ABSTRACT
Chorismate mutase (CM) enzymes have long served as model systems for benchmarking new methods and tools in computational chemistry. Despite the enzymes' prominence in the literature, the extent of the roles that activation enthalpy and entropy play in catalyzing the conversion of chorismate to prephenate is still subject to debate. Knowledge of these parameters is a key piece in fully understanding the mechanism of chorismate mutases. Within this study, we utilize EVB/MD free energy perturbation calculations at a range of temperatures, allowing us to extract activation enthalpies and entropies from an Arrhenius plot of activation free energies of the reaction catalyzed by a monofunctional Bacillus subtilis CM and the promiscuous enzyme isochorismate pyruvate lyase of Pseudomonas aeruginosa. In comparison to the uncatalyzed reaction, our results show that both enzyme-catalyzed reactions exhibit a substantial reduction in activation enthalpy, while the effect on activation entropy is relatively minor, demonstrating that enzyme-catalyzed CM reactions are enthalpically driven. Furthermore, we observe that the monofunctional CM from B. subtilis more efficiently catalyzes this reaction than its promiscuous counterpart. This is supported by a structural analysis of the reaction pathway at the transition state, from which we identified key residues explaining the enthalpically driven nature of the reactions and also the difference in efficiencies between the two enzymes.
Subject(s)
Chorismate Mutase , Chorismate Mutase/chemistry , Chorismate Mutase/metabolism , Thermodynamics , Entropy , TemperatureABSTRACT
Water/Ion NMR Detected - Phospholipid Vesicle Permeability Assay (WIND-PVPA), is presented as a novel, straightforward and automatable method to assess lipid barrier integrity in vitro. The apparent permeability constants of water- and ions across the PVPA barriers are determined in a one-pot experiment under the influence of membrane-active guest molecules. NMR spectroscopy is used to quantify the water directly (D2O) and the ions indirectly (complexed with EDTA) as a function of time. WIND-PVPA is demonstrated using four anti-microbial peptides, to show that membrane active molecules can be differentiated by their disruptive influence on the PVPA system. The results obtained are compared with explicit molecular dynamics simulations of lipid bilayers, AMPs, water and salt, where the motions of all individual water molecules relative to the lipid bilayer are monitored over the course of the simulations, allowing the calculation of theoretical apparent permeability constants of the corresponding single bilayer systems. Proof-of-principle is presented that WIND-PVPA can be used to evaluate the lipid barrier destabilizing effect of active guest molecules by measuring changes in passive water- and ion permeabilities upon exposure. The method is highly flexible in terms of barrier composition, choice of probes and membrane active compounds.
Subject(s)
Phospholipids , Water , Ion Transport , Lipid Bilayers/chemistry , Magnetic Resonance Spectroscopy , Permeability , Phospholipids/chemistryABSTRACT
The determination of the temperature dependence of enzyme catalysis has traditionally been a labourious undertaking. We have developed a new approach to the classical Arrhenius parameter estimation by fitting the change in velocity under a gradual change in temperature. The evaluation with a simulated dataset shows that the approach is valid. The approach is demonstrated as a useful tool by characterizing the Bacillus pumilus LipA enzyme. Our results for the lipase show that the enzyme is psychrotolerant, with an activation energy of 15.3 kcal/mol for the chromogenic substrate para-nitrophenyl butyrate. Our results demonstrate that this can produce equivalent curves to the traditional approach while requiring significantly less sample, labour and time. Our method is further validated by characterizing three α-amylases from different species and habitats. The experiments with the α-amylases show that the approach works over a wide range of temperatures and clearly differentiates between psychrophilic, mesophilic and thermophilic enzymes. The methodology is released as an open-source implementation in Python, available online or used locally. This method of determining the activation parameters can make studies of the temperature dependence of enzyme catalysis more widely adapted to understand how enzymes have evolved to function in extreme environments. Moreover, the thermodynamic parameters that are estimated serve as functional validations of the empirical valence bond calculations of enzyme catalysis.
ABSTRACT
Cold-adapted enzymes from psychrophilic species achieve their high catalytic efficiency at low temperature by a different partitioning of the activation free energy into its enthalpic and entropic components, compared to orthologous mesophilic enzymes. Their lower activation enthalpy, partly compensated by an increased entropic penalty, has been suggested to originate from changes in flexibility of the protein surface. Multiple sequence alignments of psychrophilic and mesophilic enzymes also show characteristic motifs located in surface loops of the protein. Here, we use computer simulations to examine the effects of a number of designed surface mutations of psychrophilic and mesophilic elastases on the temperature dependence of the catalyzed peptide cleavage reaction. For each of 14 mutant enzyme variants we report calculations of their thermodynamic activation parameters. The results show that substitution of psychrophilic loop residues into the mesophilic enzyme consistently changes both the activation parameters and loop flexibilities towards the former, and vice versa for opposite substitutions.
Subject(s)
Adaptation, Physiological , Cold Temperature , Enzymes/metabolism , Protein Engineering , Amino Acid Sequence , Animals , Biocatalysis , Enzymes/chemistry , Enzymes/genetics , Molecular Dynamics Simulation , Mutant Proteins/chemistry , Mutation/genetics , Pancreatic Elastase/chemistry , Salmon , ThermodynamicsABSTRACT
Predicting the effect of single-point mutations on protein stability or protein-ligand binding is a major challenge in computational biology. Free energy calculations constitute the most rigorous approach to this problem, though the estimation of converged values for amino acid mutations remains challenging. To overcome this limitation, we developed tailored protocols to calculate free energy shifts associated with single-point mutations. We herein describe the QresFEP protocol, which includes an extension of our recent protocols to cover all amino acids mutations, based on the latest versions of the OPLS-AA force field. QresFEP is implemented in an application programming interface framework and the graphic interface QGui, for the molecular dynamics software Q. The complete protocol is benchmarked in several model systems, optimizing a number of sampling parameters and the implementation of Zwanzig's exponential formula and Bennet's acceptance ratio methods. QresFEP shows an excellent performance on estimating the hydration free energies of amino acid side-chain mimics, including their charged analogues. We also examined its performance on a protein-ligand binding problem of pharmaceutical relevance, the antagonism of neuropeptide Y1 G protein-coupled receptor. Here, the calculations show very good agreement with the experimental effect of 16 mutations on the binding of antagonists BIBP3226, in line with our recent applications in this field. Finally, the characterization of 43 mutations of T4-lysozyme reveals the capacity of our protocol to assess variations of the thermal stability of proteins, achieving a similar performance to alternative free energy perturbation (FEP) approaches. In summary, QresFEP is a robust, versatile, and user-friendly computational FEP protocol to examine biochemical effects of single-point mutations with high accuracy.
Subject(s)
Arginine/analogs & derivatives , Automation , Molecular Dynamics Simulation , Mutant Proteins/chemistry , Receptors, Neuropeptide Y/chemistry , Software , Thermodynamics , Arginine/chemistry , Arginine/pharmacology , Ligands , Mutant Proteins/antagonists & inhibitors , Protein Stability , Receptors, Neuropeptide Y/antagonists & inhibitorsABSTRACT
The class I pancreatic elastase from Atlantic salmon is considered to be a cold-adapted enzyme in view of the cold habitat, the reduced thermostability of the enzyme, and the fact that it is faster than its mesophilic porcine counterpart at room temperature. However, no experimental characterization of its catalytic properties at lower temperatures has actually been reported. Here we use extensive computer simulations of its catalytic reaction, at different temperatures and with different peptide substrates, to compare its characteristics with those of porcine pancreatic elastase, with which it shares 67% sequence identity. We find that both enzymes have a preference for smaller aliphatic residues at the P1 position, while the reaction rate with phenylalanine at P1 is predicted to be substantially lower. With the former class of substrates, the calculated reaction rates for salmon enzyme are consistently higher than those of the porcine ortholog at all temperatures examined, and the difference is most pronounced at the lowest temperature. As observed for other cold-adapted enzymes, this is caused by redistribution of the activation free energy in terms of enthalpy and entropy and can be linked to differences in the mobility of surface-exposed loops in the two enzymes. Such mobility changes are found to be reflected by characteristic sequence conservation patterns in psychrophilic and mesophilic species. Hence, calculations of mutations in a single surface loop show that the temperature dependence of the catalytic reaction is altered in a predictable way.
Subject(s)
Adaptation, Physiological/genetics , Catalysis , Enzyme Stability , Pancreatic Elastase/chemistry , Amino Acid Sequence/genetics , Animals , Cold Temperature , Entropy , Kinetics , Pancreatic Elastase/genetics , Protein Conformation , Salmo salar/genetics , Swine/geneticsABSTRACT
The role played by entropy for the enormous rate enhancement achieved by enzymes has been debated for many decades. There are, for example, several confirmed cases where the activation free energy is reduced by around 10 kcal/mol due to entropic effects, corresponding to a rate enhancement of â¼107 compared to the uncatalyzed reaction. However, despite substantial efforts from both the experimental and theoretical side, no real consensus has been reached regarding the origin of such large entropic contributions to enzyme catalysis. Another remarkable instance of entropic effects is found in enzymes that are adapted by evolution to work at low temperatures, near the freezing point of water. These cold-adapted enzymes invariably show a more negative entropy and a lower enthalpy of activation than their mesophilic orthologs, which counteracts the exponential damping of reaction rates at lower temperature. The structural origin of this universal phenomenon has, however, remained elusive. The basic problem with connecting macroscopic thermodynamic quantities, such as activation entropy and enthalpy derived from Arrhenius plots, to the 3D protein structure is that the underlying detailed (microscopic) energetics is essentially inaccessible to experiment. Moreover, attempts to calculate entropy contributions by computer simulations have mostly focused only on substrate entropies, which do not provide the full picture. We have recently devised a new approach for accessing thermodynamic activation parameters of both enzyme and solution reactions from computer simulations, which turns out to be very successful. This method is analogous to the experimental Arrhenius plots and directly evaluates the temperature dependence of calculated reaction free energy profiles. Hence, by extensive molecular dynamics simulations and calculations of up to thousands of independent free energy profiles, we are able to extract activation parameters with sufficient precision for making direct comparisons to experiment. We show here that the agreement with the measured quantities, for both enzyme catalyzed and spontaneous solution reactions, is quite remarkable. Importantly, we can now address some of the most spectacular entropy effects in enzymes and clarify their detailed microscopic origin. Herein, we discuss as examples the conversion of cytidine to uridine catalyzed by cytidine deaminase and reactions taking place on the ribosome, namely, peptide bond formation and GTP hydrolysis by elongation factor Tu. It turns out that the large entropy contributions to catalysis in these cases can now be rationalized by our computational approach. Finally, we address the problem of cold adaptation of enzyme reaction rates and prove by computational experiments that the universal activation enthalpy-entropy phenomenon originates from mechanical properties of the outer protein surface.
Subject(s)
Cytidine Deaminase/metabolism , Binding Sites , Biocatalysis , Catalytic Domain , Entropy , Hydrolysis , Molecular Dynamics Simulation , Ribosomes/metabolism , Temperature , rab GTP-Binding Proteins/metabolismABSTRACT
The psychrophilic and mesophilic endonucleases A (EndA) from Aliivibrio salmonicida (VsEndA) and Vibrio cholera (VcEndA) have been studied experimentally in terms of the biophysical properties related to thermal adaptation. The analyses of their static X-ray structures was no sufficient to rationalize the determinants of their adaptive traits at the molecular level. Thus, we used Molecular Dynamics (MD) simulations to compare the two proteins and unveil their structural and dynamical differences. Our simulations did not show a substantial increase in flexibility in the cold-adapted variant on the nanosecond time scale. The only exception is a more rigid C-terminal region in VcEndA, which is ascribable to a cluster of electrostatic interactions and hydrogen bonds, as also supported by MD simulations of the VsEndA mutant variant where the cluster of interactions was introduced. Moreover, we identified three additional amino acidic substitutions through multiple sequence alignment and the analyses of MD-based protein structure networks. In particular, T120V occurs in the proximity of the catalytic residue H80 and alters the interaction with the residue Y43, which belongs to the second coordination sphere of the Mg2+ ion. This makes T120V an amenable candidate for future experimental mutagenesis.
Subject(s)
Bacterial Proteins/metabolism , Cold Temperature , Endodeoxyribonucleases/metabolism , Membrane Proteins/metabolism , Molecular Dynamics Simulation , Aliivibrio salmonicida/enzymology , Aliivibrio salmonicida/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites/genetics , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/genetics , Enzyme Stability , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Kinetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mutation , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Static Electricity , Thermodynamics , Vibrio cholerae/enzymology , Vibrio cholerae/geneticsABSTRACT
Enzymes are able to catalyze chemical reactions by reducing the activation free energy, yielding significant increases in the reaction rates. This can thermodynamically be accomplished by either reducing the activation enthalpy or increasing the activation entropy. The effect of remote mutations on the thermodynamic activation parameters of human purine nucleoside phosphorylase is examined using extensive molecular dynamics and free energy simulations. More than 2700 independent reaction free energy profiles for six different temperatures have been calculated to obtain high-precision computational Arrhenius plots. On the basis of these, the activation enthalpies and entropies were computed from linear regression of the plots with ΔG⧧ as a function of 1/T, and the obtained thermodynamic activation parameters are in very good agreement with those from experiments. The Arrhenius plots immediately show that the 6-oxopurines (INO and GUO) have identical slopes, whereas the 6-aminopurine (ADO) has a significantly different slope, indicating that the substrate specificity is related to the difference in thermodynamic activation parameters. Furthermore, the calculations show that the human PNP specificity for 6-oxopurines over 6-aminopurines originates from significant differences in electrostatic preorganization. The effect of the remote double mutation, K22E and H104R (E:R), has also been examined, as it alters human PNP toward the bovine PNP. These residues are situated on the protein surface, 28-35 Å from the active site, and the mutation alters the enthalpy-entropy balance with little effect on the catalytic rates. It is thus quite remarkable that the empirical valence bond method can reproduce the enthalpies and entropies induced by these long-range mutations.
Subject(s)
Molecular Dynamics Simulation , Protein Domains , Purine-Nucleoside Phosphorylase/chemistry , Thermodynamics , Adenosine/chemistry , Adenosine/metabolism , Animals , Biocatalysis , Catalytic Domain , Cattle , Guanosine/chemistry , Guanosine/metabolism , Humans , Inosine/chemistry , Inosine/metabolism , Kinetics , Linear Models , Molecular Structure , Mutation , Protein Binding , Purine-Nucleoside Phosphorylase/genetics , Purine-Nucleoside Phosphorylase/metabolism , Static Electricity , Substrate SpecificityABSTRACT
DYRK1A has emerged as a potential target for therapies of Alzheimer's disease using small molecules. On the basis of the observation of selective DYRK1A inhibition by firefly d-luciferin, we have explored static and dynamic structural properties of fragment sized variants of the benzothiazole scaffold with respect to DYRK1A using X-ray crystallography and NMR techniques. The compounds have excellent ligand efficiencies and show a remarkable diversity of binding modes in dynamic equilibrium. Binding geometries are determined in part by interactions often considered "weak", including "orthogonal multipolar" types represented by, for example, F-CO, sulfur-aromatic, and halogen-aromatic interactions, together with hydrogen bonds that are modulated by variation of electron withdrawing groups. These studies show how the benzothiazole scaffold is highly promising for the development of therapeutic DYRK1A inhibitors. In addition, the subtleties of the binding interactions, including dynamics, show how full structural studies are required to fully interpret the essential physical determinants of binding.
Subject(s)
Benzothiazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/chemistry , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/chemistry , Benzothiazoles/chemical synthesis , Benzothiazoles/chemistry , Crystallography, X-Ray , Dose-Response Relationship, Drug , Humans , Magnetic Resonance Spectroscopy , Molecular Docking Simulation , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Structure-Activity Relationship , Dyrk KinasesABSTRACT
The structural origin of enzyme adaptation to low temperature, allowing efficient catalysis of chemical reactions even near the freezing point of water, remains a fundamental puzzle in biocatalysis. A remarkable universal fingerprint shared by all cold-active enzymes is a reduction of the activation enthalpy accompanied by a more negative entropy, which alleviates the exponential decrease in chemical reaction rates caused by lowering of the temperature. Herein, we explore the role of protein surface mobility in determining this enthalpy-entropy balance. The effects of modifying surface rigidity in cold- and warm-active trypsins are demonstrated here by calculation of high-precision Arrhenius plots and thermodynamic activation parameters for the peptide hydrolysis reaction, using extensive computer simulations. The protein surface flexibility is systematically varied by applying positional restraints, causing the remarkable effect of turning the cold-active trypsin into a variant with mesophilic characteristics without changing the amino acid sequence. Furthermore, we show that just restraining a key surface loop causes the same effect as a point mutation in that loop between the cold- and warm-active trypsin. Importantly, changes in the activation enthalpy-entropy balance of up to 10 kcal/mol are almost perfectly balanced at room temperature, whereas they yield significantly higher rates at low temperatures for the cold-adapted enzyme.
Subject(s)
Enzymes/metabolism , Temperature , Animals , Catalysis , Cattle , Entropy , Molecular Dynamics Simulation , TrypsinABSTRACT
Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.
Subject(s)
Adenosine/metabolism , Guanosine/metabolism , Inosine/metabolism , Models, Molecular , Mutant Proteins/metabolism , Purine-Nucleoside Phosphorylase/metabolism , Adenosine/chemistry , Amino Acid Substitution , Biocatalysis , Catalytic Domain , Databases, Protein , Energy Transfer , Guanosine/chemistry , Humans , Hydrogen Bonding , Hydrolysis , Inosine/chemistry , Molecular Conformation , Molecular Dynamics Simulation , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutation , Purine-Nucleoside Phosphorylase/chemistry , Purine-Nucleoside Phosphorylase/genetics , Static Electricity , Substrate SpecificityABSTRACT
Structural information and activity data has increased rapidly for many protein targets during the last decades. In this paper, we present a high-throughput interface (Qgui) for automated free energy and empirical valence bond (EVB) calculations that use molecular dynamics (MD) simulations for conformational sampling. Applications to ligand binding using both the linear interaction energy (LIE) method and the free energy perturbation (FEP) technique are given using the estrogen receptor (ERα) as a model system. Examples of free energy profiles obtained using the EVB method for the rate-limiting step of the enzymatic reaction catalyzed by trypsin are also shown. In addition, we present calculation of high-precision Arrhenius plots to obtain the thermodynamic activation enthalpy and entropy with Qgui from running a large number of EVB simulations.
Subject(s)
Computer Graphics , High-Throughput Screening Assays , Models, Biological , Models, Chemical , Molecular Dynamics Simulation , User-Computer Interface , Animals , Automation , Calibration , Models, Molecular , Monte Carlo Method , Reference Standards , Thermodynamics , Trypsin/chemistryABSTRACT
We use a mathematical model to show that if symmetric stem cell division is regulated by differentiated cells, then changes in the population dynamics of the differentiated cells can lead to changes in the population dynamics of the stem cells. More precisely, the relative fitness of the stem cells can be affected by modifying the death rate of the differentiated cells. This result is interesting because stem cells are less sensitive than differentiated cells to environmental factors, such as medical therapy. Our result implies that stem cells can be manipulated indirectly by medical treatments that target the differentiated cells.
Subject(s)
Cell Differentiation , Cell Division , Stem Cells/cytology , Animals , Humans , Models, BiologicalABSTRACT
Life has effectively colonized most of our planet and extremophilic organisms require specialized enzymes to survive under harsh conditions. Cold-loving organisms (psychrophiles) express heat-labile enzymes that possess a high specific activity and catalytic efficiency at low temperatures. A remarkable universal characteristic of cold-active enzymes is that they show a reduction both in activation enthalpy and entropy, compared to mesophilic orthologs, which makes their reaction rates less sensitive to falling temperature. Despite significant efforts since the early 1970s, the important question of the origin of this effect still largely remains unanswered. Here we use cold- and warm-active trypsins as model systems to investigate the temperature dependence of the reaction rates with extensive molecular dynamics free energy simulations. The calculations quantitatively reproduce the catalytic rates of the two enzymes and further yield high-precision Arrhenius plots, which show the characteristic trends in activation enthalpy and entropy. Detailed structural analysis indicates that the relationship between these parameters and the 3D structure is reflected by significantly different internal protein energy changes during the reaction. The origin of this effect is not localized to the active site, but is found in the outer regions of the protein, where the cold-active enzyme has a higher degree of softness. Several structural mechanisms for softening the protein surface are identified, together with key mutations responsible for this effect. Our simulations further show that single point-mutations can significantly affect the thermodynamic activation parameters, indicating how these can be optimized by evolution.
Subject(s)
Trypsin/chemistry , Trypsin/metabolism , Cold Temperature , Computational Biology , Molecular Dynamics Simulation , Mutation/genetics , Protein Stability , Surface Properties , Thermodynamics , Trypsin/geneticsABSTRACT
We have in the present study explored the anticancer activity against human Burkitt's lymphoma cells (Ramos) of a series of small linear and cyclic tetrapeptides containing a ß2,2-amino acid with either two 2-naphthyl-methylene or two para-CF3-benzyl side chains, along with their interaction with the main plasma protein human serum albumin (HSA). The cyclic and more amphipathic tetrapeptides revealed a notably higher anticancer potency against Ramos cells [50% inhibitory concentration (IC50) 1170 µM] compared to the linear tetrapeptide counterparts (IC50 18.7 to >413 µM). The most potent cyclic tetrapeptide c3 had a 16.5-fold preference for Ramos cells compared to human red blood cells, whereas the cyclic tetrapeptide c1 both showed low hemolytic activity and displayed the overall highest (2.9-fold) preference for Ramos cells (IC50 23 µM) compared to healthy human lung fibroblast cells (MRC-5). Investigating the interaction of selected tetrapeptides and recently reported hexapeptides with HSA revealed that the peptides bind to drug site II of HSA in the 2228 µM range, disregarding size and overall structure. NMR and in silico molecular docking experiments identified the lipophilic residues as responsible for the interaction, but in vitro studies showed that the anticancer potency of the peptides varied in the presence of HSA and that c3 remained the most potent peptide. Based on our findings, we call for implementing serum albumin binding in development of anticancer peptides, as it may have implications for future administration and systemic distribution of peptide-based cancer drugs.
Subject(s)
Antineoplastic Agents/pharmacology , Oligopeptides/pharmacology , Peptides, Cyclic/pharmacology , Serum Albumin/metabolism , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacokinetics , Cell Line , Humans , Inhibitory Concentration 50 , Molecular Docking Simulation , Nuclear Magnetic Resonance, Biomolecular , Oligopeptides/metabolism , Oligopeptides/pharmacokinetics , Peptides, Cyclic/metabolism , Peptides, Cyclic/pharmacokinetics , Protein BindingABSTRACT
BACKGROUND: Many biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics. RESULTS: The binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the µM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs. CONCLUSIONS: The biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful consideration in antimicrobial peptide studies, as the systemic distribution can be significantly affected by HSA interactions.
Subject(s)
Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Serum Albumin/chemistry , Serum Albumin/metabolism , Binding Sites , Calorimetry , Escherichia coli/drug effects , Humans , Hydrogen Bonding , Methicillin-Resistant Staphylococcus aureus/drug effects , Molecular Docking Simulation , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Staphylococcus aureus/drug effects , ThermodynamicsABSTRACT
Several drugs interact with the major plasma proteins serum albumin and alpha-1 acid glycoprotein. Such binding may be either beneficial or disadvantageous from a pharmacokinetic perspective. In the present paper, we investigate the thermodynamics involved in the binding of a series of promising cationic antimicrobial peptides to the alpha-1 acid glycoprotein using isothermal titration calorimetry. The drug-like peptides are able to effectively destroy multiresistant bacterial strains, and members of this peptide class are currently in clinical phase II trials. Similar peptides, in a previous study, have been shown to bind to serum albumin resulting in a 10-fold reduction in the peptides ability to kill bacteria in vitro. Here, it is shown that the peptides also are ligands for alpha-1 glycoprotein with moderate binding affinities. The binding mode is investigated in detail using molecular docking, which maps the interaction to sub-pockets I, II and III of the binding site. Despite this interaction, protein binding is shown to have little or no effect on the ability of the peptides to kill bacteria in vitro, either at normal physiological or acute phase concentrations. The results show that although the peptides interact with the binding pocket of alpha-1 acid glycoprotein, the low stoichiometric binding ratio ensures that the interaction is not an obstacle for further development of these promising peptides as antimicrobial therapies.
Subject(s)
Antimicrobial Cationic Peptides , Orosomucoid/chemistry , Staphylococcus aureus/drug effects , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Binding Sites , Calorimetry , Clinical Trials, Phase II as Topic , Humans , Microbial Sensitivity Tests , Molecular Docking Simulation , Protein Binding , Staphylococcus aureus/growth & development , ThermodynamicsABSTRACT
A theoretical study of the one-photon absorption of five fluorescent proteins (FPs) is presented. The absorption properties are calculated using a polarizable embedding approach combined with density functional theory (PE-DFT) on the wild-type green fluorescent protein (wtGFP) and several of its mutants (BFP, eGFP, YFP and eCFP). The observed trends in excitation energies among the FPs are reproduced by our approach when performing calculations directly on the crystal structures or when using structures extracted from molecular dynamics simulations. However, in the former case, QM/MM geometry optimization of the chromophores within a frozen protein environment is needed in order to reproduce the experimental trends. An explicit account of polarization in the force field is not needed to yield the correct trend between the different FPs, but it is necessary for reproducing the experimentally observed red shift from vacuum to protein. This is the first computational study of a range of fluorescent proteins using a polarizable embedding potential.
Subject(s)
Green Fluorescent Proteins/chemistry , Protons , Quantum Theory , Green Fluorescent Proteins/genetics , Molecular Dynamics Simulation , Molecular Structure , MutationABSTRACT
We have recently reported a series of synthetic anticancer heptapeptides (H-KKWß(2,2) WKK-NH(2) ) containing a central achiral and lipophilic ß(2,2) -amino acid that display low toxicity against non-malignant cells and high proteolytic stability. In the present study, we have further investigated the effects of increasing the rigidity and amphipathicity of two of our lead heptapeptides by preparing a series of seven to five residue cyclic peptides containing the two most promising ß(2,2) -amino acid derivatives as part of the central lipophilic core. The peptides were tested for anticancer activity against human Burkitt's lymphoma (Ramos cells), haemolytic activity against human red blood cells (RBC) and cytotoxicity against healthy human lung fibroblast cells (MRC-5). The results demonstrated a considerable increase in anticancer potency following head-to-tail peptide cyclization, especially for the shortest derivatives lacking a tryptophan residue. High-resolution NMR studies and molecular dynamics simulations together with an annexin-V-FITC and propidium iodide fluorescent assay showed that the peptides had a membrane disruptive mode of action and that the more potent peptides penetrated deeper into the lipid bilayer. The need for new anticancer drugs with novel modes of action is demanding, and development of short cyclic anticancer peptides with an overall rigidified and amphipathic structure is a promising approach to new anticancer agents.