric regulation of RNMT by RAM. We show that RAM selects the RNMT active site conformations that are optimal for binding of substrates (AdoMet and the cap), thus enhancing their affinity. Furthermore, our results strongly suggest the likely scenario in which the cap binding promotes the subsequent AdoMet binding, consistent with the previously suggested cooperative binding model. By employing the network community analyses, we revealed the underlying long-range allosteric networks and paths that are crucial for allosteric regulation by RAM. Our findings complement and explain previous experimental data on RNMT activity. Moreover, this study provides the most complete description of the cap and AdoMet binding poses and interactions within the enzyme’s active site. This information is critical for the drug discovery efforts that consider RNMT as a promising anti-cancer target.

Cooperative Binding of Sodium Dodecyl Sulfate with Chitosan in Water–Alcohol Mixtures

The formation of polymer-colloid complexes of chitosan and anionic surfactant—sodium dodecyl sulfate—in water–alcohol mixtures is studied by potentiometry using ion-selective electrodes. The influence of the nature and content of organic cosolvent (methanol, ethanol, and 2-propanol) on the parameters of binding of surfactant with chitosan and the stability of complexes is discussed. The obtained data are analyzed in terms of the model of cooperative binding and pseudophase model. It is shown that addition of 10–20 vol % of ethanol and 2-propanol to aqueous solution intensifies cooperative binding of sodium dodecyl sulfate with chitosan, which leads to the decrease in the critical association concentration and increase in the parameters of cooperativity and stability of complexes.

A kinetic account for amphetamine-induced monoamine release.

The plasmalemmal monoamine transporters for dopamine, norepinephrine, and serotonin (SERT) are targets for amphetamines. In vivo, amphetamines elicit most, if not all, of their actions by triggering monoamine efflux. This is thought to be accomplished by an amphetamine-induced switch from the forward-transport to the substrate-exchange mode. The mechanism underlying this switch has remained elusive; available kinetic models posit that substrates and cosubstrate Na+ ions bind either in a random or in a sequential order. Neither can account for all reported experimental observations. We used electrophysiological recordings to interrogate crucial conformational transitions associated with the binding of five different substrates (serotonin, para-chloroamphetamine, and the high-affinity naphthyl-propan-amines PAL-287, PAL-1045, and PAL-1046) to human SERT expressed in HEK293 cells; specifically, we determined the relaxation kinetics of SERT from a substrate-loaded to a substrate-free state at various intracellular and extracellular Na+ concentrations. These rates and their dependence on intracellular and extracellular Na+ concentrations differed considerably between substrates. We also examined the effect of K+ on substrate affinity and found that K+ enhanced substrate dissociation. A kinetic model was developed, which allowed for random, but cooperative, binding of substrate and Na+ (or K+). The synthetic data generated by this model recapitulated the experimental observations. More importantly, the cooperative binding model accounted for the releasing action of amphetamines without any digression from alternating access. To the best of our knowledge, this model is the first to provide a mechanistic framework for amphetamine-induced monoamine release and to account for the findings that some substrates are less efficacious than others in promoting the substrate-exchange mode.

Pharmacodynamics of Interaction between Propoxazepam and a GABA-Benzodiazepine Receptor-Ionofor Complex

Effects of a propyloxy derivative of 1.4-benzodiazepine, propoxazepam, on GABA-benzodiazepine receptor complexes (GABA RCs) were examined in vitro and in vivo. The parameters of propoxazepam binding to synaptosomes from the rat brain were estimated in vitro. The Ki constant for inhibition of [3H] flumazenil binding by this agent was 3.5 ± 0.3 nM, on average. Considering the value of the GABA shift (1.9), propoxazepam can be considered as GABA-RC full agonist. On the model of picrotoxin-induced seizures in vivo, the propoxazepam average effective dose was estimated as 4.1 ± ± 0.21 mmol/kg. It was found that the parameters of myoclonic components (latent period of the onset of myoclonic seizures and their number), as well as death time of the tested animals, characterize adequately an anticonvulsant action of propoxazepam of picrotoxin-induced seizures in mice. Competitive interaction with picrotoxin is the possible mechanism of these effects at the level of GABAARCs. Significant deviations from a competitive model of monomolecular and cooperative binding of the agent at the receptor level have been found.

Effects of methyl substitution on DNA binding enthalpies of enantiopure Ru(phenanthroline)2dipyridophenazine2+ complexes.

Isothermal titration calorimetry (ITC) has been utilized to investigate the effect of methyl substituents on the intercalating dppz ligand of the enantiomers of the parent complex Ru(phen)2dppz2+ (phen = 1,10-phenanthroline; dppz = dipyrido[3,2-a:2',3'-c]phenazine) on DNA binding thermodynamics. The methylated complexes (10-methyl-dppz and 11,12-dimethyl-dppz) have large, concentration-dependent, positive heats of dilution, and a strong endothermic background is also apparent in the ITC-profiles from titration of methylated complexes into poly(dAdT)2, which make direct comparison between complexes difficult. By augmenting a simple cooperative binding model with one equilibrium for complex self-aggregation in solution and one equilibrium for complex aggregation on saturated DNA, it was possible to find an excellent global fit to the experimental data with DNA affinity parameters restricted to be equal for all Δ-enantiomers as well as for all Λ-enantiomers. In general, enthalpic differences, compared to the unsubstituted complex, were small and less than 4 kJ mol-1, except for the heat of intercalation of Δ-10-methyl-dppz (-11,6 kJ mol-1) and Λ-11,12-dimethyl-dppz (+4.3 kJ mol-1).

Population Pharmacokinetic and Pharmacodynamic Modeling of Etelcalcetide in Patients with Chronic Kidney Disease and Secondary Hyperparathyroidism Receiving Hemodialysis.

Etelcalcetide is a novel calcimimetic that binds and activates calcium-sensing receptors (CaSRs) for the treatment of secondary hyperparathyroidism (SHPT). To assess titrated dosing regimens, population pharmacokinetic (PK) and PK/pharmacodynamic (PKPD) modeling of etelcalcetide was performed using NONMEM 7.2. In this analysis, plasma etelcalcetide, serum parathyroid hormone (PTH) and calcium (Ca) concentration-time data were collected from five phase I, II, and III clinical trials following single or multiple intravenous doses of etelcalcetide ranging from 2.5 to 60mg. A semi-mechanistic model was used to describe the relationship between etelcalcetide, PTH, and Ca. This model included the role of PTH in Ca regulation, the feedback of Ca onto PTH production via the CaSR, and the activity of etelcalcetide plasma levels in increasing the sensitivity of the CaSR to Ca via the cooperative binding model. The impact of relevant covariates was evaluated by stepwise forward/backward selection. Model evaluation was based on standard goodness-of-fit plots and prediction-corrected visual predictive checks (pcVPCs). Simulation was conducted to evaluate titrated dosing regimens. The time courses of etelcalcetide, PTH, and Ca were well-described by the model. The clearance and central volume of distribution (Vc) of etelcalcetide were 0.472L/h and 49.9L, respectively, while estimates of the turnover half-lives of PTH and Ca were 0.36 and 23h, respectively. The extent of interindividual variability in model parameters was low to moderate (6-67%), and no covariates were identified as significant predictors of PK and PD variability. pcVPCs confirmed the predictive ability of the model. The current analysis confirms the putative mechanism of action of etelcalcetide as an allosteric activator of CaSR. Simulations showed that dose titration of etelcalcetide, rather than fixed dose, is needed to effectively decrease the PTH level in patient populations.

Modeling of flux, binding and substitution of urea molecules in the urea transporter dvUT

Urea transporters (UTs) are transmembrane proteins that transport urea molecules across cell membranes and play a crucial role in urea excretion and water balance. Modeling the functional characteristics of UTs helps us understand how their structures accomplish the functions at the atomic level, and facilitates future therapeutic design targeting the UTs. This study was based on the crystal structure of Desulfovibrio vulgaris urea transporter (dvUT). To model the binding behavior of urea molecules in dvUT, we constructed a cooperative binding model. To model the substitution of urea by the urea analogue N,N′-dimethylurea (DMU) in dvUT, we calculated the occupation probability of DMU along the urea pore and the ratio of the occupation probabilities of DMU at the external (Sext) and internal (Sint) binding sites, and we established the mutual substitution rule for binding and substitution of urea and DMU. Based on these calculations and modelings, together with the use of the Monte Carlo (MC) method, we further modeled the urea flux in dvUT, equilibrium urea binding to dvUT, and the substitution of urea by DMU in the dvUT. Our modeling results are in good agreement with the existing experimental functional data. Furthermore, the modelings have discovered the microscopic process and mechanisms of those functional characteristics. The methods and the results would help our future understanding of the underlying mechanisms of the diseases associated with impaired UT functions and rational drug design for the treatment of these diseases.

From conformational spread to allosteric and cooperative models of E. coli flagellar motor

Escherichia coli swims using flagella activated by rotary motors. The direction of rotation of the motors is indirectly regulated by the binding of a single messenger protein. The conformational spread model has been shown to accurately describe the equilibrium properties as well as the dynamics of the flagellar motor. In this paper we study this model from an analytic point of view. By exploiting the separation of timescales observed in experiments, we show how to reduce the conformational spread model to a coarse-grained, cooperative binding model. We show that this simplified model reproduces very well the dynamics of the motor switch.

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding

Mutations in hemoglobin can cause a wide range of phenotypic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin α chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) → Leu mutation in α1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (∼16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the α H58L replacement. Mutant α subunits containing Leu-58(E7) autoxidize ∼8 times and lose hemin ∼200 times more rapidly than native α subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the α H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O2, promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant α subunit has an ∼80,000-fold higher affinity for CO than O2, causing it to rapidly take up and retain carbon monoxide, which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.

Co-operative and Hierarchical Binding of c-FLIP and Caspase-8: A Unified Model Defines How c-FLIP Isoforms Differentially Control Cell Fate

SummaryThe death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIPL only) or anti-apoptotic (c-FLIPL/c-FLIPS) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIPL/S binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate.

The origin of cooperative solubilisation by hydrotropes.

The signature of hydrotropic solubilisation is the sigmoidal solubility curve; when plotted against hydrotrope concentration, solubility increases suddenly after the minimum hydrotrope concentration (MHC), and reaches a plateau at higher hydrotrope concentrations. This sigmoidal curve is characteristic of cooperative phenomena, yet the true molecular basis of hydrotropic cooperativity has long remained unclear. Here we develop a theory, derived from the first principles of statistical thermodynamics using partially-open ensembles, to identify the origin of hydrophobic cooperativity. Our theory bears a close resemblance to the cooperative binding model used for protein-ligand binding. The cause of cooperativity is the enhancement of the hydrotrope m-body interaction induced by the presence of the solute; m can be estimated from the experimental solubility data.

Evidence for cooperative tandem binding of hnRNP C RRMs in mRNA processing.

The human hnRNP C is a ubiquitous cellular protein involved in mRNA maturation. Recently, we have shown that this protein specifically recognizes uridine (U) pentamers through its single RNA recognition motif (RRM). However, a large fraction of natural RNA targets of hnRNP C consists of much longer contiguous uridine stretches. To understand how these extended sites are recognized, we studied the binding of the RRM to U-tracts of 8–11 bases. In vivo investigation of internal translation activation of unr (upstream of N-ras) mRNA indicates that the conservation of the entire hnRNP C binding site, UC(U)8, is required for hnRNP C-dependent IRES activation. The assays further suggest a synergistic interplay between hnRNP C monomers, dependent on the protein's ability to oligomerize. In vitro spectroscopic and thermodynamic analyses show that isolated RRMs bind to (U)11 oligomers as dimers. Structural modeling of a ternary double-RRM/RNA complex indicates additionally that two RRM copies can be accommodated on the canonical sequence UC(U)8. The proposed tandem RRM binding is in very good agreement with the transcriptome-wide recognition of extended U-tracts by full-length hnRNP C, which displays a cross-linking pattern consistent with a positively cooperative RRM dimer binding model.

Pi-pi Stacking Mediated Cooperative Mechanism for Human Cytochrome P450 3A4.

Human Cytochrome P450 3A4 (CYP3A4) is an important member of the cytochrome P450 superfamily with responsibility for metabolizing ~50% of clinical drugs. Experimental evidence showed that CYP3A4 can adopt multiple substrates in its active site to form a cooperative binding model, accelerating substrate metabolism efficiency. In the current study, we constructed both normal and cooperative binding models of human CYP3A4 with antifungal drug ketoconazoles (KLN). Molecular dynamics simulation and free energy calculation were then carried out to study the cooperative binding mechanism. Our simulation showed that the second KLN in the cooperative binding model had a positive impact on the first one binding in the active site by two significant pi-pi stacking interactions. The first one was formed by Phe215, functioning to position the first KLN in a favorable orientation in the active site for further metabolism reactions. The second one was contributed by Phe304. This pi-pi stacking was enhanced in the cooperative binding model by the parallel conformation between the aromatic rings in Phe304 and the dioxolan moiety of the first KLN. These findings can provide an atomic insight into the cooperative binding in CYP3A4, revealing a novel pi-pi stacking mechanism for drug-drug interactions.

CPSF30, a Novel Non-Classical Zinc Finger Protein that Utilizes Iron & Zinc Coordination for RNA Recognition

Zinc finger (ZF) proteins are eukaryotic proteins that are involved in transcriptional and translational regulation. ZFs utilize cysteine and histidine residues to coordinate zinc ions to fold and function properly. There are fourteen different classes of zinc finger proteins, which are classified based upon the ligands involved in zinc coordination, the ZF domain folds, and the proteins’ biological targets. Cleavage and Polyadenylation Specificity Factor 30 (CPSF30) is a ‘non-classical’ ZF protein that contains multiple Cys3His zinc binding domains and is involved in pre-mRNA regulation. CPSF30 contains 5 Cys3His domains and a Cys2HisCys zinc knuckle domain. A construct of CPSF30 construct containing only CPSF30's five Cys3His domains, denoted as CPSF305FE was prepared. This protein is predicted to have five zinc ions; however, UV-visible and ICP-MS analysis of the isolated construct revealed that CPSF305FE contains iron in addition to zinc in a 1:4:1 Fe:Zn:CPSF305FE stoichiometetry. XAS analysis identified the iron site as a 2Fe-2S cluster. CPSF30 is predicted to recognize AU-rich RNA sequences. To test this hypothesis, RNA binding studies using both electrophoretic mobility shift assays (EMSA) and fluorescence anisotropy (FA) of CPSF305FE with α-synuclein pre-mRNA (which contains an AU-rich sequence) along with a series of mutated RNA sequences were performed. These studies revealed that CPSF30 binds to α-synuclein pre-RNA with nanomolar affinity and requires the AU-rich sequence for binding. The binding interaction was best fit to a cooperative binding model, suggesting that the CPF30/RNA binding event is cooperative.

Adsorption and Vibrational Study of Folic Acid on Gold Nanopillar Structures Using Surface-Enhanced Raman Scattering Spectroscopy

This paper presents a study of adsorption and vibrational features of folic acid, using surface-enhanced Raman scattering (SERS). A gold-capped silicon nanopillar (Au NP) with a height of 600 nm and a width of 120 nm was utilized to study the vibrational features of FA molecules adsorbed on the nanopillars within the high electromagnetic field areas. The adsorption behaviour of folic acid and the band assignment of the main vibrations together with the optimized geometry of folic acid and folic acid in the presence of a cluster of 10 gold atoms were assessed using the density functional theory (B3LYP(6–31G(d))) and the scalar relativistic effective core potential with a double-zeta basis set (LANL2DZ). The vibrations obtained from the solid-state folic acid and the folic acid on a gold cluster were in accordance with those observed experimentally. The analysis of the main vibrations indicated that the interaction of folic acid with the Au NP occurred primarily through the nitrogen atoms, from their pteridine ring. Finally, the obtained adsorption isotherm for folic acid was deduced from the analysis of the SERS spectra and it followed a negative cooperative binding model.