The protein-protein interactions between SMPI and thermolysin studied by molecular dynamics and MM/PBSA calculations

Thermolysin is a zinc-metalloendopeptidase secreted by the gram-positive thermophilic bacterium Bacillus thermoproteolyticus. Thermolysin belongs to the gluzinicin family of enzymes, which is selectively inhibited by Steptomyces metalloproteinase inhibitor (SMPI). Very little is known about the interaction between SMPI and thermolysin. Knowledge about the protein-protein interactions is very important for designing new thermolysin inhibitors with possible industrial or pharmaceutical applications. In the present study, two binding modes between SMPI and thermolysin were studied by 2300 picoseconds (ps) of comparative molecular dynamics (MD) simulations and calculation of the free energy of binding using the molecular mechanics-Poisson-Boltmann surface area (MM/PBSA) method. One of the positions, the 'horizontal arrow head docking' (HAHD) was similar to the previously proposed binding mode by Tate et al. (Tate, S., Ohno, A., Seeram, S. S., Hiraga, K., Oda, K., and Kainosho, M. J. Mol. Biol. 282, 435-446 (1998)). The other position, the 'vertical arrow head docking' (VAHD) was obtained by a manual docking guided by the shape and charge distribution of SMPI and the binding pocket of thermolysin. The calculations showed that SMPI had stronger interactions with thermolysin in the VAHD than in the HAHD complex, and the VAHD complex was considered more realistic than the HAHD complex. SMPI interacted with thermolysin not only at the active site but had auxiliary binding sites contributing to proper interactions. The VAHD complex can be used for designing small molecule inhibitors mimicking the SMPI-thermolysin binding interfaces.     

Revisiting Free Energy Calculations: A Theoretical Connection to MM/PBSA and Direct Calculation of the Association Free Energy

The prediction of absolute ligand-receptor binding affinities is essential in a wide range of biophysical queries, from the study of protein-protein interactions to structure-based drug design. End-point free energy methods, such as the Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) model, have received much attention and widespread application in recent literature. These methods benefit from computational efficiency as only the initial and final states of the system are evaluated, yet there remains a need for strengthening their theoretical foundation. Here a clear connection between statistical thermodynamics and end-point free energy models is presented. The importance of the association free energy, arising from one molecule's loss of translational and rotational freedom from the standard state concentration, is addressed. A novel method for calculating this quantity directly from a molecular dynamics simulation is described. The challenges of accounting for changes in the protein conformation and its fluctuations from separate simulations are discussed. A simple first-order approximation of the configuration integral is presented to lay the groundwork for future efforts. This model has been applied to FKBP12, a small immunophilin that has been widely studied in the drug industry for its potential immunosuppressive and neuroregenerative effects.     

Predicting Binding Free Energy Change Caused by Point Mutations with Knowledge-Modified MM/PBSA Method

A new methodology termed Single Amino Acid Mutation based change in Binding free Energy (SAAMBE) was developed to predict the changes of the binding free energy caused by mutations. The method utilizes 3D structures of the corresponding protein-protein complexes and takes advantage of both approaches: sequence- and structure-based methods. The method has two components: a MM/PBSA-based component, and an additional set of statistical terms delivered from statistical investigation of physico-chemical properties of protein complexes. While the approach is rigid body approach and does not explicitly consider plausible conformational changes caused by the binding, the effect of conformational changes, including changes away from binding interface, on electrostatics are mimicked with amino acid specific dielectric constants. This provides significant improvement of SAAMBE predictions as indicated by better match against experimentally determined binding free energy changes over 1300 mutations in 43 proteins. The final benchmarking resulted in a very good agreement with experimental data (correlation coefficient 0.624) while the algorithm being fast enough to allow for large-scale calculations (the average time is less than a minute per mutation).     

Lipid Membrane Structure and Dynamics Studied by All-Atom Molecular Dynamics Simulations of Hydrated Phospholipid Bilayers

Abstract

The structure and dynamics of phosphatidylcholine bilayers are examined by reviewing the results of several nanoseconds of molecular dynamics simulations on a number of bilayer and monolayer models. The lengths of these simulations, the longest single one of which was 2 nanoseconds, were sufficiently long to effectively sample many of the longer-scale motions governing the behaviour of biomembranes. These simulations reproduce many experimental observables well and provide a degree of resolution currently unavailable experimentally.     

Cryo-Cooling Effect on DHFR Crystal Studied by Replica-Exchange Molecular Dynamics Simulations

Cryo-cooling is routinely performed before x-ray diffraction image collection to reduce the damage to crystals due to ionizing radiation. It has been suggested that although backbone structures are usually very similar between room temperature and cryo-temperature, cryo-cooling may hamper biologically relevant dynamics. In this study, the crystal of Escherichia coli dihydrofolate reductase is studied with replica-exchange molecular dynamics simulation, and the results are compared with the crystal structure determined at cryo-temperature and room temperature with the time-averaged ensemble method. Although temperature dependence of unit cell compaction and root mean-square fluctuation of Cα is found in accord with experiment, it is found that the protein structure at low temperature can be more heterogeneous than the ensemble of structures reported by using the time-averaged ensemble method, encouraging further development of the time-averaged ensemble method and indicating that data should be examined carefully to avoid overinterpretation of one average structure.     

Intrinsic Dynamics of Restriction Endonuclease EcoO109I Studied by Molecular Dynamics Simulations and X-Ray Scattering Data Analysis

EcoO109I is a type II restriction endonuclease that functions as a dimer in solution. Upon DNA binding to the enzyme, the two subunits rotate counterclockwise relative to each other, as the two catalytic domains undergo structural changes to capture the cognate DNA. Using a 150-ns molecular dynamics simulation, we investigated the intrinsic dynamics of the DNA-free enzyme in solution to elucidate the relationship between enzyme dynamics and structural changes. The simulation revealed that the enzyme is considerably flexible, and thus exhibits large fluctuations in the radius of gyration. The small-angle x-ray scattering profile calculated from the simulation, including scattering from explicit hydration water, was in agreement with the experimentally observed profile. Principal component analysis revealed that the major dynamics were represented by the open-close and counterclockwise motions: the former is required for the enzyme to access DNA, whereas the latter corresponds to structural changes upon DNA binding. Furthermore, the intrinsic dynamics in the catalytic domains were consistent with motions capturing the cognate DNA. These results indicate that the structure of EcoO109I is intrinsically flexible in the direction of its functional movement, to facilitate effective structural changes for sequence-specific DNA recognition and processing.     

Exploring the RING-Catalyzed Ubiquitin Transfer Mechanism by MD and QM/MM Calculations

Ubiquitylation is a universal mechanism for controlling cellular functions. A large family of ubiquitin E3 ligases (E3) mediates Ubiquitin (Ub) modification. To facilitate Ub transfer, RING E3 ligases bind both the substrate and ubiquitin E2 conjugating enzyme (E2) linked to Ub via a thioester bond to form a catalytic complex. The mechanism of Ub transfer catalyzed by RING E3 remains elusive. By employing a combined computational approach including molecular modeling, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations, we characterized this catalytic mechanism in detail. The three-dimensional model of dimeric RING E3 ligase RNF4 RING, E2 ligase UbcH5A, Ub and the substrate SUMO2 shows close contact between the substrate and Ub transfer catalytic center. Deprotonation of the substrate lysine by D117 on UbcH5A occurs with almost no energy barrier as calculated by MD and QM/MM calculations. Then, the side chain of the activated lysine gets close to the thioester bond via a conformation change. The Ub transfer pathway begins with a nucleophilic addition that forms an oxyanion intermediate of a 4.23 kcal/mol energy barrier followed by nucleophilic elimination, resulting in a Ub modified substrate by a 5.65 kcal/mol energy barrier. These results provide insight into the mechanism of RING-catalyzed Ub transfer guiding the discovery of Ub system inhibitors.     

Molecular Dynamics of a Schiff Base Tetramacrocycle Studied by NMR and MD Simulations

Schiff base condensation of the pentadentate tetrapodal amine 1 with 2,6-diformyl-4-methylphenol 2 leads in a [3 + 6] condensation to a previously not described macrocycle 3 which shows C₃-symmetry. X-ray analysis shows a truncated cone shape for 3. At T S 100°C, the ¹H-ROESY/EXSY spectrum reveals pairwise exchange of corresponding sites, indicative of inversion of the whole molecule in an umbrella-like fashion. Molecular dynamics simulations support this hypothesis.     

Interactions between Fengycin and Model Bilayers Quantified by Coarse-Grained Molecular Dynamics

Bacteria, particularly of the genus Bacillus, produce a wide variety of antifungal compounds. They act by affecting the lipid bilayers of fungal membranes, causing curvature-induced strain and eventual permeabilization. One class of these, known as fengycins, has been commercialized for treating agricultural infections and shows some promise as a possible antifungal pharmaceutical. Understanding the mechanism by which fengycins damage lipid bilayers could prove useful to the future development of related antifungal treatments. In this work, we present multi-microsecond-long simulations of fengycin interacting with different lipid bilayer systems. We see fengycin aggregation and uncover a clear aggregation pattern that is partially influenced by bilayer composition. We also quantify some local bilayer perturbations caused by fengycin binding, including curvature of the lipid bilayer and local electrostatic-driven reorganization.     

Interactions between Fengycin and Model Bilayers Quantified by Coarse-Grained Molecular Dynamics

Bacteria, particularly of the genus Bacillus, produce a wide variety of antifungal compounds. They act by affecting the lipid bilayers of fungal membranes, causing curvature-induced strain and eventual permeabilization. One class of these, known as fengycins, has been commercialized for treating agricultural infections and shows some promise as a possible antifungal pharmaceutical. Understanding the mechanism by which fengycins damage lipid bilayers could prove useful to the future development of related antifungal treatments. In this work, we present multi-microsecond-long simulations of fengycin interacting with different lipid bilayer systems. We see fengycin aggregation and uncover a clear aggregation pattern that is partially influenced by bilayer composition. We also quantify some local bilayer perturbations caused by fengycin binding, including curvature of the lipid bilayer and local electrostatic-driven reorganization.     

Tautomerism in 8-Nitroguanosine Studied by NMR and Theoretical Calculations

The guanine base in DNA, due to its low oxidation potential, is particularly sensitive to chemical modifications. A large number of guanine lesions have been characterized and studied in some detail due to their relationship with tissue inflammations. Nevertheless, one example of these lesions is the formation of 8-nitro-guanosine, but the NMR data of this compound was only partially interpreted. A comprehensive study of the two possible tautomeric forms, through a detailed characterization of this compound, has implications for its base pairing properties. The target compound was obtained through a synthetic sequence of five steps, where all intermediates were fully characterized using spectral data. The analysis of the two tautomers was then evaluated through NMR spectroscopy and theoretical calculations of the chemical shifts and NH coupling constants, which were also compared with the data from guanosine.     

Analysis of the Interactions between Arp2/3 Complex and an Inhibitor Arpin by Molecular Dynamics Simulation

The Arp2/3 complex is one of the main regulators of the actin cytoskeleton and a basic molecular machine that nucleates the branched actin filaments. In this work, we studied the interaction of the Arp2/3 complex with its inhibitor, arpin, and revealed the amino-acid residues that are responsible for complex formation. The free-energy calculation for arpin binding to the Arp2/3 complex was performed using umbrella sampling. It has been shown that the dissociation constant of the Arp2/3–arpin complex is higher on average than that of Arp2/3 complexes with other inhibitors. Two arpin binding sites with different affinities were identified on the surface of the Arp2/3 complex. The mechanism of the inhibition of the Arp2/3 complex by arpin is discussed.     

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.     

Enantiomerization of Allylic Trifluoromethyl Sulfoxides Studied by HPLC Analysis and DFT Calculations

Enantiomerization of allylic trifluoromethyl sulfoxides occurs spontaneously at room temperature through the corresponding allylic trifluoromethanesulfenates via a [2,3]‐sigmatropic rearrangement. Dynamic enantioselective high‐performance liquid chromatography (HPLC) analysis revealed the stereodynamics of these sulfoxides ranging from chromatographic resolution to peak coalescence at temperatures between 5 and 53 °C. The rate constant of enantiomerization and activation parameters were determined and compared with Density Functional Theory (DFT) calculations. Chirality 28:136–142, 2016. © 2015 Wiley Periodicals, Inc.     

Study of the Interactions between Neurophysin II and Dipeptide Ligand by Means of Molecular Dynamics

The nonapeptide hormones oxytocin (OT) and vasopressin (VP), while transported in the posterior pituitary, are packaged into neurosecretory granules (NSG) in the form of high associates with disulfide-rich proteins known as neurophysin I (NPI) and neurophysin II (NPII), respectively. In the NSG, neurophysins serve as carrier proteins to the hormones, until the latter are dissociated upon secretion into blood. To shed more light on molecular self-recognition between NPs, and between NPs and their ligands, we have studied their molecular association, using as a starting point the recently published solid-state structure (C^α-trace) of the neurophysin II-dipeptide complex. Another purpose of this work was the development of reliable strategies for molecular modeling, that would utilize minimal structural information (like C^α-trace and/or structural homology) yet be useful for studies of protein/ligand interactions. An initial all-atom representation of the protein-peptide complex (2:2) was obtained by the conversion of the C^α-carbon trace deposited in the Brookhaven Protein Data Bank (file 1BN2), using the InsightII/Biopolymer modules from the suite of programs supplied by Biosym Technologies, San Diego. The free NPII homodimer was obtained by removal of the dipeptide ligands from the starting structures. Both associates, after initial immersion in water, were submitted to gradual (side chains first then all atoms) minimization of energy. Subsequently, they were thermally equilibrated and submitted to the molecular dynamics (AMBER 4.0) at 300K, until the total energy was stabilized. The structures, averaged over the last 20 ps of the dynamics, were compared with the starting C^α-trace and among themselves. The protein/ligand complex, simulated in water, compares favorably with the solid-state reference. An allosteric mechanism for the NPII dimer/ligand interaction is proposed and discussed.     

Dynamics of Inter-heavy Chain Interactions in Human Immunoglobulin G (IgG) Subclasses Studied by Kinetic Fab Arm Exchange

Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange (“Fab arm exchange”). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m(c3c5*/6,24*), whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange in vivo for IgG4.     

Binding of the Bacteriophage P22 N-Peptide to the boxB RNA Motif Studied by Molecular Dynamics Simulations

Protein-RNA interactions are important for many cellular processes. The Nut-utilization site (N)-protein of bacteriophages contains an N-terminal arginine-rich motif that undergoes a folding transition upon binding to the boxB RNA hairpin loop target structure. Molecular dynamics simulations were used to investigate the dynamics of the P22 N-peptide-boxB complex and to elucidate the energetic contributions to binding. In addition, the free-energy changes of RNA and peptide conformational adaptation to the bound forms, as well as the role of strongly bound water molecules at the peptide-RNA interface, were studied. The influence of peptide amino acid substitutions and the salt dependence of interaction were investigated and showed good agreement with available experimental results. Several tightly bound water molecules were found at the RNA-binding interface in both the presence and absence of N-peptide. Explicit consideration of the waters resulted in shifts of calculated contributions during the energetic analysis, but overall similar binding energy contributions were found. Of interest, it was found that the electrostatic field of the RNA has a favorable influence on the coil-to-α-helix transition of the N-peptide already outside of the peptide-binding site. This result may have important implications for understanding peptide-RNA complex formation, which often involves coupled folding and association processes. It indicates that electrostatic interactions near RNA molecules can lead to a shift in the equilibrium toward the bound form of an interacting partner before it enters the binding pocket.