Curcumin stably interacts with DNA hairpin through minor groove binding and demonstrates enhanced cytotoxicity in combination with FdU nucleotides

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV–Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two ? H of curcumin heptadiene chain are closely positioned to the A₁₆-H8 and A₁₇-H8, while G₁₂-H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy.     

Molecular dynamics simulations of lignin peroxidase in solution

The dynamical and structural properties of lignin peroxidase and its Trp171Ala mutant have been investigated in aqueous solution using molecular dynamics (MD) simulations. In both cases, the enzyme retained its overall backbone structure and all its noncovalent interactions in the course of the MD simulations. Very interestingly, the analysis of the MD trajectories showed the presence of large fluctuations in correspondence of the residues forming the heme access channel; these movements enlarge the opening and facilitate the access of substrates to the enzyme active site. Moreover, steered molecular dynamics docking simulations have shown that lignin peroxidase natural substrate (veratryl alcohol) can easily approach the heme edge through the access channel.     

Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, III: Molecular Dynamics Simulation Incorporating a Cyanophenylalanine Spectroscopic Probe

A nitrile-derived amino acid, Phe_CN, has been used as an internal spectroscopic probe to study the binding of an inhalational anesthetic to a model membrane protein. The infrared spectra from experiment showed a blue-shift of the nitrile vibrational frequency in the presence of the anesthetic halothane. To interpret the infrared results and explore the nature of the interaction between halothane and the model protein, all-atom molecular dynamics (MD) simulations have been used to probe the structural and dynamic properties of the protein in the presence and absence of one halothane molecule. The frequency shift analyzed from MD simulations agrees well with the experimental infrared results. Decomposition of the forces acting on the nitrile probes demonstrates an indirect impact on the probes from halothane, namely a change of the protein's electrostatic local environment around the probes induced by halothane. Although the halothane remains localized within the designed hydrophobic binding cavity, it undergoes a significant amount of translational and rotational motion, modulated by the interaction of the trifluorine end of halothane with backbone hydrogens of the residues forming the cavity. This dominant interaction between halothane and backbone hydrogens outweighs the direct interaction between halothane and the nitrile groups, making it a good “spectator” probe of the halothane-protein interaction. These MD simulations provide insight into action of anesthetic molecules on the model membrane protein, and also support the further development of nitrile-labeled amino acids as spectroscopic probes within the designed binding cavity.     

Molecular dynamics insights into protein-glycosaminoglycan systems from microsecond-scale simulations

Heparin is a key player in cell signaling via its physical interactions with protein targets in the extracellular matrix. However, basic molecular level understanding of these highly biologically relevant intermolecular interactions is still incomplete. In this study, for the first time, microsecond-scale MD simulations are reported for a complex between fibroblast growth factor 1 and heparin. We rigorously analyze this molecular system in terms of the conformational space, structural, energetic, and dynamic characteristics. We reveal that the conformational selection mechanism of binding denotes a recognition specificity determinant. We conclude that the length of the simulation could be crucial for evaluation of some of the analyzed parameters. Our data provide novel significant insights into the interactions in the fibroblast growth factor 1 complex with heparin, in particular, and into the physical-chemical nature of protein-glycosaminoglycan systems in general, which have potential applicability for biomaterials development in the area of regenerative medicine.     

Computational modeling of membrane trafficking processes: From large molecular assemblies to chemical specificity

In the last decade, molecular dynamics (MD) simulations have become an essential tool to investigate the molecular properties of membrane trafficking processes, often in conjunction with experimental approaches. The combination of MD simulations with recent developments in structural biology, such as cryo-electron microscopy and artificial intelligence-based structure determination, opens new, exciting possibilities for future investigations. However, the full potential of MD simulations to provide a molecular view of the complex and dynamic processes involving membrane trafficking can only be realized if certain limitations are addressed, and especially those concerning the quality of coarse-grain models, which, despite recent successes in describing large-scale systems, still suffer from far-from-ideal chemical accuracy. In this review, we will highlight recent success stories of MD simulations in the investigation of membrane trafficking processes, their implications for future research, and the challenges that lie ahead in this specific research domain.     

Molecular Dynamics Simulations to Provide Insights into Epitopes Coupled to the Soluble and Membrane-Bound MHC-II Complexes

Epitope recognition by major histocompatibility complex II (MHC-II) is essential for the activation of immunological responses to infectious diseases. Several studies have demonstrated that this molecular event takes place in the MHC-II peptide-binding groove constituted by the α and β light chains of the heterodimer. This MHC-II peptide-binding groove has several pockets (P1-P11) involved in peptide recognition and complex stabilization that have been probed through crystallographic experiments and in silico calculations. However, most of these theoretical calculations have been performed without taking into consideration the heavy chains, which could generate misleading information about conformational mobility both in water and in the membrane environment. Therefore, in absence of structural information about the difference in the conformational changes between the peptide-free and peptide-bound states (pMHC-II) when the system is soluble in an aqueous environment or non-covalently bound to a cell membrane, as the physiological environment for MHC-II is. In this study, we explored the mechanistic basis of these MHC-II components using molecular dynamics (MD) simulations in which MHC-II was previously co-crystallized with a small epitope (P₇) or coupled by docking procedures to a large (P₂₂) epitope. These MD simulations were performed at 310 K over 100 ns for the water-soluble (MHC-II_w, MHC-II-P_7w, and MHC-II-P_22w) and 150 ns for the membrane-bound species (MHC-II_m, MHC-II-P_7m, and MHC-II-P_22m). Our results reveal that despite the different epitope sizes and MD simulation environments, both peptides are stabilized primarily by residues lining P1, P4, and P6-7, and similar noncovalent intermolecular energies were observed for the soluble and membrane-bound complexes. However, there were remarkably differences in the conformational mobility and intramolecular energies upon complex formation, causing some differences with respect to how the two peptides are stabilized in the peptide-binding groove.     

Protein flexibility using constraints from molecular dynamics simulations

Proteins are held together in the native state by hydrophobic interactions, hydrogen bonds and interactions with the surrounding water, whose strength as well as spatial and temporal distribution affects protein flexibility and hence function. We study these effects using 10 ns molecular dynamics simulations of pure water and of two proteins, the glutamate receptor ligand binding domain and barnase. We find that most of the noncovalent interactions flicker on and off over typically nanoseconds, and so we can obtain good statistics from the molecular dynamics simulations. Based on this information, a topological network of rigid bonds corresponding to a protein structure with covalent and noncovalent bonds is constructed, with account being taken of the influence of the flickering hydrogen bonds. We define the duty cycle for the noncovalent interactions as the percentage of time a given interaction is present, which we use as an input to investigate flexibility/rigidity patterns, in the algorithm FIRST which constructs and analyses topological networks.     

Peptide folding driven by Van der Waals interactions

Contrary to the widespread view that hydrogen bonding and its entropy effect play a dominant role in protein folding, folding into helical and hairpin-like structures is observed in molecular dynamics (MD) simulations without hydrogen bonding in the peptide-solvent system. In the widely used point charge model, hydrogen bonding is calculated as part of the interaction between atomic partial charges. It is removed from these simulations by setting atomic charges of the peptide and water to zero. Because of the structural difference between the peptide and water, van der Waals (VDW) interactions favor peptide intramolecular interactions and are a major contributing factor to the structural compactness. These compact structures are amino acid sequence dependent and closely resemble standard secondary structures, as a consequence of VDW interactions and covalent bonding constraints. Hydrogen bonding is a short range interaction and it locks the approximate structure into the specific secondary structure when it is included in the simulation. In contrast to standard molecular simulations where the total energy is dominated by charge-charge interactions, these simulation results will give us a new view of the folding mechanism.     

Comparison of force fields to study the zinc-finger containing protein NPL4, a target for disulfiram in cancer therapy

Molecular dynamics (MD) simulations are a powerful approach to studying the structure and dynamics of proteins related to health and disease. Advances in the MD field allow modeling proteins with high accuracy. However, modeling metal ions and their interactions with proteins is still challenging. NPL4 is a zinc-binding protein and works as a cofactor for p97 to regulate protein homeostasis. NPL4 is of biomedical importance and has been proposed as the target of disulfiram, a drug recently repurposed for cancer treatment. Experimental studies proposed that the disulfiram metabolites, bis-(diethyldithiocarbamate)‑copper and cupric ions, induce NPL4 misfolding and aggregation. However, the molecular details of their interactions with NPL4 and consequent structural effects are still elusive. Here, biomolecular simulations can help to shed light on the related structural details. To apply MD simulations to NPL4 and its interaction with copper the first important step is identifying a suitable force field to describe the protein in its zinc-bound states. We examined different sets of non-bonded parameters because we want to study the misfolding mechanism and cannot rule out that the zinc may detach from the protein during the process and copper replaces it. We investigated the force-field ability to model the coordination geometry of the metal ions by comparing the results from MD simulations with optimized geometries from quantum mechanics (QM) calculations using model systems of NPL4. Furthermore, we investigated the performance of a force field including bonded parameters to treat copper ions in NPL4 that we obtained based on QM calculations.     

Interaction of multicomponent anionic liposomes with cationic pyridylphenylene dendrimer: Does the complex behavior depend on the liposome composition?

Dendrimers are individual macromolecular compounds having a great potential for biomedical application. The key step of the cell penetration by dendrimers is the interaction with lipid bilayer. Here, the interaction between cationic pyridylphenylene dendrimer of third generation (D₃⁵⁰⁺) and multicomponent liquid (CL/POPC), solid (CL/DPPC) and cholesterol-containing (CL/POPC/30% Chol) anionic liposomes was investigated by dynamic light scattering, fluorescence spectroscopy, conductometry, calorimetric studies and molecular dynamic (MD) simulations. Microelectrophoresis and MD simulations revealed the interaction is electrostatic and reversible with only part of pyridinium groups of dendrimers involved in binding with liposomes. The ability of dendrimer molecules to migrate between liposomes was discovered by the labeling liposomes with Rhodamine B. The phase state of the lipid membrane and the incorporation of cholesterol into the lipid bilayer were found to not affect the mechanism of the dendrimer - liposome complex formation. Rigid dendrimer adsorption on liposomal surface does not induce the formation of significant defects in the lipid membrane pave the way for possible biological application of pyridylphenylene dendrimers.     

Experimental,computational and chemometrics studies of BSA-vitamin B6 interaction by UV–Vis,FT-IR,fluorescence spectroscopy,molecular dynamics simulation and hard-soft modeling methods

The interaction of pyridoxine (Vitamin B6) with bovine serum albumin (BSA) is investigated under pseudo-physiological conditions by UV–Vis, fluorescence and FTIR spectroscopy. The intrinsic fluorescence of BSA was quenched by VB6, which was rationalized in terms of the static quenching mechanism. According to fluorescence quenching calculations, the bimolecular quenching constant (k_q), dynamic quenching (K_SV) and static quenching (K_LB) at 310 K were obtained. The efficiency of energy transfer and the distance between the donor (BSA) and the acceptor (VB6) were calculated by Foster’s non-radiative energy transfer theory and were equal to 41.1% and 2.11 nm.The collected UV–Vis and fluorescence spectra were combined into a row-and column-wise augmented matrix and resolved by multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS helped to estimate the stoichiometry of interactions, concentration profiles and pure spectra for three species (BSA, VB6 and VB6-BSA complex) existed in the interaction procedure. Based on the MCR-ALS results, using mass balance equations, a model was developed and binding constant of complex was calculated using non-linear least squares curve fitting. FT-IR spectra showed that the conformation of proteins was altered in presence of VB6. Finally, the combined docking and molecular dynamics (MD) simulations were used to estimate the binding affinity of VB6 to BSA. Five-nanosecond MD simulations were performed on bovine serum albumin (BSA) to study the conformational features of its ligand binding site. From MD results, eleven BSA snapshots were extracted, at every 0.5 ns, to explore the binding affinity (GOLD score) of VB6 using a docking procedure. MD simulations indicated that there is a considerable flexibility in the structure of protein that affected ligand recognition. Structural analyses and docking simulations indicated that VB6 binds to site I and GOLD score values depend on the conformations of both BSA and ligand. Molecular modeling results showed that VB6–BSA complex formed not only on the basis of electrostatic forces, but also on the basis of π–π staking and hydrogen bond. There was an excellent agreement between the experimental and computational results. The results presented in this paper, will offer a reference for detailed and systematic studies on the biological effects and action mechanism of small molecules with proteins.     

Atomic Level Rendering of DNA-Drug Encounter

Computer simulations have been demonstrated to be important for unraveling atomic mechanisms in biological systems. In this study, we show how combining unbiased molecular dynamic simulations with appropriate analysis tools can successfully describe metal-based drug interactions with DNA. To elucidate the noncovalent affinity of cisplatin’s family to DNA, we performed extensive all-atom molecular dynamics simulations (3.7 μs total simulation length). The results show that the parent drug, cisplatin, has less affinity to form noncovalent adducts in the major groove than its aquo complexes. Furthermore, the relative position in which the drugs enter the major groove is dependent on the compound’s net charge. Based on the simulations, we estimated noncovalent binding free energies through the use of Markov state models. In addition, and to overcome the lack of experimental information, we employed two additional methods: Molecular Mechanics Poisson-Boltzmann Surface Area (MMPB-SA) and steered molecular dynamics with the Jarzynski estimator, with an overall good agreement between the three methods. All complexes show interaction energies below 3 kcal/mol with DNA but the charged hydrolysis products have slightly more favorable binding free energies than the parent drug. Moreover, this study sets the precedent for future unbiased DNA-ligand simulations of more complex binders.     

Structure of a protein–detergent complex: the balance between detergent cohesion and binding

Despite the major interest in membrane proteins at functional, genomic, and therapeutic levels, their biochemical and structural study remains challenging, as they require, among other things, solubilization in detergent micelles. The complexity of this task derives from the dependence of membrane protein structure on their anisotropic environment, influenced by a delicate balance between many different physicochemical properties. To study such properties in a small protein–detergent complex, we used fluorescence measurements and molecular dynamics (MD) simulations on the transmembrane part of glycophorin A (GpAtm) solubilized in micelles of dihexanoylphosphatidylcholine (DHPC) detergent. Fluorescence measurements show that DHPC has limited ability to solubilize the peptide, while MD provides a possible molecular explanation for this. We observe that the detergent molecules are balanced between two different types of interactions: cohesive interactions between detergent molecules that hold the micelle together, and adhesive interactions with the peptide. While the cohesive interactions are detergent mediated, the adhesion to the peptide depends on the specific interactions between the hydrophobic parts of the detergent and the topography of the peptide dictated by the amino acids. The balance between these two parameters results in a certain frustration of the system and rather slow equilibration. These observations suggest how molecular properties of detergents could influence membrane protein stabilization and solubilization.     

Insights into the thermal stabilization and conformational transitions of DNA by hyperthermophile protein Sso7d: molecular dynamics simulations and MM-PBSA analysis

In the assembly of DNA-protein complex, the DNA kinking plays an important role in nucleoprotein structures and gene regulation. Molecular dynamics (MD) simulations were performed on specific protein-DNA complexes in this study to investigate the stability and structural transitions of DNA depending on temperature. Furthermore, we introduced the molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) approach to analyze the interactions between DNA and protein in hyperthermophile. Focused on two specific Sso7d-DNA complexes (PDB codes: 1BNZ and 1BF4), we performed MD simulations at four temperatures (300, 360, 420, and 480?K) and MM-PBSA at 300 and 360?K to illustrate detailed information on the changes of DNA. Our results show that Sso7d stabilizes DNA duplex over a certain temperature range and DNA molecules undergo B-like to A-like form transitions in the binary complex with the temperature increasing, which are consistent with the experimental data. Our work will contribute to a better understanding of protein-DNA interaction.     

Can molecular dynamics simulations improve the structural accuracy and virtual screening performance of GPCR models?

The determination of G protein-coupled receptor (GPCR) structures at atomic resolution has improved understanding of cellular signaling and will accelerate the development of new drug candidates. However, experimental structures still remain unavailable for a majority of the GPCR family. GPCR structures and their interactions with ligands can also be modelled computationally, but such predictions have limited accuracy. In this work, we explored if molecular dynamics (MD) simulations could be used to refine the accuracy of in silico models of receptor-ligand complexes that were submitted to a community-wide assessment of GPCR structure prediction (GPCR Dock). Two simulation protocols were used to refine 30 models of the D₃ dopamine receptor (D₃R) in complex with an antagonist. Close to 60 μs of simulation time was generated and the resulting MD refined models were compared to a D₃R crystal structure. In the MD simulations, the receptor models generally drifted further away from the crystal structure conformation. However, MD refinement was able to improve the accuracy of the ligand binding mode. The best refinement protocol improved agreement with the experimentally observed ligand binding mode for a majority of the models. Receptor structures with improved virtual screening performance, which was assessed by molecular docking of ligands and decoys, could also be identified among the MD refined models. Application of weak restraints to the transmembrane helixes in the MD simulations further improved predictions of the ligand binding mode and second extracellular loop. These results provide guidelines for application of MD refinement in prediction of GPCR-ligand complexes and directions for further method development.     

Insights into the thermal stabilization and conformational transitions of DNA by hyperthermophile protein Sso7d: molecular dynamics simulations and MM-PBSA analysis

In the assembly of DNA-protein complex, the DNA kinking plays an important role in nucleoprotein structures and gene regulation. Molecular dynamics (MD) simulations were performed on specific protein-DNA complexes in this study to investigate the stability and structural transitions of DNA depending on temperature. Furthermore, we introduced the molecular mechanics/Poisson–Boltzmann surface area (MM-PBSA) approach to analyze the interactions between DNA and protein in hyperthermophile. Focused on two specific Sso7d-DNA complexes (PDB codes: 1BNZ and 1BF4), we performed MD simulations at four temperatures (300, 360, 420, and 480?K) and MM-PBSA at 300 and 360?K to illustrate detailed information on the changes of DNA. Our results show that Sso7d stabilizes DNA duplex over a certain temperature range and DNA molecules undergo B-like to A-like form transitions in the binary complex with the temperature increasing, which are consistent with the experimental data. Our work will contribute to a better understanding of protein-DNA interaction.     

Understanding antibody–antigen associations by molecular dynamics simulations: Detection of important intra- and inter-molecular salt bridges

1 NSec molecular dynamics (MD) simulation of anti-hen egg white antibody, HyHEL63 (HH63), complexed with HEL reveals important molecular interactions, not revealed in its X-ray crystal structure. These molecular interactions were predicted to be critical for the complex formation, based on structure–function studies of this complex and 3-other anti-HEL antibodies, HH8, HH10 and HH26, HEL complexes. All four antibodies belong to the same structural family, referred to here as HH10 family. Ala scanning results show that they recognize ‘coincident epitopes’. 1 NSec explicit, with periodic boundary condition, MD simulation of HH63-HEL reveals the presence of functionally important salt-bridges. Around 200 ps in vacuo and an additional 20 ps explicit simulation agree with the observations from 1 Nsec simulation. Intra-molecular salt-bridges predicted to play significant roles in the complex formation, were revealed during MD simulation. A very stabilizing salt-bridge network, and another intra-molecular salt-bridge, at the binding site of HEL, revealed during the MD simulation, is proposed to predipose binding site geometry for specific binding. All the revealed salt-bridges are present in one or more of the other three complexes and/or involve “hot-spot” epitope and paratope residues. Most of these charged epitope residues make large contribution to the binding free energy. The “hot spot” epitope residue Lys97_Y, which significantly contributes to the free energy of binding in all the complexes, forms an intermolecular salt-bridge in several MD conformers. Our earlier computations have shown that this inter-molecular salt-bridge plays a significant role in determining specificity and flexibility of binding in the HH8-HEL and HH26-HEL complexes. Using a robust criterion of salt-bridge detection, this inter-molecular salt-bridge was detected in the native structures of the HH8-HEL and HH26-HEL complexes, but was not revealed in the crystal structure of HH63-HEL complex. The electrostatic strength of this revealed salt-bridge was very strong. During 1 Nsec MD simulation this salt-bridge networks with another inter-molecular salt-bridge to form an inter-molecular salt-bridge triad. Participation of Lys97_Y in the formation of inter-molecular triad further validates the functional importance of Lys97_Y in HH63-HEL associations. These results demonstrate that many important structural details of biomolecular interactions can be better understood when studied in a dynamic environment, and that MD simulations can complement and expand information obtained from static X-ray structure. This study also highlights “hot-spot” molecular interactions in HyHEL63-HEL complex. The publisher or recipient acknowledges right of the U.S. Government to retain a non exclusive, royalty-free license in and to any copyright covering the article.