Comparison and Possible Binding Orientations of SARS-CoV-2 Spike N-Terminal Domain for Gangliosides GM3 and GM1.

SARS-CoV-2 spike glycoprotein is anchored by gangliosides. The sialic acid in the ganglioside headgroup is responsible for virus attachment and entry into host cells. We used coarse-grained (CG) molecular dynamics simulations to expand on our previous study of GM1 interaction with two different orientations of the SARS-CoV-2 S1 subunit N-terminal domain (NTD) and to confirm the role of sialic acid receptors in driving the viral receptor; GM3 was used as another ganglioside on the membrane. Because of the smaller headgroup, sialic acid is crucial in GM3 interactions, whereas GM1 interacts with NTD via both the sialic acid and external galactose. In line with our previous findings for NTD orientations in GM1 binding, we identified two orientations, "compact" and "distributed", comprising sugar receptor-interacting residues in GM3-embedded lipid bilayers. Gangliosides in closer proximity to the compact NTD orientation might cause relatively greater restrictions to penetrate the bilayer. However, the attachment of a distributed NTD orientation with more negative interaction energies appears to facilitate GM1/GM3 to move quickly across the membrane. Our findings likely shed some light on the orientations that the NTD receptor acquires during the early phases of interaction with GM1 and GM3 in a membrane environment.

Molecular dynamics simulations suggest Thiosemicarbazones can bind p53 cancer mutant R175H.

Cancer pathologies are associated with the unfolding and aggregation of most recurring mutations in the DNA Binding Domain (DBD) of p53 that coordinate the destabilization of protein. Substitution at the 175th codon with arginine to histidine (R175H, a mutation of large to small side-chain amino acid) destabilizes the DBD by 3 kcal/mol and triggers breasts, lung cancer, etc. Stabilizing the p53 mutant by small molecules offers an attractive drug-targeted anti-cancer therapy. The thiosemicarbazone (TSC) molecules NPC and DPT are known to act as zinc-metallochaperones to reactivate p53R175H. Here, a combination of LESMD simulations for 10 TSC conformations with a p53R175H receptor, single ligand-protein conformation MD, and ensemble docking with multiple p53R175H conformations observed during simulations is suggested to identify the potential binding site of the target protein in light of their importance for the direct TSC - p53R175H binding. NPC binds mutant R175H in the loop region L2-L3, forming pivotal hydrogen bonds with HIS175, pi­sulfur bonds with TYR163, and pi-alkyl linkages with ARG174 and PRO190, all of which are contiguous to the zinc-binding native site on p53DBD. DPT, on the other hand, was primarily targeting alternative binding sites such as the loop-helix L1/H2 region and the S8 strand. The similar structural characteristics of TSC-bound p53R175H complexes with wild-type p53DBD are thought to be attributable to involved interactions that favour binding free energy contributions of TSC ligands. Our findings may be useful in the identification of novel pockets with druggable properties.

Computational studies suggest compounds restoring function of p53 cancer mutants can bind SARS-CoV-2 spike protein.

It is reasonable to think that cancer patients undergoing chemotherapy or immunotherapy may have a more aggressive course if they are positive for the novel coronavirus disease. Their compulsive condition requires investigation into effective drugs. We applied computational techniques to a series of compounds known for restoring the function of p53 cancer mutant p53R175H and p53G245S. Two potent inhibitors, 1-(3-chlorophenyl)-3-(1, 3 -thiazol-2-yl) urea (CTU, PubChem NSC321792) with the highest binding affinity -6.92 kcal/mol followed by a thiosemicarbazone compound N'-(1-(Pyridin-2-yl)ethylidene) azetidine - 1 -carbothiohydrazide (NPC, PubChem NSC319726) with -6.75 kcal/mol were subjected to Molecular Dynamics simulation with receptor binding domain (RBD) and compared with control ligand dexamethasone. In particular, CTU adheres to pocket 1 with an average free energy of binding -21.65 ± 2.89 kcal/mol at the RBD - angiotensin-converting enzyme 2 binding region with the highest frequency of amino acid residues after reaching a local equilibrium in 100 ns MD simulation trajectory. A significant enthalpy contribution from the independent simulations unfolds the possibility of dual binding sites for NPC as shifted pocket 1 (-15.59 ± 5.98 kcal/mol) and pocket 2 (-18.90 ± 5.02 kcal/mol). The obtained results for these two compounds are in good agreement with dexamethasone (-18.45 ± 2.42 kcal/mol). Taken together our findings could facilitate the discovery of small molecules that restore the function of p53 cancer mutants newly against COVID-19 in cancer patients.Communicated by Ramaswamy H. Sarma.

Identification of possible binding modes of SARS-CoV-2 spike N-terminal domain for ganglioside GM1.

Coarse-grained molecular dynamics simulations of the lipid bilayer mixture of POPC and cholesterol were carried out in the presence and absence of ganglioside monosialo 1 (GM1) with N - terminal domain (NTD) of SARS-CoV-2 spike glycoprotein. The interactions of GM1 with two different NTD orientations were compared. NTD orientation I compactly bind GM1 predominantly through the sialic acid and the external galactose moieties providing more restriction to GM1 mobility whereas orientation II is more distributed on the lipid surface and due to the relaxed mobility of GM1 there, presumably, the NTD receptor penetrates more through the membrane.

Correction to "Aggregation of Lysozyme in the Presence of a Mixed Bilayer of POPC and POPG".

[This corrects the article DOI: 10.1021/acsomega.1c01145.].

Aggregation of Lysozyme in the Presence of a Mixed Bilayer of POPC and POPG.

Understanding the molecular mechanisms by which amyloidogenic proteins interact with membranes is a challenging task. Amyloid accumulates from many human diseases have been observed to contain membrane lipids. In this work, coarse-grained molecular dynamics simulations have been used to inspect hen egg white lysozyme (HEWL) aggregation and membrane association in the presence of a pure POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) bilayer and a POPC and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) mixed bilayer. It was observed that, in both cases, two HEWLs formed aggregates. In the presence of a mixed bilayer, after aggregation, the aggregated system started to interact with the membrane. It has been found that one of the lysozymes which came closer to the mixed bilayer unfolded more. The process of the initial insertion of an aggregated system in the mixed bilayer has been analyzed. The structural rearrangements of the protein and lipids were analyzed as well along the course of the simulation. Although with a pure POPC bilayer, aggregation was observed, the aggregated system moved away from the membrane. We believe that our study will provide considerable insights into lysozyme aggregation in the presence of a membrane environment.

Temperature dependent aggregation mechanism and pathway of lysozyme: By all atom and coarse grained molecular dynamics simulation.

Aggregation of protein causes various diseases including Alzheimer's disease, Parkinson's disease, and type II diabetes. It was found that aggregation of protein depends on many factors like temperature, pH, salt type, salt concentration, ionic strength, protein concentration, co solutes. Here we have tried to capture the aggregation mechanism and pathway of hen egg white lysozyme using molecular dynamics simulations at two different temperatures; 300 K and 340 K. Along with the all atom simulations to get the atomistic details of aggregation mechanism, we have used coarse grained simulation with MARTINI force field to monitor the aggregation for longer duration. Our results suggest that due to the aggregation, changes in the conformation of lysozyme are more at 340 K than at 300 K. The change in the conformation of the lysozyme at 300 K is mainly due to aggregation where at 340 K change in conformation of lysozyme is due to both aggregation and temperature. Also, a more compact aggregated system is formed at 340 K.

Ubiquitin folds via a flip-twist-lock mechanism.

To perform specific functional activities, the majority of proteins should fold into their distinct three-dimensional conformations. However, the biologically active conformation of a protein is generally found to be marginally stable than the other conformations that the chain can adopt. How a protein finds its native conformation from its post-synthesis unfolded structure in a complex conformational landscape is the unsolved question that still drives the protein folding community. Here, we report the folding mechanism of a globular protein, ubiquitin, from its chemically denatured state using all-atom molecular dynamics simulations. From the kinetic analysis of the simulated trajectories we show that the folding process can be described by the hydrophobic collapse mechanism, initiated by the "dewetting transition", and subsequently assisted by the origination of an N-terminal folding nucleus, and finally supported by a native salt-bridge interaction between K11 and E34. We show that ubiquitin folds via an intermediate. Finally, we confirm the presence of "biological water" and explain its role to the folding process.

Effect of Transmembrane Electric Field on GM1 Containing DMPC-Cholesterol Monolayer: A Computational Study.

Transmembrane electric potentials and membrane curvature have always provided pathways to mediate different cellular processes. We present results of molecular dynamics (MD) simulations of lipid monolayer composed of 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol (CHOL) under a transverse electric field to monitor the effect of electric field on membrane containing ganglioside monosialo 1 (GM1). Four systems were studied with membrane monolayer in the presence and absence of GM1 with and without applying electric field along the normal of the monolayer. The applied transmembrane electric field was 0.4 mV/Å which corresponds to the action potential of animal cell. Our results indicate that the electric field induces a considerable lateral stress on the monolayer in the presence of GM1, which is evident from the lateral pressure profiles. It was found that due to the application of electric field major perturbation was caused to the system containing GM1, manifested by the bending of the monolayer. We believe this study provides correlation between electric field and spontaneous membrane bending, specially based on the membrane composition. The consequences of these MD simulations provide considerable insights to different biological phenomenon and lipid membrane models.

Phase Behavior of GM1-Containing DMPC-Cholesterol Monolayer: Experimental and Theoretical Study.

Organization and distribution of lipids in cellular membranes play an important role in a diverse range of biological processes, such as membrane trafficking and signaling. Here, we present the combined experimental and simulated results to elucidate the phase behavioral features of ganglioside monosialo 1 (GM1)-containing mixed monolayer of the lipids 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and cholesterol (CHOL). Two monolayers having compositions DMPC-CHOL and GM1-DMPC-CHOL are investigated at air-water and air-solid interfaces using Langmuir-Blodgett experiments and scanning electron microscopy (SEM), respectively, to ascertain the phase behavior change of the monolayers. Surface pressure isotherms and SEM imaging of domain formation indicate that addition of GM1 to the monolayer at low surface pressure causes a fluidization of the system but once the system attains the surface pressure corresponding to its liquid-condensed phase, the monolayer becomes more ordered than the system devoid of GM1 and interacts among each other more cooperatively. Besides, the condensing effect of cholesterol on the DMPC monolayer was also verified by our experiments. Apart from these, the effects induced by GM1 on the phase behavior of the binary mixture of DMPC-CHOL were studied with and without applying liquid-expanded (LE)-liquid-condensed (LC) equilibrium surface pressure using molecular dynamics (MD) simulation. Our molecular dynamics (MD) simulation results give an atomistic-level explanation of our experimental findings and furnish a similar conclusion.

Interaction Between Luteinizing Hormone-Releasing Hormone and GM1-Doped Cholesterol/Sphingomyelin Vesicles: A Spectroscopic Study.

Understanding the role of neural membrane in translocation and action of neurohormone is of great importance. Luteinizing hormone-releasing hormone (LHRH) is a neuropeptide hormone and it acts as a final signaling molecule by stimulating the synthesis of LH and FSH to maintain reproduction in all vertebrates. The receptors of LHRH are found in breast tumors and pituitary gland in the brain. Moreover, neural plasma membrane is also found to contain specific binding site for LHRH. The mechanism by which LHRH binds to membrane before it binds to the receptors is a very critical step and can have a profound impact upon the translation of peptide across the membrane. A complex form of glycosphingolipids known as Ganglioside is an important component of plasma membrane of nerve cells and breast tumor tissues. They play an important role in various physiological membrane processes. Therefore, the interaction of ganglioside-containing membrane with LHRH might be crucial in aiding the LHRH to translate through the neural membrane and reach its receptor for binding and activation. Using CD, UV-Absorbance, and fluorescence spectroscopy, the effect of Ganglioside Monosialo 1(GM1)-induced conformational changes of LHRH in the presence of Cholesterol (CHOL)/Sphingomyelin (SM) and GM1/CHOL/SM vesicles was studied. The aforesaid spectroscopic studies show that LHRH is able to bind with both the vesicles, but GM1-containing vesicles interact more effectively than vesicles without GM1. CHOL/SM vesicles partially disturb the conformation of the peptide. Moreover, binding of LHRH to GM1/CHOL/SM vesicles induces loss of conformational rigidity and attainment of a random coil.

Effect of glycosylation on hydration behavior at the ice-binding surface of the Ocean Pout type III antifreeze protein: a molecular dynamics simulation.

Antifreeze proteins (AFPs), found in certain vertebrates, plants, fungi and bacteria have the ability to permit their survival in subzero environments by thermal hysteresis mechanism. However, the exact mechanism of ice growth inhibition is still not clearly understood. Here, four long explicit molecular dynamics (MD) simulations have been carried out at two different temperatures (277 and 298 K) with and without glycan to study the conformational rigidity of the Ocean pout type III antifreeze protein in aqueous medium and the structural arrangements of water molecules hydrating its ice-binding surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its non ice-binding surface (NonIBS) in its native and glycosylated form. Hydrophilic residues N14, T18 and Q44 are essential to antifreeze activity. Radial distribution, density distribution function and nearest neighbor orientation plots with respect to individual two surfaces confirm that density of water molecule near these binding surface in native and glycosylated form are relatively more than the nonbinding surface. The glycosylated form shows a strong peak than the native one. From rotational auto correlation function of water molecules around ice-binding sites, it is prominent that with increase in temperature, strong interaction between the water oxygen and the hydrogen bond acceptor group on the protein-binding surface decreases. This provides a possible molecular reason behind the ice-binding activity of ocean pout at the prism plane of ice.

Localization and dynamics of the anticarcinogenic curcumin with GM1 and other miceller assemblies.

Structural transitions involving shape changes play an important role in cellular physiology and enhance the bioavailability of the natural food like curcumin in surfactant aggregates. In this work, we have studied the localization, dynamics and stability of curcumin in various miceller assemblies using a combination of absorbance and fluorescence spectroscopic approaches. The measurements of absorption and fluorescence spectra of curcumin revealed that the nature of interactions of ionic and nonionic surfactants and the glycosphingolipid, GM1 with curcumin is significantly different with surfactant concentrations. At low concentrations of SDS and the GM1 the head group of SDS and GM1 binds to the central ß-diketone group of curcumin to form SDS-curcumin or GM1-curcumin complexes. At high concentrations, both formed micelles with curcumin completely solubilized inside. Cucurmin is solubilized in the stern layer of SDS micelles. Compared to spherical micelles, rod shaped micelles allow more curcumin to bind through hydrophobic interactions indicated by higher absorption and fluorescence, enhanced partition coefficient and stability. Whereas curcumin associates with GM1 micelles with lower partition coefficient, solubility and remain closer to aqueous phase decreasing its bioavailability and stability. While cucurmin is solubilized in the palisade layer of deoxycholate and octyl glucopyranoside micelles through the alkyl chains providing more hydrophobic microenvironment to curcumin with enhanced stability and bioavailability. Graphical abstract Schematic diagram of the two different types of detergent micelles and larger GM1 micelles.

Dynamics simulation of soybean agglutinin (SBA) dimer reveals the impact of glycosylation on its enhanced structural stability.

The legume lectins are widely used as a model system for studying protein-carbohydrate and protein-protein interactions. They exhibit a fascinating quaternary structure variation. Recently, it has become clear that lectins exist as oligomers. Soybean agglutinin is a tetrameric legume lectin, each of whose subunits are glycosylated. In the present study we explore the main origin for the stability of soybean agglutinin dimer. In order to understand the role of glycosylation on the dimeric interface, we have carried out normal (298K), high temperatures (380K, 500K) long explicit solvent molecular dynamics (MD) simulations and compared the structural and conformational changes between the glycosylated and non-glycosylated dimers. The study reveals that the high degree of stability at normal temperature is mostly contributed by interfacial ionic interactions (~200 kcal/mol) between polar residues like Lys, Arg, Asp, Thr, Ser, Asn and Gln (62%). It maintains its overall folded conformation due to high subunit interactions at the non-canonical interface. Mainly five important hydrogen bonds between CO of one ß sheet of one subunit with the N-H of other ß strand of the other subunit help to maintain the structural integrity. Ten inter subunit salt-bridge interactions between Arg 185-Asp192, Lys 163-Asp169, Asp 169-Lys 163 and Asp 192-Arǵ 185 at non-canonical interface appear to be important to maintain the three dimensional structure of SBA dimer. Moreover, our simulation results revealed that increase in vibrational entropy could decrease the free energy and contribute to the glycan-induced stabilization by ~45 kcal/mol at normal temperature.

Insights into the behavioral difference of water in the presence of GM1.

Studies on the structure and dynamics of interfacial water, emphasizing on the properties of water near the surface of biomolecules, are well reported, but there is a lack of evidence on the behavior of water near a comparatively rough surface containing molecules with a bulky head group like GM1. In this report we comparatively analyze the structure and dynamics of water as a function of distance from the lipid head group in GM1 containing lipid bilayers, with the lipid bilayers where GM1 is not present. This approach effectively demonstrates the behavioral difference and hence delayed convergence from bound water to bulk water in the presence of GM1 compared to a relatively smooth surface.

Impact of glycosylation on stability, structure and unfolding of soybean agglutinin (SBA): an insight from thermal perturbation molecular dynamics simulations.

Glycosylation has been recognized as one of the most prevalent and complex post-translational modifications of proteins involving numerous enzymes and substrates. Its effect on the protein conformational transitions is not clearly understood yet. In this study, we have examined the effect of glycosylation on protein stability using molecular dynamics simulation of legume lectin soybean agglutinin (SBA). Its glycosylated moiety consists of high mannose type N-linked glycan (Man9GlcNAc2). To unveil the structural perturbations during thermal unfolding of these two forms, we have studied and compared them to the experimental results. From the perspective of dynamics, our simulations revealed that the nonglycosylated monomeric form is less stable than corresponding glycosylated form at normal and elevated temperatures. Moreover, at elevated temperature thermal destabilization is more prominent in solvent exposed loops, turns and ends of distinct ß sheets. SBA maintains it folded structure due to some important saltbridges, hydrogen bonds and hydrophobic interactions within the protein. The reducing terminal GlcNAc residues interact with the protein residues VAL161, PRO182 and SER225 via hydrophobic and via hydrogen bonding with ASN 9 and ASN 75. Our simulations also revealed that single glycosylation (ASN75) has no significant effect on corresponding cis peptide angle orientation. This atomistic description might have important implications for understanding the functionality and stability of Soybean agglutinin.

In silico phase separation in the presence of GM1 in ternary and quaternary lipid bilayers.

Cell membranes are multi-component mixtures with structural and compositional heterogeneity exhibiting a complex phase behavior. Domains formed in cell membranes often known as "Rafts" are of immense importance. Using coarse grained molecular dynamics simulations, we have studied the spontaneous phase separation of the ternary (POPC [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine]/cholesterol/GM1) and quaternary (POPC/PSM[palmitoyl sphingomyelin]/cholesterol/GM1) lipid bilayers into liquid ordered (Lo) and liquid disordered (Ld) domains due to self-aggregation of GM1 molecules and co-localization of cholesterol with GM1 in accordance with experiments. It is found that GM1 molecules have the ability to associate strongly with each other which leads to the formation of ordered domains in the lipid mixture and the interactions are through the head group and unsaturated tails present in GM1. Preference of cholesterol for association with GM1 over PSM is observed, the domains consisting of GM1 and cholesterol are formed even in the presence of PSM. PSM also forms small domains with cholesterol that are randomly distributed in the Ld phase. Estimation of dynamic quantities like diffusion coefficient also shows that cholesterol has the highest diffusion rate in the Ld phase which is further attributed to its flip flop ability. It is found that in the presence of PSM, cholesterol can undergo flip flop even in the Lo phase. This is accredited to the interaction of cholesterol with PSM from which it can be concluded that in the presence of PSM, the domains formed by GM1 are less tightly packed and less stable than that in the ternary mixture.

Organization and dynamics of tryptophan residues in brain spectrin: novel insight into conformational flexibility.

Brain spectrin enjoys overall structural and sequence similarity with erythroid spectrin, but less is known about its function. We utilized the fluorescence properties of tryptophan residues to monitor their organization and dynamics in brain spectrin. Keeping in mind the functional relevance of hydrophobic binding sites in brain spectrin, we monitored the organization and dynamics of brain spectrin bound to PRODAN. Results from red edge excitation shift (REES) indicate that the organization of tryptophans in brain spectrin is maintained to a considerable extent even after denaturation. These results are supported by acrylamide quenching experiments. To the best of our knowledge, these results constitute the first report of the presence of residual structure in urea-denatured brain spectrin. We further show from REES and time-resolved emission spectra that PRODAN bound to brain spectrin is characterized by motional restriction. These results provide useful information on the differences between erythroid spectrin and brain spectrin.

Peanut protein sensitivity towards trace iron: a novel mode to ebb allergic response.

Peanut is a rich source of plant protein which is inexpensive and abundant in nature. The peanut proteins however cause hypersensitive immunogenic responses in certain individuals. A minute amount of contamination may cause strong allergic reactions and even death. Many chemical pretreatment procedures have been developed and prescribed earlier for removal of this allergenicity. In the present article we have observed trace level Fe(III) and Cu(II) complexation of the protein fractions of peanut at pH 4.8 using different spectral methods. Consequently we studied the allergic response of Fe(III) complex of the protein fractions using competitive enzyme linked immunosorbent assay (ELISA) technique and found that there were considerable losses in allergenicity of conarachin I and arachin fractions upon complexation. Immunoassay of Cu(II) complex was avoided keeping in view the Cu toxicity in living systems. The results bring up a new strategy towards reduction of allergenicity using an inexpensive and simple methodology.

Probing conformational stability and dynamics of erythroid and nonerythroid spectrin: effects of urea and guanidine hydrochloride.

We have studied the conformational stability of the two homologous membrane skeletal proteins, the erythroid and non-erythroid spectrins, in their dimeric and tetrameric forms respectively during unfolding in the presence of urea and guanidine hydrochloride (GuHCl). Fluorescence and circular dichroism (CD) spectroscopy have been used to study the changes of intrinsic tryptophan fluorescence, anisotropy, far UV-CD and extrinsic fluorescence of bound 1-anilinonapthalene-8-sulfonic acid (ANS). Chemical unfolding of both proteins were reversible and could be described as a two state transition. The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate. Fluorescence quenching, anisotropy, ANS binding and dynamic light scattering data suggest that in presence of low concentrations of the denaturants (up-to 1M) hydrogen bonding network and van der Waals interaction play a role inducing changes in quaternary as well as tertiary structures without complete dissociation of the subunits. This is the first report of two large worm like, multi-domain proteins obeying twofold rule which is commonly found in small globular proteins. The free energy of stabilization (ΔGuH20) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.