Spontaneous Dimerization and Distinct Packing Modes of Transmembrane Domains in Receptor Tyrosine Kinases.

The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF1R) are homodimeric transmembrane glycoproteins that transduce signals across the membrane on binding of extracellular peptide ligands. The structures of IR/IGF1R fragments in apo and liganded states have revealed that the extracellular subunits of these receptors adopt Lambda-shaped configurations to which are connected the intracellular tyrosine kinase (TK) domains. The binding of peptide ligands induces structural transitions in the extracellular subunits leading to potential dimerization of transmembrane domains (TMDs) and autophosphorylation in TKs. However, the activation mechanisms of IR/IGF1R, especially the role of TMDs in coordinating signal-inducing structural transitions, remain poorly understood, in part due to the lack of structures of full-length receptors in apo or liganded states. While atomistic simulations of IR/IGF1R TMDs showed that these domains can dimerize in single component membranes, spontaneous unbiased dimerization in a plasma membrane having physiologically representative lipid composition has not been observed. We address this limitation by employing coarse-grained (CG) molecular dynamics simulations to probe the dimerization propensity of IR/IGF1R TMDs. We observed that TMDs in both receptors spontaneously dimerized independent of their initial orientations in their dissociated states, signifying their natural propensity for dimerization. In the dimeric state, IR TMDs predominantly adopted X-shaped configurations with asymmetric helical packing and significant tilt relative to the membrane normal, while IGF1R TMDs adopted symmetric V-shaped or parallel configurations with either no tilt or a small tilt relative to the membrane normal. Our results suggest that IR/IGF1R TMDs spontaneously dimerize and adopt distinct dimerized configurations.

Structural models of viral insulin-like peptides and their analogs.

The insulin receptor (IR), the insulin-like growth factor-1 receptor (IGF1R), and the insulin/IGF1 hybrid receptors (hybR) are homologous transmembrane receptors. The peptide ligands, insulin and IGF1, exhibit significant structural homology and can bind to each receptor via site-1 and site-2 residues with distinct affinities. The variants of the Iridoviridae virus family show capability in expressing single-chain insulin/IGF1 like proteins, termed viral insulin-like peptides (VILPs), which can stimulate receptors from the insulin family. The sequences of VILPs lacking the central C-domain (dcVILPs) are known, but their structures in unbound and receptor-bound states have not been resolved to date. We report all-atom structural models of three dcVILPs (dcGIV, dcSGIV, and dcLCDV1) and their complexes with the receptors (µIR, µIGF1R, and µhybR), and probed the peptide/receptor interactions in each system using all-atom molecular dynamics (MD) simulations. Based on the nonbonded interaction energies computed between each residue of peptides (insulin and dcVILPs) and the receptors, we provide details on residues establishing significant interactions. The observed site-1 insulin/µIR interactions are consistent with previous experimental studies, and a residue-level comparison of interactions of peptides (insulin and dcVILPs) with the receptors revealed that, due to sequence differences, dcVILPs also establish some interactions distinct from those between insulin and IR. We also designed insulin analogs and report enhanced interactions between some analogs and the receptors.

Progress in Simulation Studies of Insulin Structure and Function.

Insulin is a peptide hormone known for chiefly regulating glucose level in blood among several other metabolic processes. Insulin remains the most effective drug for treating diabetes mellitus. Insulin is synthesized in the pancreatic ß-cells where it exists in a compact hexameric architecture although its biologically active form is monomeric. Insulin exhibits a sequence of conformational variations during the transition from the hexamer state to its biologically-active monomer state. The structural transitions and the mechanism of action of insulin have been investigated using several experimental and computational methods. This review primarily highlights the contributions of molecular dynamics (MD) simulations in elucidating the atomic-level details of conformational dynamics in insulin, where the structure of the hormone has been probed as a monomer, dimer, and hexamer. The effect of solvent, pH, temperature, and pressure have been probed at the microscopic scale. Given the focus of this review on the structure of the hormone, simulation studies involving interactions between the hormone and its receptor are only briefly highlighted, and studies on other related peptides (e.g., insulin-like growth factors) are not discussed. However, the review highlights conformational dynamics underlying the activities of reported insulin analogs and mimetics. The future prospects for computational methods in developing promising synthetic insulin analogs are also briefly highlighted.

Selection of start codon during mRNA scanning in eukaryotic translation initiation.

Accurate and high-speed scanning and subsequent selection of the correct start codon are important events in protein synthesis. Eukaryotic mRNAs have long 5' UTRs that are inspected for the presence of a start codon by the ribosomal 48S pre-initiation complex (PIC). However, the conformational state of the 48S PIC required for inspecting every codon is not clearly understood. Here, atomistic molecular dynamics (MD) simulations and energy calculations suggest that the scanning conformation of 48S PIC may reject all but 4 (GUG, CUG, UUG and ACG) of the 63 non-AUG codons, and initiation factor eIF1 is crucial for this discrimination. We provide insights into the possible role of initiation factors eIF1, eIF1A, eIF2α and eIF2ß in scanning. Overall, the study highlights how the scanning conformation of ribosomal 48S PIC acts as a coarse selectivity checkpoint for start codon selection and scans long 5' UTRs in eukaryotic mRNAs with accuracy and high speed.

Structures and interactions of insulin-like peptides from cone snail venom.

The venomous insulin-like peptides released by certain cone snails stimulate hypoglycemic shock to immobilize fish and catch the prey. Compared to human insulin (hIns), the cone snail insulins (Con-Ins) are typically monomeric and shorter in sequence, yet they exhibit moderate hIns-like biological activity. We have modeled six variants of Con-Ins (G3, K1, K2, T1A, T1B, and T2) and carried out explicit-solvent molecular dynamics (MD) simulations of eight types of insulins, two with known structures (hIns and Con-Ins-G1) and six Con-Ins with modeled structures, to characterize key residues of each insulin that interact with the truncated human insulin receptor (µIR). We show that each insulin/µIR complex is stable during explicit-solvent MD simulations and hIns interactions indicate the highest affinity for the "site 1" of IR. The residue contact maps reveal that each insulin preferably interacts with the αCT peptide than the L1 domain of IR. Through analysis of the average nonbonded interaction energy contribution of every residue of each insulin for the µIR, we probe the residues establishing favorable interactions with the receptor. We compared the interaction energy of each residue of every Con-Ins to the µIR and observed that γ-carboxylated glutamate (Gla), His, Thr, Tyr, Tyr/His, and Asn in Con-Ins are favorable substitutions for GluA4, AsnA21, ValB12, LeuB15, GlyB20, and ArgB22 in hIns, respectively. The identified insulin analogs, although lacking the last eight residues of the B-chain of hIns, bind strongly to µIR. Our findings are potentially useful in designing potent fast-acting therapeutic insulin.

Mechanistic insights into the effects of key mutations on SARS-CoV-2 RBD-ACE2 binding.

Some recent SARS-CoV-2 variants appear to have increased transmissibility compared to the original strain. An underlying mechanism could be the improved ability of the variants to bind receptors on the target cells and infect them. In this study, we provide atomic-level insights into the binding of the receptor binding domain (RBD) of the wild-type SARS-CoV-2 spike protein and its single (N501Y), double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants to the human ACE2 receptor. Using extensive all-atom molecular dynamics simulations and advanced free energy calculations, we estimate the associated binding affinities and binding hotspots. We observe significant secondary structural changes in the RBD of the mutants, which lead to different binding affinities. We find higher binding affinities for the double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants than for the wild type and the N501Y variant, which could contribute to the higher transmissibility of recent variants containing these mutations.

Electric Field-Mediated Fibronectin-Hydroxyapatite Interaction: A Molecular Insight.

In experimental research-driven biomaterials science, the influence of different material properties (elastic stiffness, surface energy, etc.) and, to a relatively lesser extent, biophysical stimulation (electric/magnetic) on cell-material interactions has been extensively investigated. Despite the central importance of protein adsorption on cell-material interactions, the quantitative analysis to probe into the role of physicochemical factors in protein adsorption remains largely unexplored in biomaterials science. In recent studies, the critical role of electric field stimulation toward the modulation of cell functionality in implantable biomaterials has been experimentally demonstrated. Given this background, we investigated the influence of external electric field stimulation (upto 1.00 V/nm) on fibronectin (FN) adsorption on a hydroxyapatite (HA) (001) surface at 300 K using the all-atom molecular dynamics (MD) simulation method. FN adsorption was found to be governed by attractive electrostatic interactions, which changed with the electric field strength. Nonmonotonous changes in the structural integrity of FN were recorded with the change in the field strength and direction. This can be attributed to the spatial rearrangement of the positions of local charges and the global structural changes of proteins. The dipole moment vectors of FN, water, and HA quantitatively exhibited a similar pattern of orienting themselves parallel to the field direction, with field strength-dependent increase in their magnitudes. No significant change has been recorded for the radial distribution function of water surrounding FN. Field-dependent variation in the salt bridge nets and the number of hydrogen bonds between FN and HA were also examined. One of the important results in the context of cell-material interaction is that the RGD (Arg-Gly-Asp) sequence of FN was exposed to the solvent side when the field was applied along an outward direction perpendicular to the HA (001) surface. In summary, the present study provides molecular insights into the influence of electric field stimulation on phenomenological interactions involved in FN adsorption on the HA surface.

Concerted Interactions between Multiple gp41 Trimers and the Target Cell Lipidome May Be Required for HIV-1 Entry.

The HIV-1 envelope glycoprotein gp41 mediates the fusion between viral and host cell membranes leading to virus entry and target cell infection. Despite years of research, important aspects of this process such as the number of gp41 trimers involved and how they orchestrate the rearrangement of the lipids in the apposed membranes along the fusion pathway remain obscure. To elucidate these molecular underpinnings, we performed coarse-grained molecular dynamics simulations of HIV-1 virions pinned to the CD4 T cell membrane by different numbers of gp41 trimers. We built realistic cell and viral membranes by mimicking their respective lipid compositions. We found that a single gp41 was inadequate for mediating fusion. Lipid mixing between membranes, indicating the onset of fusion, was efficient when three or more gp41 trimers pinned the membranes. The gp41 trimers interacted strongly with many different lipids in the host cell membrane, triggering lipid configurational rearrangements, exchange, and mixing. Simpler membranes, comprising fewer lipid types, displayed strong resistance to fusion, revealing the crucial role of the lipidomes in HIV-1 entry. Performing simulations at different temperatures, we estimated the free energy barrier to lipid mixing, and hence membrane stalk formation, with three and four tethering gp41 trimers to be ∼6.2 kcal/mol, a >4-fold reduction over estimates without gp41. Together, these findings present molecular-level, quantitative insights into the early stages of gp41-mediated HIV-1 entry. Preventing the requisite gp41 molecules from tethering the membranes or altering membrane lipid compositions may be potential intervention strategies.

Extrapolation of hydroxytyrosol and its analogues as potential anti-inflammatory agents.

Discovery of potential lead molecule is a challenging, and complex process which require lots of money, patience, and manpower. Human beings are using natural products, predominantly secondary metabolites, for this purpose since ancient time and they are still working on them as a potent source for drug discovery due to their wide structural diversity. Phenolic phytochemicals such as hydroxytyrosol and tyrosol are natural antioxidant and involved in many biological disease cure. Herein, we have carried out the quantum chemical calculations for conformational analysis, geometry optimization and computation of electronic as well as optical properties of hydroxytyrosol and its analogues (1a-1k) in terms of density functional theory by using Gaussian 09 program suite. The eventual charge transfer within the molecules has been confirmed by the analysis of frontier molecular orbitals. The molecular docking studies of 1a-1k with cyclooxygenase-2 showed the noticeable binding affinity as compared to other nonsteroidal anti-inflammatory drugs viz. aspirin, naproxen and celecoxib. Computation of pharmacokinetics and pharmacological properties confirmed the lead/drug like potential of these screened molecules. Furthermore, the molecular dynamics simulation of best three docked ligands (1f, 1h and 1k)-receptor complex and their binding free energy calculations reveals that these molecules bind in the catalytic cavity of cyclooxygenase-2 and found stable during the 100 ns of simulation. Communicated by Ramaswamy H. Sarma.

Prediction and validation of HIV-1 gp41 ecto-transmembrane domain post-fusion trimeric structure using molecular modeling.

The glycoproteins on the surface of human immunodeficiency virus (HIV) undergoes cascade of conformational transitions to evade the human immune system. The virus replicates inside the host and infects the T-cells instigating acquired immunodeficiency syndrome (AIDS). The glycoprotein 41 (gp41) of HIV helps to mediate the fusion of virus and host membranes. The detailed mechanism of host cell invasion by virus remains obscure due to the unavailability of experimental structure of complete gp41. In the current study, the post-fusion (PoF) trimeric structure of ecto-domain including transmembrane domain of gp41 was modeled using multiple homologous templates of Simian immunodeficiency virus (SIV) and HIV-1. In order to validate the gp41 model, interactions of three peptide inhibitors: T20, C37 and C34; were studied using all-atom molecular dynamics (MD) simulations, binding free-energy calculation and per-residue energy decomposition analysis. The binding free energy calculated using MM-PBSA (Molecular Mechanics Poisson-Boltzmann surface area) method predicts maximum affinity for C34 and minimum by T20 for gp41, which is in good agreement with the available computational and experimental studies. The van der Waals interaction is a dominant contributor for the peptide-gp41 complexes. The per-residue decomposition of energy confirmed the role of Trp117, Trp120 and Ile124, present in C34 and C37, for the strong hydrophobic interactions with the deep pocket localized around the N-terminal of gp41, which is lacking in T20. The HIV-1 gp41 structure developed in this work can be used in future study to gain insight into the mechanism of virus invasion and probing potent inhibitor to eliminate AIDS.Communicated by Ramaswamy H. Sarma.

Synthesis of 8',11'-dihydrospiro[cyclohexane-1,2'-oxepino[2,3-h] chromen]-4'(3'H)-ones with ring closing metathesis as a key step.

A series of novel hybrid molecular entities incorporating various spiro chromanone scaffolds onto the benzannulated oxepine core moiety were synthesised using allylation, Claisen rearrangement, Kabbe condensation and Ring Closing Metathesis (RCM) as a key step. During the synthesis we found that the nitrogen functionality in the substrate influences significantly the catalyst load due to electronic effects. Several iterations have been carried out to achieve complete conversion to products 6a-6e.

Delineating residues for haemolytic activities of snake venom cardiotoxin 1 from Naja naja as probed by molecular dynamics simulations and in vitro validations.

Cardiotoxins (CTXs) are single polypeptide chain consisting of 59-62 amino acids with four disulfide bridges and globular proteins of simple ß-sheet folds. The CTXs are one of principal toxic components causing haemolysis and damaging various cells and belong to three-finger toxin (TFT) superfamily of snake venoms. However, there is no natural or synthetic small molecular inhibitor to the protein toxins to date. In the present study, modes of interaction of cardiotoxin 1 (CTX1) from Indian cobra (Naja naja) with heterogeneous erythrocyte membrane (EM) model system have been extensively examined by using all-atom molecular dynamics (MD) simulations in near physiological conditions and comprehensive analyses of the MD data revealed two distinct principal regions ('head groove' and 'loop groove') of the protein toxin for establishing structural interactions with the EM system. Moreover, combined analyses of data from high-throughput virtual screening of NCI small molecular database, in vitro haemolytic assays for top-hits of the chemical compounds against crude venom of Naja naja and as well CTXs purified from the venom and pharmacokinetic examinations on the chemical compounds retarding haemolytic activities of CTXs suggested that Etidronic acid and Zoledronic acid are promising prototypic chemical inhibitors to CTXs of snake venoms.

Putative membrane lytic sites of P-type and S-type cardiotoxins from snake venoms as probed by all-atom molecular dynamics simulations.

Cardiotoxins (CTXs) belonging to the three-finger toxin superfamily of snake venoms are one of principal toxic components and the protein toxins exhibit membrane lytic activities when the venoms are injected into victims. In the present study, complex formations between CTX VI (a P-type CTX from Naja atra) and CTX1 (an S-type CTX from Naja naja) on zwitterionic POPC bilayers (a major lipid component of cell membranes) have been studied in near physiological conditions for a total dynamic time scale of 1.35 µs using all-atom molecular dynamics (MD) simulations. Comprehensive analyses of the MD data revealed that residues such as Leu1, Lys2, Tyr11, Lys31, Asp57 and Arg58 of CTX VI, and Ala16, Lys30 and Arg58 of CTX1 were crucial for establishing interactions with the POPC bilayer. Moreover, loop I, along with globular head and loop II of CTX VI, and loop II of CTX1 were found to be the structural regions chiefly governing complex formation of the respective proteins with POPC. Rationalizations for the differential binding modes of CTXs and implications of the findings for designing small molecular inhibitors to the toxins are also discussed. Graphical Abstract Binding modes of a P-type CTX and an S-type CTX towards the POPC bilayer.

Unfolding stabilities of two structurally similar proteins as probed by temperature-induced and force-induced molecular dynamics simulations.

Unfolding stabilities of two homologous proteins, cardiotoxin III and short-neurotoxin (SNTX) belonging to three-finger toxin (TFT) superfamily, have been probed by means of molecular dynamics (MD) simulations. Combined analysis of data obtained from steered MD and all-atom MD simulations at various temperatures in near physiological conditions on the proteins suggested that overall structural stabilities of the two proteins were different from each other and the MD results are consistent with experimental data of the proteins reported in the literature. Rationalization for the differential structural stabilities of the structurally similar proteins has been chiefly attributed to the differences in the structural contacts between C- and N-termini regions in their three-dimensional structures, and the findings endorse the 'CN network' hypothesis proposed to qualitatively analyse the thermodynamic stabilities of proteins belonging to TFT superfamily of snake venoms. Moreover, the 'CN network' hypothesis has been revisited and the present study suggested that 'CN network' should be accounted in terms of 'structural contacts' and 'structural strengths' in order to precisely describe order of structural stabilities of TFTs.

Unfolding stabilities of two paralogous proteins from Naja naja naja (Indian cobra) as probed by molecular dynamics simulations.

Structurally similar but functionally different two paralogous proteins, CTX1 (a cardiotoxin) and LNTX2 (an alpha-neurotoxin), from venom of Naja naja naja have been homology modeled and subjected to molecular dynamics (MD) simulations at four different temperatures (298 K, 310 K, 373 K & 473 K) under close quarters of physiological conditions. Each MD simulation was performed for 25 ns and trajectory structures stored at every 25 ps were used to probe various structural events occurring in the temperature-induced unfolding of the proteins. Notwithstanding their similar scaffolds, the two proteins are drastically differing in their unfolding stabilities from each other. The structural orders of flexibilities for the CTX1 and LNTX2 were found to be loop II > loop III > loop I > C-terminal and C-terminal > loop I > loop III > loop II, respectively. Based on the comprehensive analyses of the simulation data and studies on the various structural interactions of all cardiotoxins (CTXs) and alpha-neurotoxins (NTXs) for which three-dimensional structures determined by experimental techniques are available to date, we have herein proposed a hypothesis ('CN network') rationalizing the differential stabilities of the CTXs and NTXs belonging to a three-finger toxin superfamily of snake venoms.

Screening efficient BH3-mimetics to hBcl-B by means of peptidodynmimetic method.

The crucial residues of hBaxBH3 peptide for interaction with hBcl-B, an anti-apoptotic protein, were identified using molecular docking studies on the polypeptides and temperature-specific molecular dynamic simulations performed for the protein-peptide complex at near-physiological conditions (pH 7.0, 1 atmospheric pressure and 0.1 M NaCl). The data from the methods were examined by a 'strong residue contacts' filter strategy and the data analyses of the former and latter methods identified 10 (Q52, K57, S60, L63, K64, R65, G67, D68, D71 & S72) and 3 (S60, E61 & K64) crucial residues of the hBaxBH3 peptide for interacting with the protein, respectively. We have herein demonstrated that BH3-chemical mimetics screened using the pharmacophoric residues of hBaxBH3 obtained from the 'peptidodynmimetic method' were superior in terms of ligand efficiencies, bioavailability and pharmacokinetic properties vis-à-vis that of small molecule BH3-mimetics retrieved using the conventional 'peptidomimetic method'. The unique advantages of the 'peptidodynmimetic method' to identify efficient BH3-mimetics for modulating interfaces (composed of a large number of amino acids) of other anti-apoptotic proteins-BH3-only peptides have also been discussed in detail.

In silico methods for designing antagonists to anti-apoptotic members of Bcl-2 family proteins.

Designing antagonists to anti-apoptotic proteins of Bcl-2 family has become an important strategy in cancer chemotherapy. Using experimental techniques and computational methods, a few numbers of lead inhibitors to the antiapoptotic proteins have been reported in the literature and a few of them are under clinical trials. In this review, the lead inhibitors designed using in silico methodologies are exclusively covered, systematically organized and critically evaluated. An orchestrated in silico strategy for screening and identifying efficient antagonists to the anti-apoptotic proteins has also been brought into fore.