Explicit Water Molecular Dynamics Research Articles

Implicit Solvent with Explicit Ions Generalized Born Model in Molecular Dynamics: Application to DNA.

The ion atmosphere surrounding highly charged biomolecules, such as nucleic acids, is crucial for their dynamics, structure, and interactions. Here, we develop an approach for the explicit treatment of ions within an implicit solvent framework suitable for atomistic simulations of biomolecules. The proposed implicit solvent/explicit ions model, GBION, is based on a modified generalized Born (GB) model; it includes separate, modified GB terms for solute-ion and ion-ion interactions. The model is implemented in the AMBER package (version 24), and its performance is thoroughly investigated in atomistic molecular dynamics (MD) simulations of double-stranded DNA on a microsecond time scale. The aggregate characteristics of monovalent (Na+ and K+) and trivalent (Cobalt Hexammine, CoHex3+) counterion distributions around double-stranded DNA predicted by the model are in reasonable agreement with the experiment (where available), all-atom explicit water MD simulations, and the expectation from the Manning condensation theory. The radial distributions of monovalent cations around DNA are reasonably close to the ones obtained using the explicit water model: expressed in units of energy, the maximum deviations of local ion concentrations from the explicit solvent reference are within 1 kBT, comparable to the corresponding deviations expected between different established explicit water models. The proposed GBION model is able to simulate DNA fragments in a large volume of solvent with explicit ions with little additional computational overhead compared with the fully implicit GB treatment of ions. Ions simulated using the developed model explore conformational space at least 2 orders of magnitude faster than in the explicit solvent. These advantages allowed us to observe and explore an unexpected "stacking" mode of DNA condensation in the presence of trivalent counterions (CoHex3+) that was revealed by recent experiments.

Exploring the Functional Landscape of the p53 Regulatory Domain: The Stabilizing Role of Post-Translational Modifications.

This study focuses on the intrinsically disordered regulatory domain of p53 and the impact of post-translational modifications. Through fully atomistic explicit water molecular dynamics simulations, we show the wealth of information and detailed understanding that can be obtained by varying the number of phosphorylated amino acids and implementing a restriction in the conformational entropy of the N-termini of that intrinsically disordered region. The take-home message for the reader is to achieve a detailed understanding of the impact of phosphorylation with respect to (1) the conformational dynamics and flexibility, (2) structural effects, (3) protein interactivity, and (4) energy landscapes and conformational ensembles. Although our model system is the regulatory domain p53 of the tumor suppressor protein p53, this study contributes to understanding the general effects of intrinsically disordered phosphorylated proteins and the impact of phosphorylated groups, more specifically, how minor changes in the primary sequence can affect the properties mentioned above.

Influence of the Dielectric Constant on the Ionic Current Rectification of Bipolar Nanopores.

In this paper, we investigate how the dielectric constant, ϵ, of an electrolyte solvent influences the current rectification characteristics of bipolar nanopores. It is well recognized that bipolar nanopores with two oppositely charged regions rectify current when exposed to an alternating electric potential difference. Here, we consider dilute electrolytes with NaCl only and with a mixture of NaCl and charged nanoparticles. These systems are studied using two levels of description, all-atom explicit water molecular dynamics (MD) simulations and coarse-grained implicit solvent MD simulations. The charge density and electric potential profiles and current-voltage relationship predicted by the implicit solvent simulations with ϵ = 11.3 show good agreement with the predictions from the explicit water simulations. Under nonequilibrium conditions, the predictions of the implicit solvent simulations with a dielectric constant closer to the one of bulk water are significantly different from the predictions obtained with the explicit water model. These findings are closely aligned with experimental data on the dielectric constant of water when confined to nanometric spaces, which suggests that ϵ decreases significantly compared to its value in the bulk. Moreover, the largest electric current rectification is observed in systems containing nanoparticles when ϵ = 78.8. Using enhanced sampling, we have shown that this larger rectification arises from the presence of a significantly deeper minimum in the free energy of the system with a larger ϵ, and when a negative voltage bias is applied. Since implicit solvent models and mean-field continuum theories are often used to design Janus membranes based on bipolar nanopores, this work highlights the importance of properly accounting for the effects of confinement on the dielectric constant of the electrolyte solvent. The results presented here indicate that the dielectric constant in implicit solvent simulations may be used as an adjustable parameter to approximately account for the effects of nanometric confinement on aqueous electrolyte solvents.

Effect of caffeine on the aggregation of amyloid-β-A 3D RISM study.

Alzheimer's disease is a detrimental neurological disorder caused by the formation of amyloid fibrils due to the aggregation of amyloid-β peptide. The primary therapeutic approaches for treating Alzheimer's disease are targeted to prevent this amyloid fibril formation using potential inhibitor molecules. The discovery of such inhibitor molecules poses a formidable challenge to the design of anti-amyloid drugs. This study investigates the effect of caffeine on dimer formation of the full-length amyloid-β using a combined approach of all-atom, explicit water molecular dynamics simulations and the three-dimensional reference interaction site model theory. The change in the hydration free energy of amyloid-β dimer, with and without the inhibitor molecules, is calculated with respect to the monomeric amyloid-β, where the hydration free energy is decomposed into energetic and entropic components, respectively. Dimerization is accompanied by a positive change in the partial molar volume. Dimer formation is spontaneous, which implies a decrease in the hydration free energy. However, a reverse trend is observed for the dimer with inhibitor molecules. It is observed that the negatively charged residues primarily contribute for the formation of the amyloid-β dimer. A residue-wise decomposition reveals that hydration/dehydration of the side-chain atoms of the charged amino acid residues primarily contribute to dimerization.

Analyzing the binding affinity of potential homodimers produced by the C55 portion of the amyloid precursor protein, a molecular dynamics study.

Alzheimer's disease is a neurodegenerative disease that affects millions of people in the United States alone. Agglomeration of amyloid beta (Aβ) peptides is a potential cause of the disease and the loss of mental acuity it induces. Aβ is created via the cleavage of the amyloid precursor protein (APP). When APP is cleaved, part of it—approximately the C99 fragment—remains in the lipid membrane, with its N-terminus being the Aβ peptide. The dimerization of C99 is theorized to limit its further cleavage and thereby limit the production of Aβ. However, this has yet to be proven experimentally. The first 55 residues of the C99 fragment (called C55) contains both the transmembrane domain and Aβ. In this work, we explored multiple potential conformations of C55 dimers (starting from the PDB ID 2LP1) and calculated their binding affinity to better understand the viability of homodimerization. The homodimer conformations were explored using both implicit and explicit water Molecular Dynamics simulations. We used an Extended Adaptive Biasing Force method to calculate the binding affinity of these dimers in a POPC lipid membrane. This methodology is characterized by the use of a continuously updated biasing force that cancels all force along a specified reaction coordinate and, in this way, enables uniform sampling on a flat free-energy surface. Applying this methodology using collective variables for root-mean-square deviation, spin, tilt, and Euclidean distance for both the bulk (monomer) and bound (dimer) states, we compute a binding affinity of roughly −4 kcal/mol for one of our dimer conformations. This lends in silica credence to the hypothesis that the dimerization of C55 limits the formation of Aβ, as the dimer appears energetically viable.

Aggregation propensities of proteins with varying degrees of disorder.

The hydration thermodynamics of a globular protein (AcP), three intrinsically disordered protein regions (1CD3, 1MVF, 1F0R) and a fully disordered protein (α-synuclein) is studied by an approach that combines an all-atom explicit water molecular dynamics simulations and three-dimensional reference interaction site model (3D-RISM) theory. The variation in hydration free energy with percentage disorder of the selected proteins is investigated through its nonelectrostatic and electrostatic components. A decrease in hydration free energy is observed with an increase in percentage disorder, indicating favorable interactions of the disordered proteins with the solvent. This confirms the role of percentage disorder in determining the aggregation propensity of proteins which is measured in terms of the hydration free energy in addition to their respective mean net charge and mean hydrophobicity. The hydration free energy is decoupled into energetic and entropic terms. A residue-wise decomposition analysis of the hydration free energy for the selected proteins is evaluated. The decomposition shows that the disordered regions contribute more than the ordered ones for the intrinsically disordered protein regions. The dominant role of electrostatic interactions is confirmed from the residue-wise decomposition of the hydration free energy. The results depict that the negatively charged residues contribute more to the total hydration free energy for the proteins with negative mean net charge, while the positively charged residues contribute more for proteins with positive mean net charge.

Capturing the Recognition Dynamics of para-Sulfonato-calix[4]arenes by Cytochrome c: Toward a Quantitative Free Energy Assessment.

Calix[n]arenes' selective recognition of protein surfaces covers a broad range of timely applications, from controlling protein assembly and crystallization to trapping partially disordered proteins. Here, the interaction of para-sulfonated calix-[4]-arenes with cytochrome c is investigated through all-atom, explicit water molecular dynamics simulations which allow characterization of two binding sites in quantitative agreement with experimental evidence. Free energy calculations based on the MM-PBSA and the attach-pull-release (APR) methods highlight key residues implicated in the recognition process and provide binding free energy results in quantitative agreement with isothermal titration calorimetry. Our study emphasizes the role of MD simulations to capture and describe the "walk" of sulfonated calix-[4]-arenes on the cytochrome c surface, with the arginine R13 as a pivotal interacting residue. Our MD investigation allows, through the quasi-harmonic multibasin (QHMB) method, probing an allosteric reinforcement of several per-residue interactions upon calixarene binding, which suggests a more complex mode of action of these supramolecular auxiliaries.

Exploration of Somatostatin Binding Mechanism to Somatostatin Receptor Subtype 4.

Somatostatin (also named as growth hormone-inhibiting hormone or somatotropin release-inhibiting factor) is a regulatory peptide important for the proper functioning of the endocrine system, local inflammatory reactions, mood and motor coordination, and behavioral responses to stress. Somatostatin exerts its effects via binding to G-protein-coupled somatostatin receptors of which the fourth subtype (SSTR4) is a particularly important receptor mediating analgesic, anti-inflammatory, and anti-depressant effects without endocrine actions. Thus, SSTR4 agonists are promising drug candidates. Although the knowledge of the atomic resolution-binding modes of SST would be essential for drug development, experimental elucidation of the structures of SSTR4 and its complexes is still awaiting. In the present study, structures of the somatostatin–SSTR4 complex were produced using an unbiased, blind docking approach. Beyond the static structures, the binding mechanism of SST was also elucidated in the explicit water molecular dynamics (MD) calculations, and key binding modes (external, intermediate, and internal) were distinguished. The most important residues on both receptor and SST sides were identified. An energetic comparison of SST binding to SSTR4 and 2 offered a residue-level explanation of receptor subtype selectivity. The calculated structures show good agreement with available experimental results and indicate that somatostatin binding is realized via prerequisite binding modes and an induced fit mechanism. The identified binding modes and the corresponding key residues provide useful information for future drug design targeting SSTR4.

Adaptive Synthesis of Functional Amphiphilic Dendrons as a Novel Approach to Artificial Supramolecular Objects.

Supramolecular structures, such as micelles, liposomes, polymerosomes or dendrimerosomes, are widely studied and used as drug delivery systems. The behavior of amphiphilic building blocks strongly depends on their spatial distribution and shape of polar and nonpolar component. This report is focused on the development of new versatile synthetic protocols for amphiphilic carbosilane dendrons (amp-CS-DDNs) capable of self-assembly to regular micelles and other supramolecular objects. The presented strategy enables the fine modification of amphiphilic structure in several ways and also enables the facile connection of a desired functionality. DLS experiments demonstrated correlations between structural parameters of amp-CS-DDNs and the size of formed nanoparticles. For detailed information about the organization and spatial distribution of amp-CS-DDNs assemblies, computer simulation models were studied by using molecular dynamics in explicit water.

Non-Markovian modeling of protein folding

We extract the folding free energy landscape and the time-dependent friction function, the two ingredients of the generalized Langevin equation (GLE), from explicit-water molecular dynamics (MD) simulations of the α-helix forming polypeptide [Formula: see text] for a one-dimensional reaction coordinate based on the sum of the native H-bond distances. Folding and unfolding times from numerical integration of the GLE agree accurately with MD results, which demonstrate the robustness of our GLE-based non-Markovian model. In contrast, Markovian models do not accurately describe the peptide kinetics and in particular, cannot reproduce the folding and unfolding kinetics simultaneously, even if a spatially dependent friction profile is used. Analysis of the GLE demonstrates that memory effects in the friction significantly speed up peptide folding and unfolding kinetics, as predicted by the Grote-Hynes theory, and are the cause of anomalous diffusion in configuration space. Our methods are applicable to any reaction coordinate and in principle, also to experimental trajectories from single-molecule experiments. Our results demonstrate that a consistent description of protein-folding dynamics must account for memory friction effects.

Individual Ion Activity Coefficients in Aqueous Electrolytes from Explicit-Water Molecular Dynamics Simulations.

We compute individual ion activity coefficients (IIACs) in aqueous NaCl, KCl, NaF, and KF solutions from explicit-water molecular dynamics simulations. Free energy changes are obtained from insertion of single ions-accompanied by uniform neutralizing backgrounds-into solution by gradually turning on first Lennard-Jones interactions, followed by Coulombic interactions using Ewald electrostatics. Simulations are performed at multiple system sizes, and all results are extrapolated to the thermodynamic limit, thus eliminating any possible artifacts from the neutralizing backgrounds. Because of controversies associated with measurements of IIACs from electrochemical cells with ion-selective electrodes, the reported experimental data are not widely accepted; thus there remains a knowledge gap with respect to the contributions of individual ions to solution nonidealities. Our results are in good qualitative agreement with these reported measurements, though significantly larger in magnitude. In particular, the relative positioning for the activity coefficients of anions and cations matches the experimental ordering for all four systems. This work establishes a robust thermodynamic framework, without a need to invoke extra hypotheses, that sheds light on the behavior of individual ions and their contributions to nonidealities of aqueous electrolyte solutions.

Explanation of the Formation of Complexes between Representatives of Oxazolidinones and HDAS-β-CD Using Molecular Modeling as a Complementary Technique to cEKC and NMR.

Molecular modeling (MM) results for tedizolid and radezolid with heptakis-(2,3-diacetyl-6-sulfo)-β-cyclodextrin (HDAS-β-CD) are presented and compared with the results previously obtained for linezolid and sutezolid. The mechanism of interaction of chiral oxazolidinone ligands belonging to a new class of antibacterial agents, such as linezolid, tedizolid, radezolid, and sutezolid, with HDAS-β-CD based on capillary electrokinetic chromatography (cEKC), nuclear magnetic resonance (NMR) spectroscopy, and MM methods was described. Principles of chiral separation of oxazolidinone analogues using charged single isomer derivatives of cyclodextrin by the cEKC method were presented, including the selection of the optimal chiral selector and separation conditions, complex stoichiometry, and binding constants, which provided a comprehensive basis for MM studies. In turn, NMR provided, where possible, direct information on the geometry of the inclusion complexes and also provided the necessary structural information to validate the MM calculations. Consequently, MM contributed to the understanding of the structure of diastereomeric complexes, the thermodynamics of complexation, and the visualization of their structures. The most probable mean geometries of the studied supramolecular complexes and their dynamics (geometry changes over time) were determined by molecular dynamics methods. Oxazolidinone ligands have been shown to complex mainly the inner part of cyclodextrin, while the external binding is less privileged, which is consistent with the conclusions of the NMR studies. Enthalpy values of binding of complexes were calculated using long-term molecular dynamics in explicit water as well as using molecular mechanics, the Poisson–Boltzmann or generalized Born, and surface area continuum solvation (MM/PBSA and MM/GBSA) methods. Computational methods predicted the effect of changes in pH and composition of the solution on the strength and complexation process, and it adapted the conditions selected as optimal during the cEKC study. By changing the dielectric constant in the MM/PBSA and MM/GBSA calculations, the effect of changing the solution to methanol/acetonitrile was investigated. A fairly successful attempt was made to predict the chiral separation of the oxazolidinones using the modified cyclodextrin by computational methods.

CuFeO2–Water Interface under Illumination: Structural, Electronic, and Catalytic Implications for the Hydrogen Evolution Reaction

CuFeO2 is a p-type semiconductor that has been recently identified as a promising photocathode material for photoelectrochemical water splitting. CuFeO2 can absorb solar light and promote the hydrogen evolution reaction (HER), even though the photocurrents achieved so far are still well below the theoretical upper limit. While several experimental and theoretical works have provided a detailed characterization of the bulk properties of this material, surfaces have been largely unexplored. In this work, we perform first-principles simulations based on DFT to investigate the structure, electronic properties, and thermodynamic stability of CuFeO2 surfaces both in vacuum and in an electrochemical environment. To estimate the alignment of the band edges on the electrochemical scale, we perform ab initio molecular dynamics in explicit water, unraveling the structure of the solid/liquid interface for various surface terminations. We consider the system both in the dark and under illumination, showing that light absorption can induce partial reduction of the surface, giving rise to states in the gap that can pin the Fermi level, in agreement with recent measurements. Using the free energy of adsorption of atomic hydrogen as a descriptor of the catalytic activity for the HER, we show that hydride species formed at oxygen vacancies can be highly active and could therefore be an intermediate of reaction.

Hydration Thermodynamics of the N-Terminal FAD Mutants of Amyloid-β.

The hydration thermodynamics of amyloid-β (Aβ) and its pathogenic familial Alzheimer's disease (FAD) mutants such as A2V, Taiwan (D7H), Tottori (D7N), and English (H6R) and the protective A2T mutant is investigated by a combination of all-atom, explicit water molecular dynamics (MD) simulations and the three-dimensional reference interaction site model (3D-RISM) theory. The change in the hydration free energy on mutation is decomposed into the energetic and entropic components, which comprise electrostatic and nonelectrostatic contributions. An increase in the hydration free energy is observed for A2V, D7H, D7N, and H6R mutations that increase the aggregation propensity of Aβ and lead to an early onset of Alzheimer's disease, while a reverse trend is noted for the protective A2T mutation. An antiphase correlation is found between the change in the hydration energy and the internal energy of Aβ upon mutation. A residue-wise decomposition analysis shows that the change in the hydration free energy of Aβ on mutation is primarily due to the hydration/dehydration of the side-chain atoms of the negatively charged residues. The decrease in the hydration of the negatively charged residues on mutation may decrease the solubility of the mutant, which increases the observed aggregation propensity of the FAD mutants. Results obtained from the theory show an excellent match with the experimentally reported data.

Identification of Tractable Drug-like eIF4Al Inhibitors with Potent Anti-Tumor Activity

Clinical efficacy of targeted signaling inhibitors for hematologic malignancies is limited bynoutgrowth of subpopulations with alternative pathways independent of the drug target. The eIF4F complex responsible for translation initiation is a convergence point for cancer-promoting signaling pathways and its inhibition leads to decreased expression of key oncoproteins and apoptosis. Lymphomas and leukemias show particular dependence on constitutive eIF4F activation. Indeed, natural compounds targeting the eIF4F enzymatic component, eIF4A1, demonstrate activities in vitro and in vivo against lymphoma and leukemia model systems, among other tumor types. The natural compound silvestrol is a potent inhibitor of eIF4A1, results in cancer cell cytotoxicity, and has an established therapeutic window in vivo. Silvestrol shows potent antitumor activity against 924 pan-cancer tumor cell lines with 830/924 (90%) sensitive at IC50 <100nM with lymphoma and leukemia cell lines being particularly sensitive. Silvestrol and other natural compounds, however, lack core drug-like properties and synthetic tractability. To discover new, specific and tractable inhibitors of eIF4A1 that are more drug-like, we have constructed several molecular models that we used to virtually screen more than 20 million compounds. eIF4A1 is the founding member of the DEAD-box RNA helicases, which include its paralogs eIF4A2 (91% amino-acid identity with eIF4A1) and eIF4A3 (60% identity). All DEAD-box helicases contain two RecA-like domains separated by a flexible linker. The cleft between these domains is lined with helicase motifs that mediate nucleotide binding and hydrolysis. In an absence of RNA or nucleotide, eIF4A proteins adopt diffuse open conformations; binding of RNA and ATP triggers transition to a more stable closed state. Modeling small-molecule interactions in the nucleotide cleft of eIF4A1 therefore assesses ability of molecules to lock eIF4A1 in a conformation unable to cycle through ATPase and helicase activities. A new crystal structure of eIF4A1 has become available (2019) with a resolution of 2 angstroms. The protein is co-crystallized with ANP in the nucleotide binding site at the interface of the N and C-terminal domains and with known inhibitor, Rocaglamide, bound to the interface of the eIF4A1and a polypurine RNA. We used this high-resolution crystal structure to build models predicting interactions of small molecules in the interdomain nucleotide-binding cleft. We then performed all-atom explicit-water molecular dynamics (MD) simulations for 500-700 ns to study conformational dynamics and atomic interactions of ATP-bound and ATP-unbound states. Extended molecular dynamic simulations confirm the hypothesis that rocaglamide stabilizes the interaction between the helicase and a polypurine sequence on RNA, thus preventing further ATPase activity and RNA unwinding. Pooling these results, we constructed two homology models of human eIF4A1 with both open and closed conformations as structural templates. Over 50 compounds identified as hits in silicowere ordered and tested thus far in our biochemical and cell-based validation platform. Using our machine learning and virtual screening approach targeted to the ATP binding site of eIF4A1, we identified a promising piperazine-amide fragment scaffold (UM107; ~300 MW) with similar electronics to nucleotide triphosphates. UM107 caused cellular toxicities with an LD50 of 50 uM and was weakly active in the biochemical screen against eIF4A1 with an IC50 of 250 uM. We will increase molecular weight by adding more groups to maximize hydrogen bond interactions in the active site. These analogs will be synthesized and screened virtually building on the core using established medicinal chemistry optimization tools followed by biochemical and cellular validation. We therefore have developed an accurate and novel in silico models of eIF4A1 highly useful in assessing interactions of small-molecule ATPase inhibitors, with focus on the ATP-binding cleft. Disclosures No relevant conflicts of interest to declare.

Aqueous Nanoclusters Govern Ion Partitioning in Dense Polymer Membranes.

The uptake and sorption of charged molecules by responsive polymer membranes and hydrogels in aqueous solutions is of key importance for the development of soft functional materials. Here, we investigate the partitioning of simple monatomic (Na+, K+, Cs+, Cl–, I–) and one molecular ion (4-nitrophenolate; NP–) within a dense, electroneutral poly(N-isopropylacrylamide) membrane using explicit-water molecular dynamics simulations. Inside the predominantly hydrophobic environment, water distributes in a network of polydisperse water nanoclusters. The average cluster size determines the mean electrostatic self-energy of the simple ions, which preferably reside deeply inside them; we therefore find substantially larger partition ratios K ≃10–1 than expected from a simple Born picture using a uniform dielectric constant. Despite their irregular shapes, we observe that the water clusters possess a universal negative electrostatic potential with respect to their surroundings, as is known for aqueous liquid–vapor interfaces. This potential, which we find concealed in cases of symmetric monatomic salts, can dramatically impact the transfer free energies of larger charged molecules because of their weak hydration and increased affinity to interfaces. Consequently, and in stark contrast to the simple ions, the molecular ion NP– can have a partition ratio much larger than unity, K ≃10–30 (depending on the cation type) or even 103 in excess of monovalent salt, which explains recent observations of enhanced reaction kinetics of NP– reduction catalyzed within dense polymer networks. These results also suggest that ionizing a molecule can even enhance the partitioning in a collapsed, rather hydrophobic gel, which strongly challenges the traditional simplistic reasoning.

Explicit Characterization of the Free Energy Landscape of pKID-KIX Coupled Folding and Binding.

The most fundamental aspect of the free energy landscape of proteins is that it is globally funneled such that protein folding is energetically biased. Then, what are the distinctive characteristics of the landscape of intrinsically disordered proteins, apparently lacking such energetic bias, that nevertheless fold upon binding? Here, we address this fundamental issue through the explicit characterization of the free energy landscape of the paradigmatic pKID–KIX system (pKID, phosphorylated kinase-inducible domain; KIX, kinase interacting domain). This is done based on unguided, fully atomistic, explicit-water molecular dynamics simulations with an aggregated simulation time of >30 μs and on the computation of the free energy that defines the landscape. We find that, while the landscape of pKID before binding is considerably shallower than the one for a protein that autonomously folds, it becomes progressively more funneled as the binding of pKID with KIX proceeds. This explains why pKID is disordered in a free state, and the binding of pKID with KIX is a prerequisite for pKID’s folding. In addition, we observe that the key event in completing the pKID–KIX coupled folding and binding is the directed self-assembly where pKID is docked upon the KIX surface to maximize the surface electrostatic complementarity, which, in turn, require pKID to adopt the correct folded structure. This key process shows up as the free energy barrier in the pKID landscape separating the intermediate nonspecific complex state and the specific complex state. The present work not only provides a detailed molecular picture of the coupled folding and binding of pKID but also expands the funneled landscape perspective to intrinsically disordered proteins.

Cross-linker effect on solute adsorption in swollen thermoresponsive polymer networks.

The selective solute partitioning within a polymeric network is of key importance to applications in which controlled release or uptake of solutes in a responsive hydrogel is required. In this work we investigate the impact of cross-links on solute adsorption in a swollen polymer network by means of all-atom, explicit-water molecular dynamics simulations. We focus on a representative network subunit consisting of poly(N-isopropylacrylamide) (PNIPAM) and N,N'-methylenebisacrylamide (BIS/MBA) cross-linker types. Our studied system consists of one BIS-linker with four atactic PNIPAM chains attached in a tetrahedral geometry. The adsorption of several representative solutes of different polarity in the low concentration limit at the linker region is examined. We subdivide the solute adsorption regions and distinguish between contributions stemming from polymer chains and cross-link parts. In comparison to a single polymer chain, we observe that the adsorption of the solutes to the cross-link region can significantly differ, with details depending on the specific compounds' size and polarity. In particular, for solutes that have already a relatively large affinity to PNIPAM chains the dense cross-link region (where many-body attractions are at play) amplifies the local adsorption by an order of magnitude. We also find that the cross-link region can serve as a seed for the aggregation of mutually attractive solutes at higher solute concentrations. Utilizing the microscopic adsorption coefficients in a mean-field model of an idealized macroscopic polymer network, we extrapolate these results to the global solute partitioning in a swollen hydrogel and predict that these adsorption features may lead to non-monotonic partition ratios as a function of the cross-link density.

Computational Identification of Tractable Drug-like Inhibitors of eIF4A1

Targeted signaling inhibitors for hematologic malignancies often lead to limited clinical efficacy due to the outgrowth of subpopulations with alternative pathways independent of the drug target. The eIF4F complex responsible for translation initiation is a convergence point for many cancer-promoting signaling pathways and its inhibition leads to decreased expression of key oncoproteins and apoptosis. Lymphomas and leukemias show particular dependence on constitutive eIF4F activation. Indeed, natural compounds targeting the eIF4F enzymatic component, eIF4A1, demonstrate activities both in vitro and in vivo against lymphoma and leukemia model systems, among other tumor types. eIF4A1 is a noteworthy target for hematologic malignancies based on the finding that BCR stimulation leads to increased mRNA translation primary CLL patient samples. Additionally, eIF4F components eIF4A1 and eIF4G1 had increased expression upon IgM-induced BCR activation. The natural compound silvestrol is a potent inhibitor of eIF4A1, results in cancer cell cytotoxicity, and has an established therapeutic window in vivo. Silvestrol shows potent antitumor activity against 924 pan-cancer tumor cell lines with 830/924 (90%) sensitive at IC50 <100nM with lymphoma and leukemia cell lines being particularly sensitive. Silvestrol and other natural compounds, however, lack core drug-like properties and synthetic tractability.To discover new, specific and tractable inhibitors of eIF4A1 that are more drug-like, we have constructed several molecular models that we used to virtually screen more than 20 million compounds. eIF4A1 is the founding member of the DEAD-box RNA helicases, which include its paralogs eIF4A2 (91% amino-acid identity with eIF4A1) and eIF4A3 (60% identity). All DEAD-box helicases contain two RecA-like domains separated by a flexible linker. The cleft between these domains is lined with helicase motifs that mediate nucleotide binding and hydrolysis. In an absence of RNA or nucleotide, eIF4A proteins adopt diffuse open conformations; binding of RNA and ATP triggers transition to a more stable closed state. Modeling small-molecule interactions in the nucleotide cleft of eIF4A1 therefore assesses ability of molecules to lock eIF4A1 in a conformation unable to cycle through ATPase and helicase activities. Although no experimentally derived structure of human eIF4A1 co-crystalized with ATP exists, crystal structures of other DEAD-box family members with similar motifs permit detailed studies of nucleotide and ligand-binding and the development of homology models. We have used four available high-resolution crystal structures to build models predicting interactions of small molecules in the interdomain nucleotide-binding cleft. We identified nucleotide binding-site residues and accurately reproduced ATP interactions for all four models (derived from PDB: 2J0S, 1FUU, 2VSO, 2DB3). We then performed all-atom explicit-water molecular dynamics (MD) simulations for 500-700 ns to study conformational dynamics and atomic interactions of ATP-bound and ATP-unbound states. Yeast eif4A crystal structure (PDB:1FUU) in the open state, for example, illustrated closure of the two RecA domains upon ATP binding. As expected, ATP makes strong interactions with the N-terminal, while phosphate groups extend to the C-terminal interacting with arginines, bringing the two RecA domains together. In contrast, we did not observe domain closure in the same simulation with 1FUU without ATP bound. We also assessed 2J0S, a crystal structure of human closed eIF4A3 bound to ANP. ATP docked to this active site followed by 500 ns MD held the protein in the closed state with several interdomain interactions. Upon nucleotide removal, marked RecA separation occurred. We observed similar domain closure and opening for PDB: 2VSO and 2DB3. Pooling these results, we constructed two homology models of human eIF4A1 with both open (2VSO, 1FUU) and closed conformations (2J0S) as structural templates. We therefore have developed accurate and novel in silico models of eIF4A1 highly useful in assessing interactions of small-molecule ATPase inhibitors, with focus on the ATP-binding cleft. DisclosuresNo relevant conflicts of interest to declare.

The Hypervariable Loops of Free TCRs Sample Multiple Distinct Metastable Conformations in Solution.

CD4+ and CD8+ αβ T cell antigen recognition is determined by the interaction between the TCR Complementarity Determining Region (CDR) loops and the peptide-presenting MHC complex. These T cells are known for their ability to recognize multiple pMHC complexes, and for a necessary promiscuity that is required for their selection and function in the periphery. Crystallographic studies have previously elucidated the role of structural interactions in TCR engagement, but our understanding of the dynamic process that occurs during TCR binding is limited. To better understand the dynamic states that exist for TCR CDR loops in solution, and how this relates to their states when in complex with pMHC, we simulated the 2C T cell receptor in solution using all-atom molecular dynamics in explicit water and constructed a Markov State Model for each of the CDR3α and CDR3β loops. These models reveal multiple metastable states for the CDR3 loops in solution. Simulation data and metastable states reproduce known CDR3β crystal conformations, and reveal several novel conformations suggesting that CDR3β bound states are the result of search processes from nearby pre-existing equilibrium conformational states. Similar simulations of the invariant, Type I Natural Killer T cell receptor NKT15, which engages the monomorphic, MHC-like CD1d ligand, demonstrate that iNKT TCRs also have distinct states, but comparatively restricted degrees of motion.