Protein-protein association: investigation of factors influencing association rates by brownian dynamics simulations

The rate of protein-protein association limits the response time due to protein-protein interactions. The bimolecular association rate may be diffusion-controlled or influenced, and in such cases, Brownian dynamics simulations of protein-protein diffusional association may be used to compute association rates. Here, we report Brownian dynamics simulations of the diffusional association of five different protein-protein pairs: barnase and barstar, acetylcholinesterase and fasciculin-2, cytochrome c peroxidase and cytochrome c, the HyHEL-5 antibody and hen egg lysozyme (HEL), and the HyHEL-10 antibody and HEL. The same protocol was used to compute the diffusional association rates for all the protein pairs in order to assess, by comparison to experimentally measured rates, whether the association of these proteins can be explained solely on the basis of diffusional encounter. The simulation protocol is similar to those previously derived for simulation of the association of barnase and barstar, and of acetylcholinesterase and fasciculin-2; these produced results in excellent agreement with experimental data for these protein pairs, with changes in association rate due to mutations reproduced within the limits of expected computational and modeling errors. Here, we find that for all protein pairs, the effects of mutations can be well reproduced by the simulations, even though the degree of the electrostatic translational and orientational steering varies widely between the cases. However, the absolute values of association rates for the acetylcholinesterase: fasciculin-2 and HyHEL-10 antibody: HEL pairs are overestimated. Comparison of bound and unbound protein structures shows that this may be due to gating resulting from protein flexibility in some of the proteins. This may lower the association rates compared to their bimolecular diffusional encounter rates.     

Real time kinetic studies of the interaction between folded antisense and target RNAs using surface plasmon resonance

Antisense RNAs interact with their complementary target RNAs as folded structures. The formation of early binding intermediates is the most important step in determining the overall rates of stable complex formation in vitro and the efficiency of control in vivo. In the case of CopA and CopT (antisense/target RNA pair of plasmid R1), recent studies have identified a four-way junction structure as the major binding intermediate. Previously, the kinetics of antisense/target RNA interaction was studied by indirect methods. Here we have used surface plasmon resonance to follow the binding of CopI (a truncated variant of CopA) to CopT in real time. A protocol was developed that permitted the determination of association and dissociation rate constants for wild-type and mutant CopI-CopT pairs. The K(D)-values calculated from these rate constants were in good agreement with the results obtained by indirect methods. In comparison to earlier model studies of interactions between simple complementary nucleic acids, we observe a different temperature dependence for dissociation rate constants. This may be indicative of the complexity of the steps required for interacting folded RNAs; intramolecular structure competes with intermolecular helix progression during complex formation. The association rate constants were not significantly dependent on temperature. The analysis presented shows that the stability of a kissing complex is not the primary determinant of the rate of stable CopA/CopT complex formation.     

Switches of hydrogen bonds during ligand–protein association processes determine binding kinetics

Revealing the processes of ligand–protein associations deepens our understanding of molecular recognition and binding kinetics. Hydrogen bonds (H‐bonds) play a crucial role in optimizing ligand–protein interactions and ligand specificity. In addition to the formation of stable H‐bonds in the final bound state, the formation of transient H‐bonds during binding processes contributes binding kinetics that define a ligand as a fast or slow binder, which also affects drug action. However, the effect of forming the transient H‐bonds on the kinetic properties is little understood. Guided by results from coarse‐grained Brownian dynamics simulations, we used classical molecular dynamics simulations in an implicit solvent model and accelerated molecular dynamics simulations in explicit waters to show that the position and distribution of the H‐bond donor or acceptor of a drug result in switching intermolecular and intramolecular H‐bond pairs during ligand recognition processes. We studied two major types of HIV‐1 protease ligands: a fast binder, xk263, and a slow binder, ritonavir. The slow association rate in ritonavir can be attributed to increased flexibility of ritonavir, which yields multistep transitions and stepwise entering patterns and the formation and breaking of complex H‐bond pairs during the binding process. This model suggests the importance of conversions of spatiotemporal H‐bonds during the association of ligands and proteins, which helps in designing inhibitors with preferred binding kinetics. Copyright © 2014 John Wiley & Sons, Ltd.     

Computation of FRAP recovery times for linker histone – chromatin binding on the basis of Brownian dynamics simulations

BackgroundFluorescence recovery after photobleaching (FRAP) studies can provide kinetic information about proteins in cells. Single point mutations can significantly affect the binding kinetics of proteins and result in variations in the recovery half time (t₅₀) measured in FRAP experiments. FRAP measurements of linker histone (LH) proteins in the cell nucleus have previously been reported by Brown et al. (2006) and Lele et al. (2006).MethodsWe performed Brownian dynamics (BD) simulations of the diffusional association of the wild-type and 38 single or double point mutants of the globular domain of mouse linker histone H1.0 (gH1.0) to a nucleosome. From these simulations, we calculated the bimolecular association rate constant (k_on), the Gibbs binding free energy (ΔG) and the dissociation rate constant (k_off) related to formation of a diffusional encounter complex between the nucleosome and the gH1.0.ResultsWe used these parameters, after application of a correction factor to account for the effects of the crowded environment of the nucleus, to compute FRAP recovery times and curves that are in good agreement with previously published, experimentally measured FRAP recovery time courses.ConclusionsOur computational analysis suggests that BD simulations can be used to predict the relative effects of single point mutations on FRAP recovery times related to protein binding.General SignificanceBD simulations assist in providing a detailed molecular level interpretation of FRAP data.     

Dynamics of flagellum- and pilus-mediated association of Pseudomonas aeruginosa with contact lens surfaces

Flagella and pili are appendages that modulate attachment of Pseudomonas aeruginosa to solid surfaces. However, previous studies have mostly reported absolute attachment. Neither the dynamic roles of these appendages in surface association nor those of attachment phenotypes have been quantified. We used video microscopy to address this issue. Unworn, sterile, soft contact lenses were placed in a laminar-flow optical chamber. Initial lens association kinetics for P. aeruginosa strain PAK were assessed in addition to lens-surface association phenotypes. Comparisons were made to strains with mutations in flagellin (fliC) or pilin (pilA) or those in flagellum (motAB) or pilus (pilU) function. PAK and its mutants associated with the contact lens surface at a constant rate according to first-order kinetics. Nonswimming mutants associated ～30 to 40 times slower than the wild type. PAK and its pilA mutant associated at similar rates, but each ～4 times faster than the pilU mutant. Lens attachment by wild-type PAK induced multiple phenotypes (static, lateral, and rotational surface movement), each showing only minor detachment. Flagellin (fliC) and flagellar-motility (motAB) mutants did not exhibit surface rotation. Conversely, strains with mutations in pilin (pilA) and pilus retraction (pilU) lacked lateral-surface movement but displayed enhanced surface rotation. Slower surface association of swimming-incapable P. aeruginosa mutants was ascribed to lower convective-diffusion-arrival rates, not to an inability to adhere. Flagellum function (swimming) enhanced lens association, attachment, and rotation; hyperpiliation hindered lens association. P. aeruginosa bound through three different adhesion sites: flagellum, pili, and body. Reduction of bacterial attachment to contact lenses thus requires blockage of multiple adhesion phenotypes.     

Correlations between relatives for acrocentric association frequency

Summary Acrocentric association was investigated in peripheral blood lymphocytes of twins (10 monozygotic and 11 dizygotic pairs), newborns and both parents (30 families), and spouses (51 pairs). Seventy two hour cultures were G-banded and scored for both absolute and relative acrocentric association frequency, except in the case of the spouse pairs where only the absolute frequency was measured. Both relative and absolute parameters of acrocentric association show positive correlations between relatives with the values being highest and most consistent in monozygotic twins, intermediate in parent and offspring, and most variable in dizygotic twins. Husband and wife pairs from our family collections show a positive correlation for the absolute parameter but not for relative parameters. The environmental factors responsible have not been identified.A rough estimate of broad sense heritability (0.81) has been made for the relative parameters. It probably contains a large component due to genetic dominance. Heritability of the absolute parameters is probably lower than for the relative parameters though estimation of its value is complicated by inconsistent results. A model is proposed to account for the variation in satellite association frequency which contains two elements: (i) The genotype determines the ratio of one chromosome type to another in the population of associated chromosomes. (ii) All other environmental factors influence the absolute frequency of association without altering this basic ratio.     

Improved hidden Markov models for molecular motors, part 1: basic theory

Hidden Markov models (HMMs) provide an excellent analysis of recordings with very poor signal/noise ratio made from systems such as ion channels which switch among a few states. This method has also recently been used for modeling the kinetic rate constants of molecular motors, where the observable variable—the position—steadily accumulates as a result of the motor's reaction cycle. We present a new HMM implementation for obtaining the chemical-kinetic model of a molecular motor's reaction cycle called the variable-stepsize HMM in which the quantized position variable is represented by a large number of states of the Markov model. Unlike previous methods, the model allows for arbitrary distributions of step sizes, and allows these distributions to be estimated. The result is a robust algorithm that requires little or no user input for characterizing the stepping kinetics of molecular motors as recorded by optical techniques.     

Kinetics of GPIbalpha-vWF-A1 tether bond under flow: effect of GPIbalpha mutations on the association and dissociation rates

The interaction between platelet glycoprotein (GP) Ib-IX-V complex and von Willebrand factor (vWF) is the first step of the hemostatic response to vessel injury. In platelet-type von Willebrand disease, two mutations, G233V and M239V, have been described within the Cys209-Cys248 disulfide loop of GPIbalpha that compromise hemostasis by increasing the affinity for vWF. We have earlier shown that converting other residues in this region to valine alters the affinity of GPIbalpha for vWF, with mutations K237V and Q232V, respectively, showing the greatest increase and decrease in affinity. Here, we investigated further the effect of these two mutations on the kinetics of the GPIbalpha interaction with the vWF-A1 domain under dynamic flow conditions. We measured the cellular on- and off-rate constants of Chinese hamster ovary cells expressing GPIb-IX complexes containing wild-type or mutant GPIbalpha interacting with vWF-A1-coated surfaces at different shear stresses. We found that the gain-of-function mutant, K237V, rolled very slowly and continuously on vWF-A1 surface while the loss-of-function mutant, Q232V, showed fast, saltatory movement compared to the wild-type (WT). The off-rate constants, calculated based on the analysis of lifetimes of transient tethers formed on surfaces coated with limiting densities of vWF-A1, revealed that the Q232V and K237V dissociated 1.25-fold faster and 2.2-fold slower than the WT. The cellular on-rate constant of WT, measured in terms of tethering frequency, was threefold more and threefold less than Q232V and K237V, respectively. Thus, the gain- and loss-of-function mutations in GPIbalpha affect both the association and dissociation kinetics of the GPIbalpha-vWF-A1 bond. These findings are in contrast to the functionally similar selectin bonds where some of the mutations have been reported to affect only the dissociation rate.     

Species dependence of enzyme-substrate encounter rates for triose phosphate isomerases

Triose phosphate isomerase (TIM) is a diffusion-controlled enzyme whose rate is limited by the diffusional encounter of the negatively charged substrate glyceraldehyde 3-phosphate (GAP) with the homodimeric enzyme's active sites. Translational and orientational steering of GAP toward the active sites by the electrostatic field of chicken muscle TIM has been observed in previous Brownian dynamics (BD) simulations. Here we report simulations of the association of GAP with TIMs from four species with net charges at pH 7 varying from -12e to +12e. Computed second-order rate constants are in good agreement with experimental data. The BD simulations and computation of average Boltzmann factors of substrate–protein interaction energies show that the protein electrostatic potential enhances the rates for all the enzymes. There is much less variation in the computed rates than might be expected on the basis of the net charges. Comparison of the electrostatic potentials by means of similarity indices shows that this is due to conservation of the local electrostatic potentials around the active sites which are the primary determinants of electrostatic steering of the substrate. Proteins 31:406–416, 1998. © 1998 Wiley-Liss, Inc.     

Kinetics of association of anti-lysozyme monoclonal antibody D44.1 and hen-egg lysozyme

Association rate constants for antigen/antibody associations have been computed by Brownian Dynamics simulations of D. L. Ermak and J. A. McCammon, J. Chem. Phys. 69:1352-1360, 1978. The model of monoclonal antibody (mAb) D44.1 is based on crystallographic data (B. C. Braden et al., J. Mol. Biol. 243:767-781, 1994). Electrostatic forces that steer the antigen to the antibody-combining site are computed by solving the linearized Poisson-Boltzmann equation. D44. 1-HEL complex displays very similar association motifs to a related anti-lysozyme antibody, HyHEL-5-HEL system. The computed association rate constants are comparable in the two systems, although the experimental affinity constants differ by three orders of magnitude (D. Tello et al., Biochem. Soc. Trans. 21:943-946, 1993; K. A. Hibbits et al., Biochemistry. 33:3584-3590, 1994). Simulations suggest that the origin of the differences in the affinity come from dissociation rate constants. We have also carried out simulation experiments on a number of mutant antibody fragment-HEL associations to address the role of electrostatics and, to a limited extent, the orientational aspects of association.     

The association between a negatively charged ligand and the electronegative binding pocket of its receptor.

Many examples exist of charged amino acids that play a role in attracting or holding a charged ligand toward or inside an oppositely charged binding pocket of the protein. For example, the enzymes superoxide dismutase, triose-phosphate isomerase, and acetylcholinesterase can steer ligands toward their oppositely charged binding pockets or gorges. Interestingly, in our Brownian dynamics simulations of a phosphate-binding protein, we discovered that negatively charged phosphate (HPO(2-)(4)) could make its way into the negatively charged binding pocket. In fact, the phosphate-binding protein exhibits counterintuitive kinetics of association. That is, one would expect that the rate of association would increase on increases to the ionic strength since the interaction between the ligand, with a charge of -2, and the electronegative binding pocket would be repulsive and greater screening should reduce this repulsion and increase the rate of association. However, the opposite is seen-i.e., the rate of association decreases on increases in the ionic strength. We used Brownian dynamics techniques to compute the diffusion limited association rate constants between the negatively charged phosphate ligand and several open forms of PBP (wild-type and several mutants based on an x-ray structure of open-form PBP, mutant T141D). With the appropriate choices of reaction criteria and molecular parameters, the ligand was able to diffuse into the binding pocket. A number of residues influence binding of the ligand within the pocket via hydrogen bonds or salt bridges. Arg135 partially neutralizes the charges on the HPO(2-)(4) ligand in the binding pocket, allowing it to enter. It is also found that the positive electrostatic patches above and below the binding entrance of PBP contribute the major attractive forces that direct the ligand toward the surface of the protein near the binding site.     

Synaptobrevin Transmembrane Domain Dimerization Studied by Multiscale Molecular Dynamics Simulations

Synaptic vesicle fusion requires assembly of the SNARE complex composed of SNAP-25, syntaxin-1, and synaptobrevin-2 (sybII) proteins. The SNARE proteins found in vesicle membranes have previously been shown to dimerize via transmembrane (TM) domain interactions. While syntaxin homodimerization is supposed to promote the transition from hemifusion to complete fusion, the role of synaptobrevin’s TM domain association in the fusion process remains poorly understood. Here, we combined coarse-grained and atomistic simulations to model the homodimerization of the sybII transmembrane domain and of selected TM mutants. The wild-type helix is shown to form a stable, right-handed dimer with the most populated helix-helix interface, including key residues predicted in a previous mutagenesis study. In addition, two alternative binding interfaces were discovered, which are essential to explain the experimentally observed higher-order oligomerization of sybII. In contrast, only one dimerization interface was found for a fusion-inactive poly-Leu mutant. Moreover, the association kinetics found for this mutant is lower as compared to the wild-type. These differences in dimerization between the wild-type and the poly-Leu mutant are suggested to be responsible for the reported differences in fusogenic activity between these peptides. This study provides molecular insight into the role of TM sequence specificity for peptide aggregation in membranes.     

How optimal are the binding energetics of barnase and barstar?

The extracellular ribonuclease barnase and its intracellular inhibitor barstar bind fast and with high affinity. Although extensive experimental and theoretical studies have been carried out on this system, it is unclear what the relative importance of different contributions to the high affinity is and whether binding can be improved through point mutations. In this work, we first applied Poisson-Boltzmann electrostatic calculations to 65 barnase-barstar complexes with mutations in both barnase and barstar. The continuum electrostatic calculations with a van der Waals surface dielectric boundary definition result in the electrostatic interaction free energy providing the dominant contribution favoring barnase-barstar binding. The results show that the computed electrostatic binding free energy can be improved through mutations at W44/barstar and E73/barnase. Furthermore, the determinants of binding affinity were quantified by applying COMparative BINding Energy (COMBINE) analysis to derive quantitative structure-activity relationships (QSARs) for the 65 complexes. The COMBINE QSAR model highlights approximately 20 interfacial residue pairs as responsible for most of the differences in binding affinity between the mutant complexes, mainly due to electrostatic interactions. Based on the COMBINE model, together with Brownian dynamics simulations to compute diffusional association rate constants, several mutants were designed to have higher binding affinities than the wild-type proteins.     

Association Rate Constants of Ras-Effector Interactions Are Evolutionarily Conserved

Evolutionary conservation of protein interaction properties has been shown to be a valuable indication for functional importance. Here we use homology interface modeling of 10 Ras-effector complexes by selecting ortholog proteins from 12 organisms representing the major eukaryotic branches, except plants. We find that with increasing divergence time the sequence similarity decreases with respect to the human protein, but the affinities and association rate constants are conserved as predicted by the protein design algorithm, FoldX. In parallel we have done computer simulations on a minimal network based on Ras-effector interactions, and our results indicate that in the absence of negative feedback, changes in kinetics that result in similar binding constants have strong consequences on network behavior. This, together with the previous results, suggests an important biological role, not only for equilibrium binding constants but also for kinetics in signaling processes involving Ras-effector interactions. Our findings are important to take into consideration in system biology approaches and simulations of biological networks.     

The association−dissociation behavior of the ApoE proteins: kinetic and equilibrium studies

The apolipoprotein E family consists of three major protein isoforms: apolipoprotein E4 (ApoE4), ApoE3, and ApoE2. The isoforms, which contain 299 residues, differ only by single-amino acid changes, but of the three, only ApoE4 is a risk factor for Alzheimer’s disease. At micromolar concentrations, lipid-free ApoE exists predominantly as tetramers. In more dilute solutions, lower-molecular mass species predominate. Using fluorescence correlation spectroscopy (FCS), intermolecular fluorescence resonance energy transfer (FRET), and sedimentation methods, we found that the association?dissociation reaction of ApoE can be modeled with a monomer?dimer?tetramer process. Equilibrium constants have been determined from the sedimentation data, while the individual rate constants for association and dissociation were determined by measurement of the kinetics of dissociation of ApoE and are in agreement with the equilibrium constants. Dissociation kinetics as measured by intermolecular FRET show two phases reflecting the dissociation of tetramer to dimer and of dimer to monomer, with dissociation from tetramer to dimer being more rapid than the dissociation from dimer to monomer. The rate constants differ for the different ApoE isoforms, showing that the association?dissociation process is isoform specific. Strikingly, the association rate constants are almost 2 orders of magnitude slower than expected for a diffusion-controlled process. Dissociation kinetics were also monitored by tryptophan fluorescence in the presence of acrylamide and the data found to be consistent with the monomer?dimer?tetramer model. The approach combining multiple methods establishes the reaction scheme of ApoE self-association.     

Site-directed mutagenesis of beta-lactamase I. Single and double mutants of Glu-166 and Lys-73.

Two single mutants and the corresponding double mutant of beta-lactamase I from Bacillus cereus 569/H were constructed and their kinetics investigated. The mutants have Lys-73 replaced by arginine (K73R), or Glu-166 replaced by aspartic acid (E166D), or both (K73R + E166D). All four rate constants in the acyl-enzyme mechanism were determined for the E166D mutant by the methods described by Christensen, Martin & Waley [(1990) Biochem. J. 266, 853-861]. Both the rate constants for acylation and deacylation for the hydrolysis of benzylpenicillin were decreased about 2000-fold in this mutant. In the K73R mutant, and in the double mutant, the rate constants for acylation were decreased about 100-fold and 10,000-fold respectively. All three mutants also had lowered values for the rate constants for the formation and dissociation of the non-covalent enzyme-substrate complex. The specificities of the mutants did not differ greatly from those of wild-type beta-lactamase, but the hydrolysis of cephalosporin C by the K73R mutant gave 'burst' kinetics.     

Regulatory behavior of Escherichia coli aspartate transcarbamylase altered by site-specific mutation of Tyr240----Phe in the catalytic chain

Isotopic exchange kinetics at chemical equilibrium have been used to identify changes in the regulatory properties of aspartate transcarbamylase (ATCase) caused by site-specific mutation of Tyr240----Phe (Y240F) in the catalytic chain. With both wild-type and the mutant enzymes, ATP activates both [14C]Asp in equilibrium N-carbamyl-L-aspartate (C-Asp) and the [32P]carbamyl phosphate (C-P) in equilibrium Pi exchanges. In contrast, with wild-type enzyme, CTP inhibits both exchanges, but with Y240F mutant enzyme CTP inhibits Asp in equilibrium C-Asp exchange and activates C-P in equilibrium Pi exchange. The bisubstrate analog N-(phosphonacetyl-L-aspartate), PALA, activates Asp in equilibrium C-Asp at a lower concentration with the Y240F enzyme, but the extent of activation is decreased, relative to wild-type enzyme. PALA activation of C-P in equilibrium Pi observed with wild-type enzyme disappears completely with the Y240F mutant enzyme. Analysis of perturbations of exchange rates by ATP and CTP were carried out by systematic methods plus computer-based simulations with the ISOBI program. These analyses indicate that (a) ATP increases the rates of association and dissociation for both C-P and Asp, but (b) CTP differentially increases the rate of C-P association to a greater degree than dissociation, but also decreases the rates for Asp association and dissociation in equal proportion. In addition, Arrhenius plots for Y240F ATCase suggest that ATP and CTP act by different mechanisms: ATP increases Vmax (decreases delta G not equal to) uniformly at all temperatures, whereas CTP does not alter either Vmax (delta G not equal to) or the Arrhenius slope (delta H not equal to).     

Development of a physiologically based toxicokinetic model for butadiene and four major metabolites in humans: global sensitivity analysis for experimental design issues

1,3-Butadiene (BD) is metabolized in humans and rodents to mutagenic and carcinogenic species. Our previous work has focused on developing a physiologically based toxicokinetic (PBTK) model for BD to estimate its metabolic rate to 1,2-epoxy-3-butene (EB), using exhaled breath BD concentrations in human volunteers exposed by inhalation. In this paper, we extend our BD model to describe the kinetics of its four major metabolites EB, 1,2:3,4-diepoxybutane (DEB), 3-butene-1,2-diol (BDD), and 3,4-epoxy-1,2-butanediol (EBD), and to test whether the extended model and experimental data (to be collected for BD and metabolites in humans) are together adequate to estimate the metabolic rate constants of each of the above chemicals. Global sensitivity analyses (GSA) were conducted to evaluate the relative importance of the model parameters on model outputs during the 20min of exposure and the 40min after exposure ended. All model parameters were studied together with various potentially measurable model outputs: concentrations of BD and EB in exhaled air, concentrations of BD and all metabolites in venous blood, and cumulated amounts of urinary metabolites excreted within 24h. Our results show that pulmonary absorption of BD and subsequent distribution and metabolism in the well-perfused tissues compartment are the critical processes in the toxicokinetics of BD and metabolites. In particular, three parameters influence numerous outputs: the blood:air partition coefficient for BD, the metabolic rate of BD to EB, and the volume of the well-perfused tissues. Other influential parameters include other metabolic rates, some partition coefficients, and parameters driving the gas exchanges (in particular, for BD outputs). GSA shows that the impact of the metabolic rate of BD to EB on the BD concentrations in exhaled air is greatly increased if a few of the model's important parameters (such as the blood:air partition coefficient for BD) are measured experimentally. GSA also shows that all the transformation pathways described in the PBTK model may not be estimable if only data on the studied outputs are collected, and that data on a specific output for a chemical may not inform all the transformations involving that chemical.     

Thermodynamics and kinetics of actin filament nucleation.

We have performed computer simulations and free energy calculations to determine the thermodynamics and kinetics of actin nucleation and thus identify a probable nucleation pathway and critical nucleus size. The binding free energies of structures along the nucleation pathway are found through a combination of electrostatic calculations and estimates of the entropic and surface area contributions. The association kinetics for the formation of each structure are determined through a series of Brownian dynamics simulations. The combination of the binding free energies and the association rate constants determines the dissociation rate constants, allowing for a complete characterization of the nucleation and polymerization kinetics. The results indicate that the trimer is the size of the critical nucleus, and the rate constants produce polymerization plots that agree very well with experimental results over a range of actin monomer concentrations.