Microsecond Molecular Dynamics Simulations Research Articles

Insights into the Allosteric Effect of SENP1 Q597A Mutation on the Hydrolytic Reaction of SUMO1 via an Integrated Computational Study.

Small ubiquitin-related modifier (SUMO)-specific protease 1 (SENP1) is a cysteine protease that catalyzes the cleavage of the C-terminus of SUMO1 for the processing of SUMO precursors and deSUMOylation of target proteins. SENP1 is considered to be a promising target for the treatment of hepatocellular carcinoma (HCC) and prostate cancer. SENP1 Gln597 is located at the unstructured loop connecting the helices α4 to α5. The Q597A mutation of SENP1 allosterically disrupts the hydrolytic reaction of SUMO1 through an unknown mechanism. Here, extensive multiple replicates of microsecond molecular dynamics (MD) simulations, coupled with principal component analysis, dynamic cross-correlation analysis, community network analysis, and binding free energy calculations, were performed to elucidate the detailed mechanism. Our MD simulations showed that the Q597A mutation induced marked dynamic conformational changes in SENP1, especially in the unstructured loop connecting the helices α4 to α5 which the mutation site occupies. Moreover, the Q597A mutation caused conformational changes to catalytic Cys603 and His533 at the active site, which might impair the catalytic activity of SENP1 in processing SUMO1. Moreover, binding free energy calculations revealed that the Q597A mutation had a minor effect on the binding affinity of SUMO1 to SENP1. Together, these results may broaden our understanding of the allosteric modulation of the SENP1−SUMO1 complex.

Singular Interface Dynamics of the SARS-CoV-2 Delta Variant Explained with Contact Perturbation Analysis.

Emerging SARS-CoV-2 variants raise concerns about our ability to withstand the Covid-19 pandemic, and therefore, understanding mechanistic differences of those variants is crucial. In this study, we investigate disparities between the SARS-CoV-2 wild type and five variants that emerged in late 2020, focusing on the structure and dynamics of the spike protein interface with the human angiotensin-converting enzyme 2 (ACE2) receptor, by using crystallographic structures and extended analysis of microsecond molecular dynamics simulations. Dihedral angle principal component analysis (PCA) showed the strong similarities in the spike receptor binding domain (RBD) dynamics of the Alpha, Beta, Gamma, and Delta variants, in contrast with those of WT and Epsilon. Dynamical perturbation networks and contact PCA identified the peculiar interface dynamics of the Delta variant, which cannot be directly imputable to its specific L452R and T478K mutations since those residues are not in direct contact with the human ACE2 receptor. Our outcome shows that in the Delta variant the L452R and T478K mutations act synergistically on neighboring residues to provoke drastic changes in the spike/ACE2 interface; thus a singular mechanism of action eventually explains why it dominated over preceding variants.

In Silico Drug Design of Benzothiadiazine Derivatives Interacting with Phospholipid Cell Membranes.

The use of drugs derived from benzothiadiazine, a bicyclic heterocyclic benzene derivative, has become a widespread treatment for diseases such as hypertension, low blood sugar or the human immunodeficiency virus, among others. In this work we have investigated the interactions of benzothiadiazine and four of its derivatives designed in silico with model zwitterionic cell membranes formed by dioleoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoserine and cholesterol at the liquid–crystal phase inside aqueous potassium chloride solution. We have elucidated the local structure of benzothiadiazine by means of microsecond molecular dynamics simulations of systems including a benzothiadiazine molecule or one of its derivatives. Such derivatives were obtained by the substitution of a single hydrogen site of benzothiadiazine by two different classes of chemical groups, one of them electron-donating groups (methyl and ethyl) and another one by electron-accepting groups (fluorine and trifluoromethyl). Our data have revealed that benzothiadiazine derivatives have a strong affinity to stay at the cell membrane interface although their solvation characteristics can vary significantly—they can be fully solvated by water in short periods of time or continuously attached to specific lipid sites during intervals of 10–70 ns. Furthermore, benzothiadiazines are able to bind lipids and cholesterol chains by means of single and double hydrogen-bonds of characteristic lengths between 1.6 and 2.1 Å.

Halogenated Baicalein as a Promising Antiviral Agent toward SARS-CoV-2 Main Protease.

The coronavirus disease pandemic is a constant reminder that global citizens are in imminent danger of exposure to emerging infectious diseases. Therefore, developing a technique for inhibitor discovery is essential for effective drug design. Herein, we proposed fragment molecular orbital (FMO)-based virtual screening to predict the molecular binding energy of potential severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease inhibitors. The integration of quantum mechanical approaches and trajectory analysis from a microsecond molecular dynamics simulation was used to identify potential inhibitors. We identified brominated baicalein as a potent inhibitor of the SARS-CoV-2 main protease and confirmed its inhibitory activity in an in vitro assay. Brominated baicalein did not demonstrate significant toxicity in either in vitro or in vivo studies. The pair interaction energy from FMO-RIMP2/PCM and inhibitory constants based on the protease enzyme assay suggested that the brominated baicalein could be further developed into novel SARS-CoV-2 protease inhibitors.

Mechanistic insight into impact of phosphorylation on dynamics of farnesyltransferase: targeting the protein under hyperinsulinemia

Farnesyltransferase (FTase) is a heterodimeric enzyme that adds the farnesyl group to target proteins such as Ras, thus modulating its function in the cell. The phosphorylation of the α-subunit of FTase has been shown to increase the activity of the protein under hyperinsulinemia, which is prevalent in cancer; however, molecular mechanism has remained elusive. To address this, we performed a total of 18 microseconds molecular dynamics simulations using the systems that are included in the farnesylation process: i)FTase+farnesylpyrophosphate(FPP), ii)FTase+FPP+Kras-peptide(substrate), iii)FTase+farnesylated Kras-peptide(product), which are named as binary, ternary and product, respectively.

Annealing metallic glasses above Tg in order to accelerate the relaxation process in molecular dynamics simulations

To bridge the gap between nano- and micro-seconds molecular dynamics simulations and milliseconds timescale phenomena in metallic glasses remains an area of active research. Through systematic control of the annealing parameters, we have been able to simulate metallic glasses resembling structures usually obtained by quenching at cooling rates used in the melt spinning process. Density, local order, and local entropy calculations predict metallic glasses with structures prepared at cooling rates orders of magnitude lower than those typically realized in atomistic modeling. Hence, annealing above Tg is an alternative to melt quenching simulations and offers the prospect of modeling well relaxed glassy structures that were not achievable before in molecular dynamics simulations.

Microsecond molecular dynamics simulations revealed the inhibitory potency of amiloride analogs against SARS-CoV-2 E viroporin.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes small envelope protein (E) that plays a major role in viral assembly, release, pathogenesis, and host inflammation. Previous studies demonstrated that pyrazine ring containing amiloride analogs inhibit this protein in different types of coronavirus including SARS-CoV-1 small envelope protein E (SARS-CoV-1 E). SARS-CoV-1 E has 93.42% sequence identity with SARS-CoV-2 E and shared a conserved domain NS3/small envelope protein (NS3_envE). Amiloride analog hexamethylene amiloride (HMA) can inhibit SARS-CoV-1 E. Therefore, we performed molecular docking and dynamics simulations to explore whether amiloride analogs are effective in inhibiting SARS-CoV-2 E. To do so, SARS-CoV-1 E and SARS-CoV-2 E proteins were taken as receptors while HMA and 3-amino-5-(azepan-1-yl)-N-(diaminomethylidene)-6-pyrimidin-5-ylpyrazine-2-carboxamide (3A5NP2C) were selected as ligands. Molecular docking simulation showed higher binding affinity scores of HMA and 3A5NP2C for SARS-CoV-2 E than SARS-CoV-1 E. Moreover, HMA and 3A5NP2C engaged more amino acids in SARS-CoV-2 E. Molecular dynamics simulation for 1 μs (1,000 ns) revealed that these ligands could alter the native structure of the proteins and their flexibility. Our study suggests that suitable amiloride analogs might yield a prospective drug against coronavirus disease 2019.

Inactive-to-Active Transition of Human Thymidine Kinase 1 Revealed by Molecular Dynamics Simulations.

Despite its importance in the nucleoside (and nucleoside prodrug) metabolism, the structure of the active conformation of human thymidine kinase 1 (hTK1) remains elusive. We perform microsecond molecular dynamics simulations of the inactive enzyme form bound to a bisubstrate inhibitor that was shown experimentally to activate another TK1-like kinase, Thermotoga maritima TK (TmTK). Our results are in excellent agreement with the experimental findings for the TmTK closed-to-open state transition. We show that the inhibitor induces an increase of the enzyme radius of gyration due to the expansion on one of the dimer interfaces; the structural changes observed, including the active site pocket volume increase and the decrease in the monomer–monomer buried surface area and of the number of hydrogen bonds (as compared to the inactive enzyme control simulation), indicate that the catalytically competent (open) conformation of hTK1 can be assumed in the presence of an activating ligand.

Supramolecular Organization of SARS-CoV and SARS-CoV-2 Virions Revealed by Coarse-Grained Models of Intact Virus Envelopes.

The coronavirus disease 19 (COVID-19) pandemic is causing a global health crisis and has already caused a devastating societal and economic burden. The pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has a high sequence and architecture identity with SARS-CoV, but far more people have been infected by SARS-CoV-2. Here, combining the structural data from cryo-electron microscopy and structure prediction, we constructed bottom-up Martini coarse-grained models of intact SARS-CoV and SARS-CoV-2 envelopes. Microsecond molecular dynamics simulations were performed, allowing us to explore their dynamics and supramolecular organization. Both SARS-CoV and SARS-CoV-2 envelopes present a spherical morphology, with structural proteins forming multiple string-like islands in the membrane and clusters between the heads of spike proteins. Critical differences between the SARS-CoV and SARS-CoV-2 envelopes are the interaction pattern between the spike proteins and the flexibility of the spike proteins. Our models provide structural and dynamic insights into the SARS virus envelopes and could be used for further investigation, such as drug design and membrane fusion and fission processes.

Small molecule interactions with the SARS-CoV-2 main protease: In silico all-atom microsecond MD simulations, PELE Monte Carlo simulations, and determination of in vitro activity inhibition

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the ongoing COVID-19 pandemic. With some notable exceptions, safe and effective vaccines, which are now being widely distributed globally, have largely begun to stabilise the situation. However, emerging variants of concern and vaccine hesitancy are apparent obstacles to eradication. Therefore, the need for the development of potent antivirals is still of importance. In this context, the SARS-CoV-2 main protease (Mpro) is a critical target and numerous clinical trials, predominantly in the private domain, are currently in progress. Here, our aim was to extend our previous studies, with hypericin and cyanidin-3-O-glucoside, as potential inhibitors of the SARS-CoV-2 Mpro. Firstly, we performed all-atom microsecond molecular dynamics simulations, which highlight the stability of the ligands in the Mpro active site over the duration of the trajectories. We also invoked PELE Monte Carlo simulations which indicate that both hypericin and cyanidin-3-O-glucoside preferentially interact with the Mpro active site and known allosteric sites. For further validation, we performed an in vitro enzymatic activity assay that demonstrated that hypericin and cyanidin-3-O-glucoside inhibit Mpro activity in a dose-dependent manner at biologically relevant (μM) concentrations. However, both ligands are much less potent than the well-known covalent antiviral GC376, which was used as a positive control in our experiments. Nevertheless, the biologically relevant activity of hypericin and cyanidin-3-O-glucoside is encouraging. In particular, a synthetic version of hypericin has FDA orphan drug designation, which could simplify potential clinical evaluation in the context of COVID-19.

Unraveling the Allosteric Mechanism of Four Cancer-related Mutations in the Disruption of p53-DNA Interaction.

The p53 protein plays active roles in the physiological regulation of cell cycle as well as in cancer developments. In more than half of human cancers, the protein is inactivated by mutations located primarily in its DNA-binding domain (DBD), and some mutations located in the β-sandwich region of DBD are reported to decrease p53-DNA binding affinities. To understand the long-range correlation between p53 β-sandwich and DNA, and the allosteric mechanism of β-sandwich mutations in the disruption of p53-DNA interactions, we first identify three regions with a strong correlation with DNA based on microsecond molecular dynamics (MD) simulations of wild-type p53-DNA complex and then perform multiple MD simulations on four cancer-related mutants L145Q, P151S, Y220C, and G266R, which are located in these three regions. Our simulations show that these mutations allosterically destabilize the structural stability of the DNA-binding groove in p53 and disrupt the p53-DNA interactions. Network analyses reveal optimal correlation paths through which the mutation-induced allosteric signal passes to DNA, and the disturbance effect of these mutations on the global connectivity and dynamical correlation of the p53-DNA complex. This work paves the way for the in-depth understanding of the mutation-induced loss in p53's DNA-recognition ability and the pathological mechanism of cancer development.

In-silico screening and microsecond molecular dynamics simulations to identify single point mutations that destabilize β-hexosaminidase A causing Tay-Sachs disease.

β-hexosaminidase A (HexA) protein is responsible for the degradation of GM2 gangliosides in the central and peripheral nervous systems. Tay-Sachs disease occurs when HexA within Hexosaminidase does not properly function and harmful GM2 gangliosides begin to build up within the neurons. In this study, in silico methods such as SIFT, PolyPhen-2, PhD-SNP, and MutPred were utilized to analyze the effects of nonsynonymous single nucleotide polymorphisms (nsSNPs) on HexA in order to identify possible pathogenetic and deleterious variants. Molecular dynamics (MD) simulations showed that two mutants, P25S and W485R, experienced an increase in structural flexibility compared to the native protein. Particularly, there was a decrease in the overall number and frequencies of hydrogen bonds for the mutants compared to the wildtype. MM/GBSA calculations were performed to help assess the change in binding affinity between the wildtype and mutant structures and a mechanism-based inhibitor, NGT, which is known to help increase the residual activity of HexA. Both of the mutants experienced a decrease in the binding affinity from -23.8kcal/mol in wildtype to -20.9 and -18.7kcal/mol for the P25S and W485R variants of HexA, respectively.

In silico investigation to identify potential small molecule inhibitors of the RNA-dependent RNA polymerase (RdRp) nidovirus RdRp-associated nucleotidyltransferase domain

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) is a promising target for antiviral drugs. In this study, a chemical library (n = 300) was screened against the nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain. Blind docking was performed using a selection of 30 compounds and nine ligands were chosen based on their docking scores, safety profile, and availability. Using cluster analysis on a 10 microsecond molecular dynamics simulation trajectory (from D.E. Shaw Research), the compounds were docked to the different conformations. On the basis of our modelling studies, oleuropein was identified as a potential lead compound.

Reverse Protonation of Buried Ion-Pairs in Staphylococcal Nuclease Mutants.

Although buried titratable residues in protein cavities are often of major functional importance, it is generally challenging to understand their properties such as the ionization state and factors of stabilization based on experimental studies alone. A specific set of examples involve buried Glu-Lys pairs in a series of variants of Staphylococcal nuclease, for which recent structural and thermodynamic studies appeared to suggest that both the stability and the ionization state of the buried Glu-Lys pair are sensitive to its orientation (i.e., Glu23-Lys36 vs Lys23-Glu36). To further clarify the situation, especially ionization states of the buried Glu-Lys pairs, we have conducted extensive molecular dynamics simulations and free energy computations. Microsecond molecular dynamics simulations show that the hydration level of the cavity depends on the orientation of the buried ion-pair therein as well as its ionization state; free energy simulations recapitulate the relative stability of Glu23-Lys36 (EK) vs Lys23-Glu36 (KE) mutants measured experimentally, although the difference is similar in magnitude regardless of the ionization state of the Glu-Lys pair. A complementary set of free energy simulations strongly suggests that, in contrast to the original suggestion in the experimental analysis, the Glu and Lys residues prefer to adopt their charge-neutral rather than the ionized states. This result is consistent with the low dielectric constant computed for water in the cavity, which makes it difficult for the protein cavity to stabilize a pair of charged Glu-Lys residues, even with water penetration. The current study highlights the role of free energy simulations in understanding the ionization state of buried titratable residues and the relevant energetic contributions, forming the basis for the rational design of buried charge networks in proteins.

Conformational dynamics of Tetracenomycin aromatase/cyclase regulate polyketide binding and enzyme aggregation propensity

BackgroundThe N-terminal domain of Tetracenomycin aromatase/cyclase (TcmN), an enzyme derived from Streptomyces glaucescens, is involved in polyketide cyclization, aromatization, and folding. Polyketides are a diverse class of secondary metabolites produced by certain groups of bacteria, fungi, and plants with various pharmaceutical applications. Examples include antibiotics, such as tetracycline, and anticancer drugs, such as doxorubicin. Because TcmN is a promising enzyme for in vitro production of polyketides, it is important to identify conditions that enhance its thermal resistance and optimize its function. MethodsTcmN unfolding, stability, and dynamics were evaluated by fluorescence spectroscopy, circular dichroism, nuclear magnetic resonance 15N relaxation experiments, and microsecond molecular dynamics (MD) simulations. ResultsTcmN thermal resistance was enhanced at low protein and high salt concentrations, was pH-dependent, and denaturation was irreversible. Conformational dynamics on the μs-ms timescale were detected for residues in the substrate-binding cavity, and two predominant conformers representing opened and closed cavity states were observed in the MD simulations. ConclusionBased on the results, a mechanism was proposed in which the thermodynamics and kinetics of the TcmN conformational equilibrium modulate enzyme function by favoring ligand binding and avoiding aggregation. General significanceUnderstanding the principles underlying TcmN stability and dynamics may help in designing mutants with optimal properties for biotechnological applications.

Dissecting the Conformational Free Energy of a Small Peptide in Solution.

The free energy surface of a small peptide was analyzed based on an unbiased microsecond molecular dynamics simulation. The peptide sampled disordered conformational ensembles of distinct compactness, and its free energy was decomposed into separate contributions from the intramolecular potential energy, conformational entropy, and solvation free energy. The latter was further broken down into enthalpic and entropic contributions due to peptide-water and water-water interactions. This decomposition was enabled by a generalized linear response relation between the peptide-water interaction energy and the solvation free energy, which was empirically parametrized by explicit solvation free energy calculations for representative peptide conformations. This full dissection of the peptide free energy identifies individual contributions that stabilize and destabilize compact and extended peptide conformational ensembles and reveals the origin of a free energy barrier associated with transitions between them.

Bcl-xL Dynamics under the Lens of Protein Structure Networks.

Understanding the finely orchestrated interactions leading to or preventing programmed cell death (apoptosis) is of utmost importance in cancer research because the failure of these systems could eventually lead to the onset of the disease. In this regard, the maintenance of a delicate balance between the promoters and inhibitors of mitochondrial apoptosis is crucial, as demonstrated by the interplay among the Bcl-2 family members. In particular, B-cell lymphoma extra-large (Bcl-xL) is a target of interest due to the forefront role of its dysfunctions in cancer development. Bcl-xL prevents apoptosis by binding both the pro-apoptotic BH3-only proteins, like PUMA, and the noncanonical partners, such as p53, at different sites. An allosteric communication between the BH3-only protein binding pocket and the p53 binding site, mediating the release of p53 from Bcl-xL upon PUMA binding, has been postulated and supported by nuclear magnetic resonance and other biophysical data. The molecular details of this mechanism, especially at the residue level, remain unclear. In this work, we investigated the distal communication between these two sites in Bcl-xL in its free state and when bound to PUMA. We also evaluated how missense mutations of Bcl-xL found in cancer samples might impair this communication and therefore the allosteric mechanism. We employed all-atom explicit solvent microsecond molecular dynamics simulations, analyzed through a Protein Structure Network approach and integrated with calculations of changes in free energies upon cancer-related mutations identified by genomics studies. We found a subset of candidate residues responsible for both maintaining protein stability and for conveying structural information between the two binding sites and hypothesized possible communication routes between specific residues at both sites.

Characterizing the function of domain linkers in regulating the dynamics of multi-domain fusion proteins by microsecond molecular dynamics simulations and artificial intelligence.

Multi-domain proteins are not only formed through natural evolution but can also be generated by recombinant DNA technology. Because many fusion proteins can enhance the selectivity of cell targeting, these artificially produced molecules, called multi-specific biologics, are promising drug candidates, especially for immunotherapy. Moreover, the rational design of domain linkers in fusion proteins is becoming an essential step toward a quantitative understanding of the dynamics in these biopharmaceutics. We developed a computational framework to characterize the impacts of peptide linkers on the dynamics of multi-specific biologics. Specifically, we first constructed a benchmark containing six types of linkers that represent various lengths and degrees of flexibility and used them to connect two natural proteins as a test system. We then projected the microsecond dynamics of these proteins generated from Anton onto a coarse-grained conformational space. We further analyzed the similarity of dynamics among different proteins in this low-dimensional space by a neural-network-based classification model. Finally, we applied hierarchical clustering to place linkers into different subgroups based on the classification results. The clustering results suggest that the length of linkers, which is used to spatially separate different functional modules, plays the most important role in regulating the dynamics of this fusion protein. Given the same number of amino acids, linker flexibility functions as a regulator of protein dynamics. In summary, we illustrated that a new computational strategy can be used to study the dynamics of multi-domain fusion proteins by a combination of long timescale molecular dynamics simulation, coarse-grained feature extraction, and artificial intelligence.

Targeting multiple conformations of SARS-CoV2 Papain-Like Protease for drug repositioning: An in-silico study

Papain-Like Protease (PLpro) is a key protein for SARS-CoV-2 viral replication which is the cause of the emerging COVID-19 pandemic. Targeting PLpro can suppress viral replication and provide treatment options for COVID-19. Due to the dynamic nature of its binding site loop, PLpro multiple conformations were generated through a long-range 1 micro-second molecular dynamics (MD) simulation. Clustering the MD trajectory enabled us to extract representative structures for the conformational space generated. Adding to the MD representative structures, X-ray structures were involved in an ensemble docking approach to screen the FDA approved drugs for a drug repositioning endeavor. Guided by our recent benchmarking study of SARS-CoV-2 PLpro, FRED docking software was selected for such a virtual screening task. The results highlighted potential consensus binders to many of the MD clusters as well as the newly introduced X-ray structure of PLpro complexed with a small molecule. For instance, three drugs Benserazide, Dobutamine and Masoprocol showed a superior consensus enrichment against the PLpro conformations. Further MD simulations for these drugs complexed with PLpro suggested the superior stability and binding of dobutamine and masoprocol inside the binding site compared to Benserazide. Generally, this approach can facilitate identifying drugs for repositioning via targeting multiple conformations of a crucial target for the rapidly emerging COVID-19 pandemic.

Contrasting conformational dynamics of β-sheet A and helix F with implications in neuroserpin inhibition and aggregation

Neuroserpin (NS) is an inhibitory protein of serpin super family, its shutter region variants have high propensity to aggregate leading to pathological disorders like familial encephalopathy with NS inclusion bodies (FENIB). Helix F and β-sheet A of NS participate in the tissue plasminogen activator (tPA) inhibition but the mechanism is not yet completely understood. A microsecond (μs) molecular dynamics simulation of the helix F and strand 3A variants showed predominant fluctuations in the loop connecting the strands of β-sheet A. Therefore to understand the role of helix F and strand 3A of β-sheet A, cysteine was incorporated at the position N182 in stand 3A (N182C) and position W154 (W154C) in the helix F using site-directed mutagenesis. Purified variants were further labeled with Alexa Fluor488 C5 maleimide dye. Temperature dependent study using non-denaturing PAGE showed the formation of large aggregates of helix F variant W154C but not the strand 3A N182C variant. Interestingly tPA inhibition was found to be decreased in the labeled N182C with decreased tPA-complex formation as compared to labeled W154C NS variant. The fluorescence emission intensity of the labeled helix F variant W154C decreased in the presence of an increasing concentration of tPA, whereas an increase in emission intensity was observed in labeled strand 3A variant N182C, indicating more exposure of strand 3A and shielding of helix F. Taken together the data shows that helix F has a predominant role in the aggregation but a minor role in the inhibition mechanism.