Rates Of Ligand Binding Research Articles

Adrenodoxin allosterically alters human cytochrome P450 11B enzymes to accelerate substrate binding and decelerate release.

Two human mitochondrial membrane CYP11B enzymes play a pivotal role in steroidogenesis. CYP11B1 generates the major glucocorticoid cortisol, while CYP11B2 catalysis yields the primary mineralocorticoid aldosterone. Catalysis by both requires electron delivery by a soluble iron-sulfur adrenodoxin redox partner. However recent studies have shown that adrenodoxin/CYP11B interaction alone allosterically increases substrate and inhibitor affinity as exhibited by decreased dissociation constant (K d) values. The current study moves beyond such equilibrium studies, by defining adrenodoxin effects on the rates of P450 ligand binding and release separately. Stopped-flow data clearly demonstrate that adrenodoxin interaction with the P450 proximal surfaces increases ligand binding in both P450 CYP11B active sites by increasing the on rate constant and decreasing the off rate constant. As substrate entry and exit from the sequestered P450 active site requires conformational changes on the distal side of the P450 enzyme, a likely explanation is that adrenodoxin binding allosterically modulates CYP11B conformational changes. The 93% identical CYP11B enzymes can bind and hydroxylate each other's native substrates differing only by a hydroxyl. However, CYP11B1 exhibits monophasic substrate binding and CYP11B2 biphasic substrate binding, even when the substrates are swapped. This indicates that small differences in amino acid sequence between human CYP11B1 and CYP11B2 enzymes are more functionally important in ligand binding and could suggest avenues for more selective inhibition of these drug targets. Both protein/protein interactions and protein/substrate interactions are most likely to act by modulating CYP11B conformational dynamics.

PH Regulates Ligand Binding to an Enzyme Active Site by Modulating Intermediate Populations.

Understanding the mechanism of ligands binding to their protein targets and the influence of various factors governing the binding thermodynamics is essential for rational drug design. The solution pH is one of the critical factors that can influence ligand binding to a protein cavity, especially in enzymes whose function is sensitive to the pH. Using computer simulations, we studied the pH effect on the binding of a guanidinium ion (Gdm+) to the active site of hen egg-white lysozyme (HEWL). HEWL serves as a model system for enzymes with two acidic residues in the active site and ligands with Gdm+ moieties, which can bind to the active sites of such enzymes and are present in several approved drugs treating various disorders. The computed free energy surface (FES) shows that Gdm+ binds to the HEWL active site using two dominant binding pathways populating multiple intermediates. We show that the residues close to the active site that can anchor the ligand could play a critical role in ligand binding. Using a Markov state model, we quantified the lifetimes and kinetic pathways connecting the different states in the FES. The protonation and deprotonation of the acidic residues in the active site in response to the pH change strongly influence the Gdm+ binding. There is a sharp jump in the ligand-binding rate constant when the pH approaches the largest pKa of the acidic residue present in the active site. The simulations reveal that, at most, three Gdm+ can bind at the active site, with the Gdm+ bound in the cavity of the active site acting as a scaffold for the other two Gdm+ ions binding. These results can aid in providing greater insights into designing novel molecules containing Gdm+ moieties that can have high binding affinities to inhibit the function of enzymes with acidic residues in their active site.

Estimation of binding rates and affinities from multiensemble Markov models and ligand decoupling.

Accurate and efficient simulation of the thermodynamics and kinetics of protein-ligand interactions is crucial for computational drug discovery. Multiensemble Markov Model (MEMM) estimators can provide estimates of both binding rates and affinities from collections of short trajectories but have not been systematically explored for situations when a ligand is decoupled through scaling of non-bonded interactions. In this work, we compare the performance of two MEMM approaches for estimating ligand binding affinities and rates: (1) the transition-based reweighting analysis method (TRAM) and (2) a Maximum Caliber (MaxCal) based method. As a test system, we construct a small host-guest system where the ligand is a single uncharged Lennard-Jones (LJ) particle, and the receptor is an 11-particle icosahedral pocket made from the same atom type. To realistically mimic a protein-ligand binding system, the LJ ϵ parameter was tuned, and the system was placed in a periodic box with 860 TIP3P water molecules. A benchmark was performed using over 80 µs of unbiased simulation, and an 18-state Markov state model was used to estimate reference binding affinities and rates. We then tested the performance of TRAM and MaxCal when challenged with limited data. Both TRAM and MaxCal approaches perform better than conventional Markov state models, with TRAM showing better convergence and accuracy. We find that subsampling of trajectories to remove time correlation improves the accuracy of both TRAM and MaxCal and that in most cases, only a single biased ensemble to enhance sampled transitions is required to make accurate estimates.

Dissecting the Protein Dynamics Coupled Ligand Binding with Kinetic Models and Single-Molecule FRET.

Protein-ligand interactions are crucial to many biological processes. Ligand binding and dissociation are the basic steps that allow proteins to function. Protein conformational dynamics have been shown to play important roles in ligand binding and dissociation. However, it is challenging to determine the ligand binding kinetics of dynamic proteins. Here, we undertook comprehensive single-molecule FRET (smFRET) measurements and kinetic model analysis to characterize the conformational dynamics coupled ligand binding of glutamine-binding protein (GlnBP). We showed that hinge and T118A mutations of GlnBP affect its conformational dynamics as well as the ligand binding affinity. Based on smFRET measurements, the kinetic model of ligand-GlnBP interactions was constructed. Using experimentally measured parameters, we solved the rate equations of the model and obtained the undetectable parameters of the model which allowed us to understand the ligand binding kinetics fully. Our results demonstrate that modulation of the conformational dynamics of GlnBP affects the ligand binding and dissociation rates. This study provides insights into the binding kinetics of ligands, which are related to the protein function itself.

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

Unusually Fast bis-Histidyl Coordination in a Plant Hemoglobin.

The recently identified nonsymbiotic hemoglobin gene MtGlb1-2 of the legume Medicago truncatula possesses unique properties as it generates four alternative splice forms encoding proteins with one or two heme domains. Here we investigate the ligand binding kinetics of MtGlb1-2.1 and MtGlb1-2.4, bearing two hemes and one heme, respectively. Unexpectedly, the overall time-course of ligand rebinding was unusually fast. Thus, we complemented nanosecond laser flash photolysis kinetics with data collected with a hybrid femtosecond–nanosecond pump–probe setup. Most photodissociated ligands are rebound geminately within a few nanoseconds, which leads to rates of the bimolecular rebinding to pentacoordinate species in the 108 M−1s−1 range. Binding of the distal histidine to the heme competes with CO rebinding with extremely high rates (kh ~ 105 s−1). Histidine dissociation from the heme occurs with comparable rates, thus resulting in moderate equilibrium binding constants (KH ~ 1). The rate constants for ligation and deligation of distal histidine to the heme are the highest reported for any plant or vertebrate globin. The combination of microscopic rates results in unusually high overall ligand binding rate constants, a fact that contributes to explaining at the mechanistic level the extremely high reactivity of these proteins toward the physiological ligands oxygen, nitric oxide and nitrite.

Requirements for efficient ligand-gated co-transcriptional switching in designed variants of the B. subtilis pbuE adenine-responsive riboswitch in E. coli.

Riboswitches, generally located in the 5’-leader of bacterial mRNAs, direct expression via a small molecule-dependent structural switch that informs the transcriptional or translational machinery. While the structure and function of riboswitch effector-binding (aptamer) domains have been intensely studied, only recently have the requirements for efficient linkage between small molecule binding and the structural switch in the cellular and co-transcriptional context begun to be actively explored. To address this aspect of riboswitch function, we have performed a structure-guided mutagenic analysis of the B. subtilis pbuE adenine-responsive riboswitch, one of the simplest riboswitches that employs a strand displacement switching mechanism to regulate transcription. Using a cell-based fluorescent protein reporter assay to assess ligand-dependent regulatory activity in E. coli, these studies revealed previously unrecognized features of the riboswitch. Within the aptamer domain, local and long-range conformational dynamics influenced by sequences within helices have a significant effect upon efficient regulatory switching. Sequence features of the expression platform including the pre-aptamer leader sequence, a toehold helix and an RNA polymerase pause site all serve to promote strong ligand-dependent regulation. By optimizing these features, we were able to improve the performance of the B. subtilis pbuE riboswitch in E. coli from 5.6-fold induction of reporter gene expression by the wild type riboswitch to over 120-fold in the top performing designed variant. Together, these data point to sequence and structural features distributed throughout the riboswitch required to strike a balance between rates of ligand binding, transcription and secondary structural switching via a strand exchange mechanism and yield new insights into the design of artificial riboswitches.

Multiscale Simulations Examining Glycan Shield Effects on Drug Binding to Influenza Neuraminidase

Influenza neuraminidase is an important drug target. Glycans are present on neuraminidase and are generally considered to inhibit antibody binding via their glycan shield. In this work, we studied the effect of glycans on the binding kinetics of antiviral drugs to the influenza neuraminidase. We created all-atom in silico systems of influenza neuraminidase with experimentally derived glycoprofiles consisting of four systems with different glycan conformations and one system without glycans. Using Brownian dynamics simulations, we observe a two- to eightfold decrease in the rate of ligand binding to the primary binding site of neuraminidase due to the presence of glycans. These glycans are capable of covering much of the surface area of neuraminidase, and the ligand binding inhibition is derived from glycans sterically occluding the primary binding site on a neighboring monomer. Our work also indicates that drugs preferentially bind to the primary binding site (i.e., the active site) over the secondary binding site, and we propose a binding mechanism illustrating this. These results help illuminate the complex interplay between glycans and ligand binding on the influenza membrane protein neuraminidase.

The Interaction of Curcumin and Rosmarinic Acid with Non‐Amyloid‐Component Domain of Alpha‐Synuclein: A Molecular Dynamics Study

AbstractOne notable phenomenon in the Parkinson's disease (PD) treatment is the identification of inhibitors of the self‐assembly of Alpha‐Synuclein (AS) protein. Here, the interactions of two herbal compounds of Curcumin (CUR) and Rosmarinic‐acid (RA) with the NAC domain of AS were investigated by the molecular dynamics (MD) simulation method. More specifically, the purpose of this study was to investigate the molecular aspects and the inhibition mechanism of the considered molecules, as well as evaluating their effectiveness in preventing the AS aggregation process. The trajectories of MD simulations were used to evaluate the structural properties of the simulated systems and to infer qualitative and quantitative facts regarding the interactions between the considered ligands and the NAC domains. In addition to the MD studies, the energetics of the ligand‐NAC domain interactions were studied by MM‐PBSA method. The free energy of binding and various contributions to it, as modeled in the MM‐PBSA method, were evaluated and analyzed. The results were also analyzed in terms of the rate and strength of ligand binding to the domains. These analyses showed that RA could bind to the NAC domain faster, as compared to CUR, while the latter binds to the domain more strongly than the former does. However, overall, the results imply that both CUR and RA could effectively bind to NAC domains; so, they could be considered as candidates to reduce the aggregation of AS or as potential drugs for PD.

Competition between Ligand Binding and Transcription Rate Modulates Riboswitch-Mediated Regulation of Transcription

Riboswitches are genetic elements mostly found in the 5’ untranslated region of bacterial mRNAs, which act to control gene expression through small molecule-mediated conformational changes. In particular, the fluoride riboswitch, found in Bacillus cereus, regulates transcription upon binding of the small negatively charged ion, fluoride. Binding of the ligand to the riboswitch prevents the formation of a terminator hairpin, which in turn promotes active transcription of the downstream crcBgene. Previous studies have determined the structure of the riboswitch alone and in its transcriptional elongation complex (EC), but little is known about the impact of co-transcriptional folding of the riboswitch on termination of transcription. Using in vitro transcription assays, we found that fluoride and the transcription rate each modulate the termination efficiency in dependence of the riboswitch while competing with each other. Interestingly, we found that a downstream transcriptional pause of RNAP is modulated by the presence of fluoride, leading to efficient termination when the ligand is absent. Here, we used Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to probe the conformational dynamics of the terminator stem as a function of ligand binding and transcription rate. Interestingly, the terminator stem in a paused corresponding nascent run-off transcript is more stable, showing that the presence of RNAP negatively impacts this particular RNA structure at the pause site. Finally, we observed that a fast transcription rate negatively impacts the terminator stem formation showing the profound impact of the transcription process into riboswitch-mediated regulation of gene expression. These results will be presented in light of future works using transcription factors that affect the transcription rate. This work provides unprecedented insights into the powerful mechanism by which RNA structure can modulate the function of RNA polymerase and vice versa.

Role of conserved arginine in the heme distal site of HutZ from Vibrio cholerae in the heme degradation reaction

HutZ from Vibrio cholerae is a dimeric enzyme that catalyzes degradation of heme. The highly conserved Arg92 residue in the HutZ family is proposed to interact with an iron-bound water molecule in the distal heme pocket. To clarify the specific role of Arg92 in the heme degradation reaction, the residue was substituted with alanine, leucine, histidine or lysine to modulate electrostatic interactions with iron-bound ligand. All four Arg92 mutants reacted with hydrogen peroxide to form verdoheme, a prominent intermediate in the heme degradation process. However, when ascorbic acid was used as an electron source, iron was not released even at pH 6.0 despite a decrease in the Soret band, indicating that non-enzymatic heme degradation occurred. Comparison of the rates of heme reduction, ligand binding and verdoheme formation suggested that proton transfer to the reduced oxyferrous heme, a potential rate-limiting step of heme degradation in HutZ, is hampered by mutation. In our previous study, we found that the increase in the distance between heme and Trp109 from 16 to 18 Å upon lowering the pH from 8.0 to 6.0 leads to activation of ascorbic acid-assisted heme degradation by HutZ. The distance in Arg92 mutants was >19 Å at pH 6.0, suggesting that subunit-subunit interactions at this pH are not suitable for heme degradation, similar to Asp132 and His63 mutants. These results suggest that interactions of Arg92 with heme-bound ligand induce alterations in the distance between subunits, which plays a key role in controlling the heme degradation activity of HutZ.

Cover Feature: Ligand Binding Rate Constants in Heme Proteins Using Markov State Models and Molecular Dynamics Simulations (ChemPhysChem 19/2019)

Cover Feature: Ligand Binding Rate Constants in Heme Proteins Using Markov State Models and Molecular Dynamics Simulations (ChemPhysChem 19/2019)

Ligand Binding Rate Constants in Heme Proteins Using Markov State Models and Molecular Dynamics Simulations.

Computer simulation studies of the molecular basis for ligand migration in proteins allow the description of key events such as the transition between docking sites, displacement of existing ligands and solvent molecules, and open/closure of specific "gates", among others. In heme proteins, ligand migration from the solvent to the active site preludes the binding to the heme iron and triggers different functions. In this work, molecular dynamics simulations, a Markov State Model of migration and empirical kinetic equations are combined to study the migration of O2 and NO in two truncated hemoglobins of Mycobacterium tuberculosis (Mt-TrHbN and Mt-TrHbO). For Mt-TrHbN, we show that the difference in the association constant in the oxy and deoxy states relies mainly in the displacement of water molecules anchored in the distal cavity in the deoxy form. The results here provide a valuable approach to study ligand migration in globins.

Predicting ligand binding affinity using on- and off-rates for the SAMPL6 SAMPLing challenge.

Interest in ligand binding kinetics has been growing rapidly, as it is being discovered in more and more systems that ligand residence time is the crucial factor governing drug efficacy. Many enhanced sampling methods have been developed with the goal of predicting ligand binding rates ([Formula: see text]) and/or ligand unbinding rates ([Formula: see text]) through explicit simulation of ligand binding pathways, and these methods work by very different mechanisms. Although there is not yet a blind challenge for ligand binding kinetics, here we take advantage of experimental measurements and rigorously computed benchmarks to compare estimates of [Formula: see text] calculated as the ratio of two rates: [Formula: see text]. These rates were determined using a new enhanced sampling method based on the weighted ensemble framework that we call "REVO": Reweighting of Ensembles by Variance Optimization. This is a further development of the WExplore enhanced sampling method, in which trajectory cloning and merging steps are guided not by the definition of sampling regions, but by maximizing trajectory variance. Here we obtain estimates of [Formula: see text] and [Formula: see text] that are consistent across multiple simulations, with an average log10-scale standard deviation of 0.28 for on-rates and 0.56 for off-rates, which is well within an order of magnitude and far better than previously observed for previous applications of the WExplore algorithm. Our rank ordering of the three host-guest pairs agrees with the reference calculations, however our predicted [Formula: see text] values were systematically lower than the reference by an average of 4.2kcal/mol. Using tree network visualizations of the trajectories in the REVO algorithm, and conformation space networks for each system, we analyze the results of our sampling, and hypothesize sources of discrepancy between our [Formula: see text] values and the reference. We also motivate the direct inclusion of [Formula: see text] and [Formula: see text] challenges in future iterations of SAMPL, to further develop the field of ligand binding kinetics prediction and modeling.

Electrostatic Tuning of the Ligand Binding Mechanism by Glu27 in Nitrophorin 7

Nitrophorins (NP) 1–7 are NO-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. The isoform NP7 displays peculiar properties, such as an abnormally high isoelectric point, the ability to bind negatively charged membranes, and a strong pH sensitivity of NO affinity. A unique trait of NP7 is the presence of Glu in position 27, which is occupied by Val in other NPs. Glu27 appears to be important for tuning the heme properties, but its influence on the pH-dependent NO release mechanism, which is assisted by a conformational change in the AB loop, remains unexplored. Here, in order to gain insight into the functional role of Glu27, we examine the effect of Glu27 → Val and Glu27 → Gln mutations on the ligand binding kinetics using CO as a model. The results reveal that annihilation of the negative charge of Glu27 upon mutation reduces the pH sensitivity of the ligand binding rate, a process that in turn depends on the ionization of Asp32. We propose that Glu27 exerts a through-space electrostatic action on Asp32, which shifts the pKa of the latter amino acid towards more acidic values thus reducing the pH sensitivity of the transition between open and closed states.

Biophysical Interactions of Direct AMPK Activators.

Protein-ligand interactions can be evaluated by a number of different biophysical methods. Here we describe some of the experimental methods that we have used to generate AMPK protein reagents and characterize its interactions with direct synthetic activators. Recombinant heterotrimeric AMPK complexes were generated using standard molecular biology methods by expression either in insect cells via infection with three different viruses or more routinely in Escherichia coli with a tricistronic expression vector. Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry was used to probe protein conformational changes and potential binding sites of activators on AMPK. X-ray crystallographic studies were carried out on crystals of AMPK with bound ligands to reveal detailed molecular interactions formed by AMPK activators at near-atomic resolution. In order to gain insights into the mechanism of enzyme activation and to probe the effects of AMPK activators on kinetic parameters such as Michaelis-Menten constant (K m ) or maximal reaction velocity (V max), we performed classical enzyme kinetic studies using radioactive 33P-ATP-based filter assay. Equilibrium dissociation constants (K D ) and on and off rates of ligand binding were obtained by application of surface plasmon resonance (SPR) technique.

Preparation and in vivo study of liposomes mediated by glycyrrhetinic acid derivative ligand 18-GA-Gly

Based on the research of active liver targeting liposomes mediated by glycyrrhetinic acid ligand at home and abroad, this paper focuses on the liver targeting effect of liposomes mediated with 18-GA-Gly, a kind of glycyrrhetinic acid ligand. salvianolic acid B(Sal B)-tanshinone ⅡA (TSN)liposomes mediated by 18-GA-Gly as well as the liposomes with unmodified ligands were prepared by film dispersion-high pressure homogenization method, and then the particle size, potential, encapsulation efficiency and ligand binding rate were detected. Plasma samples of the heart, liver, spleen, lung and kidney tissues were taken at different time points after tail vein injections. The contents of Sal B and TSN in each sample were determined with UPLC methods and the liver targeting effect of 18-GA-Gly ligands was evaluated. The results showed that the particle size, potential, encapsulation efficiency and ligand binding rate met the basic requirements; in vivo targeting investigation results showed no difference between GLY-TS-Lip group and TS-Lip group. The liposomes mediated by glycyrrhetinic acid derivative ligand 18-GA-Gly can increase the peak concentration of Sal B and TSN in liver, but showed no significant liver targeting effect.

Kinetics of Ligand Binding Through Advanced Computational Approaches: A Review.

Ligand residence times and binding rates have been found to be useful quantities to consider during drug design. The underlying structural and dynamic determinants of these kinetic quantities are difficult to discern. Driven by developments in computational hardware and simulation methodologies, molecular dynamics (MD) studies on full binding and unbinding pathways have emerged recently, showing these structural and dynamic determinants in atomic detail. However, the long timescales related to drug binding and release are still prohibitive to conventional MD simulation. Here we discuss a suite of enhanced sampling methods that have been applied to the study of full ligand binding or unbinding pathways, and reveal the kinetics of drug binding and/or release. We divide these sampling methods into three families (trajectory parallelization, metadynamics, and temperature- based methods), and discuss recent applications of each, as well as their basic theoretical underpinnings including how kinetic information is extracted. We then present an outlook for how the field could evolve, and how the rich variety of sampling methods discussed here can be leveraged in the future for computationally driven drug design.

Substrate Binding Primes Human Tryptophan 2,3-Dioxygenase for Ligand Binding.

The human heme enzyme tryptophan 2,3-dioxygenase (hTDO) catalyzes the insertion of dioxygen into its cognate substrate, l-tryptophan (l-Trp). Its active site structure is highly dynamic, and the mechanism of enzyme-substrate-ligand complex formation and the ensuing enzymatic reaction is not yet understood. Here we have studied complex formation in hTDO by using time-resolved optical and infrared spectroscopy with carbon monoxide (CO) as a ligand. We have observed that both substrate-free and substrate-bound hTDO coexist in two discrete conformations with greatly different ligand binding rates. In the fast rebinding hTDO conformation, there is facile ligand access to the heme iron, but it is greatly hindered in the slowly rebinding conformation. Spectroscopic evidence implicates active site solvation as playing a crucial role for the observed kinetic differences. Substrate binding shifts the conformational equilibrium markedly toward the fast species and thus primes the active site for subsequent ligand binding, ensuring that formation of the ternary complex occurs predominantly by first binding l-Trp and then the ligand. Consequently, the efficiency of catalysis is enhanced because O2 binding prior to substrate binding, resulting in nonproductive oxidation of the heme iron, is greatly suppressed.

Quantitative estimation of kinetic rates of ligand binding to heme proteins from molecular dynamics simulations

Quantitative estimation of kinetic rates of ligand binding to heme proteins from molecular dynamics simulations