Effects of the RNA-Polymerase Inhibitors Remdesivir and Favipiravir on the Structure of Lipid Bilayers-An MD Study.

The structure and dynamics of membranes are crucial to ensure the proper functioning of cells. There are some compounds used in therapeutics that show nonspecific interactions with membranes in addition to their specific molecular target. Among them, two compounds recently used in therapeutics against COVID-19, remdesivir and favipiravir, were subjected to molecular dynamics simulation assays. In these, we demonstrated that the compounds can spontaneously bind to model lipid membranes in the presence or absence of cholesterol. These findings correlate with the corresponding experimental results recently reported by our group. In conclusion, insertion of the compounds into the membrane is observed, with a mean position close to the phospholipid head groups.

Atomistic insight into the essential binding event of ACE2-derived peptides to the SARS-CoV-2 spike protein.

The pathogenic agent of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters into human cells through the interaction between the receptor binding domain (RBD) of its spike glycoprotein and the angiotensin-converting enzyme 2 (ACE2) receptor. Efforts have been made towards finding antivirals that block this interaction, therefore preventing infection. Here, we determined the binding affinity of ACE2-derived peptides to the RBD of SARS-CoV-2 experimentally and performed MD simulations in order to understand key characteristics of their interaction. One of the peptides, p6, binds to the RBD of SARS-CoV-2 with nM affinity. Although the ACE2-derived peptides retain conformational flexibility when bound to SARS-CoV-2 RBD, we identified residues T27 and K353 as critical anchors mediating the interaction. New ACE2-derived peptides were developed based on the p6-RBD interface analysis and expecting the native conformation of the ACE2 to be maintained. Furthermore, we found a correlation between the helicity in trifluoroethanol and the binding affinity to RBD of the new peptides. Under the hypothesis that the conservation of peptide secondary structure is decisive to the binding affinity, we developed a cyclized version of p6 which had more helicity than p6 and approximately half of its KD value.

Interaction of the small-molecule kinase inhibitors tofacitinib and lapatinib with membranes.

Lapatinib and tofacitinib are small-molecule kinase inhibitors approved for the treatment of advanced or metastatic breast cancer and rheumatoid arthritis, respectively. So far, the mechanisms which are responsible for their activities are not entirely understood. Here, we focus on the interaction of these drug molecules with phospholipid membranes, which has not yet been investigated before in molecular detail. Owing to their lipophilic characteristics, quantitatively reflected by large differences of the partition equilibrium between water and octanol phases (expressed by logP values), rather drastic differences in the membrane interaction of both molecules have to be expected. Applying experimental (nuclear magnetic resonance, fluorescence and ESR spectroscopy) and theoretical (molecular dynamics simulations) approaches, we found that lapatinib and tofacitinib bind to lipid membranes and insert into the lipid-water interface of the bilayer. For lapatinib, a deeper embedding into the membrane bilayer was observed than for tofacitinib implying different impacts of the molecules on the bilayer structure. While for tofacitinib, no influence to the membrane structure was found, lapatinib causes a membrane disturbance, as concluded from an increased permeability of the membrane for polar molecules. These data may contribute to a better understanding of the cellular uptake mechanism(s) and the side effects of the drugs.

An adipose tissue galectin controls endothelial cell function via preferential recognition of 3-fucosylated glycans.

Upon overnutrition, adipocytes activate a homeostatic program to adjust anabolic pressure. An inflammatory response enables adipose tissue (AT) expansion with concomitant enlargement of its capillary network, and reduces energy storage by increasing insulin resistance. Galectin-12 (Gal-12), an endogenous lectin preferentially expressed in AT, plays a key role in adipocyte differentiation, lipolysis, and glucose homeostasis. Here, we reveal biochemical and biophysical determinants of Gal-12 structure, including its preferential recognition of 3-fucosylated structures, a unique feature among members of the galectin family. Furthermore, we identify a previously unanticipated role for this lectin in the regulation of angiogenesis within AT. Gal-12 showed preferential localization within the inner side of lipid droplets, and its expression was upregulated under hypoxic conditions. Through glycosylation-dependent binding to endothelial cells, Gal-12 promoted in vitro angiogenesis. Moreover, analysis of in vivo AT vasculature showed reduced vascular networks in Gal-12-deficient (Lgals12-/-) compared to wild-type mice, supporting a role for this lectin in AT angiogenesis. In conclusion, this study unveils biochemical, topological, and functional features of a hypoxia-regulated galectin in AT, which modulates endothelial cell function through recognition of 3-fucosylated glycans. Thus, glycosylation-dependent programs may control AT homeostasis by modulating endothelial cell biology with critical implications in metabolic disorders and inflammation.

The Structural Biology of Galectin-Ligand Recognition: Current Advances in Modeling Tools, Protein Engineering, and Inhibitor Design.

Galectins (formerly known as "S-type lectins") are a subfamily of soluble proteins that typically bind ß-galactoside carbohydrates with high specificity. They are present in many forms of life, from nematodes and fungi to animals, where they perform a wide range of functions. Particularly in humans, different types of galectins have been described differing not only in their tissue expression but also in their cellular location, oligomerization, fold architecture and carbohydrate-binding affinity. This distinct yet sometimes overlapping distributions and physicochemical attributes make them responsible for a wide variety of both intra- and extracellular functions, including tremendous importance in immunity and disease. In this review, we aim to provide a general description of galectins most important structural features, with a special focus on the molecular determinants of their carbohydrate-recognition ability. For that purpose, we structurally compare the human galectins, in light of recent mutagenesis studies and novel X-ray structures. We also offer a detailed description on how to use the solvent structure surrounding the protein as a tool to get better predictions of galectin-carbohydrate complexes, with a potential application to the rational design of glycomimetic inhibitory compounds. Finally, using Gal-1 and Gal-3 as paramount examples, we review a series of recent advances in the development of engineered galectins and galectin inhibitors, aiming to dissect the structure-activity relationship through the description of their interaction at the molecular level.

Lacking catalase, a protistan parasite draws on its photosynthetic ancestry to complete an antioxidant repertoire with ascorbate peroxidase.

BACKGROUND: Antioxidative enzymes contribute to a parasite's ability to counteract the host's intracellular killing mechanisms. The facultative intracellular oyster parasite, Perkinsus marinus, a sister taxon to dinoflagellates and apicomplexans, is responsible for mortalities of oysters along the Atlantic coast of North America. Parasite trophozoites enter molluscan hemocytes by subverting the phagocytic response while inhibiting the typical respiratory burst. Because P. marinus lacks catalase, the mechanism(s) by which the parasite evade the toxic effects of hydrogen peroxide had remained unclear. We previously found that P. marinus displays an ascorbate-dependent peroxidase (APX) activity typical of photosynthetic eukaryotes. Like other alveolates, the evolutionary history of P. marinus includes multiple endosymbiotic events. The discovery of APX in P. marinus raised the questions: From which ancestral lineage is this APX derived, and what role does it play in the parasite's life history? RESULTS: Purification of P. marinus cytosolic APX activity identified a 32 kDa protein. Amplification of parasite cDNA with oligonucleotides corresponding to peptides of the purified protein revealed two putative APX-encoding genes, designated PmAPX1 and PmAPX2. The predicted proteins are 93% identical, and PmAPX2 carries a 30 amino acid N-terminal extension relative to PmAPX1. The P. marinus APX proteins are similar to predicted APX proteins of dinoflagellates, and they more closely resemble chloroplastic than cytosolic APX enzymes of plants. Immunofluorescence for PmAPX1 and PmAPX2 shows that PmAPX1 is cytoplasmic, while PmAPX2 is localized to the periphery of the central vacuole. Three-dimensional modeling of the predicted proteins shows pronounced differences in surface charge of PmAPX1 and PmAPX2 in the vicinity of the aperture that provides access to the heme and active site. CONCLUSIONS: PmAPX1 and PmAPX2 phylogenetic analysis suggests that they are derived from a plant ancestor. Plant ancestry is further supported by the presence of ascorbate synthesis genes in the P. marinus genome that are similar to those in plants. The localizations and 3D structures of the two APX isoforms suggest that APX fulfills multiple functions in P. marinus within two compartments. The possible role of APX in free-living and parasitic stages of the life history of P. marinus is discussed.

Anti-Hemagglutinin Antibody Derived Lead Peptides for Inhibitors of Influenza Virus Binding.

Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/Mute Swan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.

Modulation of the pH Stability of Influenza Virus Hemagglutinin: A Host Cell Adaptation Strategy.

Proteins undergo dynamic structural changes to function within the range of physical and chemical conditions of their microenvironments. Changes in these environments affect their activity unless the respective mutations preserve their proper function. Here, we examine the influenza A virus spike protein hemagglutinin (HA), which undergoes a dynamic conformational change that is essential to the viral life cycle and is dependent on endosomal pH. Since the cells of different potential hosts exhibit different levels of pH, the virus can only cross species barriers if HA undergoes mutations that still permit the structural change to occur. This key event occurs after influenza A enters the host cell via the endocytic route, during its intracellular transport inside endosomes. The acidic pH inside these vesicles triggers a major structural transition of HA that induces fusion of the viral envelope and the endosomal membrane, and permits the release of the viral genome. HA experiences specific mutations that alter its pH stability and allow the conformational changes required for fusion in different hosts, despite the differences in the degree of acidification of their endosomes. Experimental and theoretical studies over the past few years have provided detailed insights into the structural aspects of the mutational changes that alter its susceptibility to different pH thresholds. We will illustrate how such mutations modify the protein's structure and consequently its pH stability. These changes make HA an excellent model of the way subtle structural modifications affect a protein's stability and enable it to function in diverse environments.

Impact of human galectin-1 binding to saccharide ligands on dimer dissociation kinetics and structure.

Endogenous lectins can control critical biological responses, including cell communication, signaling, angiogenesis and immunity by decoding glycan-containing information on a variety of cellular receptors and the extracellular matrix. Galectin-1 (Gal-1), a prototype member of the galectin family, displays only one carbohydrate recognition domain and occurs in a subtle homodimerization equilibrium at physiologic concentrations. Such equilibrium critically governs the function of this lectin signaling by allowing tunable interactions with a preferential set of glycosylated receptors. Here, we used a combination of experimental and computational approaches to analyze the kinetics and mechanisms connecting Gal-1 ligand unbinding and dimer dissociation processes. Kinetic constants of both processes were found to differ by an order of magnitude. By means of steered molecular dynamics simulations, the ligand unbinding process was followed monitoring water occupancy changes. By determining the water sites in a carbohydrate binding place during the unbinding process, we found that rupture of ligand-protein interactions induces an increase in energy barrier while ligand unbinding process takes place, whereas the entry of water molecules to the binding groove and further occupation of their corresponding water sites contributes to lowering of the energy barrier. Moreover, our findings suggested local asymmetries between the two subunits in the dimer structure detected at a nanosecond timescale. Thus, integration of experimental and computational data allowed a more complete understanding of lectin ligand binding and dimerization processes, suggesting new insights into the relationship between Gal-1 structure and function and renewing the discussion on the biophysics and biochemistry of lectin-ligand lattices.

Solvent structure improves docking prediction in lectin-carbohydrate complexes.

Recognition and complex formation between proteins and carbohydrates is a key issue in many important biological processes. Determination of the three-dimensional structure of such complexes is thus most relevant, but particularly challenging because of their usually low binding affinity. In silico docking methods have a long-standing tradition in predicting protein-ligand complexes, and allow a potentially fast exploration of a number of possible protein-carbohydrate complex structures. However, determining which of these predicted complexes represents the correct structure is not always straightforward. In this work, we present a modification of the scoring function provided by AutoDock4, a widely used docking software, on the basis of analysis of the solvent structure adjacent to the protein surface, as derived from molecular dynamics simulations, that allows the definition and characterization of regions with higher water occupancy than the bulk solvent, called water sites. They mimic the interaction held between the carbohydrate -OH groups and the protein. We used this information for an improved docking method in relation to its capacity to correctly predict the protein-carbohydrate complexes for a number of tested proteins, whose ligands range in size from mono- to tetrasaccharide. Our results show that the presented method significantly improves the docking predictions. The resulting solvent-structure-biased docking protocol, therefore, appears as a powerful tool for the design and optimization of development of glycomimetic drugs, while providing new insights into protein-carbohydrate interactions. Moreover, the achieved improvement also underscores the relevance of the solvent structure to the protein carbohydrate recognition process.

Conformational states of 2'-C-methylpyrimidine nucleosides in single and double nucleic acid stranded structures.

The hybridization performance of a set of 12-mer RNA:RNA duplexes containing 2'-C-methyluridine, 5-bromo-2'-C-methyluridine, or (2'S)-2'-deoxy-2'-C-methyluridine was analyzed. Melting point temperatures of the modified duplexes showed an important ΔT(m) decrease (-8.9 to -12.5 °C), while circular dichroism experiments indicated that the helix was still A-type, suggesting a localized disturbance disorder. Molecular dynamics simulations using AMBER were carried out in order to gain structural knowledge about the effect of the 2'-C-methyl modification in double stranded environments. On the other hand, in an attempt to explain the behavior of the 2'-deoxy-2'-C-methyl nucleosides in single stranded environments, like the 10-23 DNAzyme core, molecular dynamic simulations were performed, incorporating the modified analogues into single stranded reported stem-loop structures, studding the sugar conformations along the MD trajectories. It was observed that, despite their preferential conformational states, the 2'-C-methyl analogues are flexible enough to adopt a different puckering in single stranded environments.

When galectins recognize glycans: from biochemistry to physiology and back again.

In the past decade, increasing efforts have been devoted to the study of galectins, a family of evolutionarily conserved glycan-binding proteins with multifunctional properties. Galectins function, either intracellularly or extracellularly, as key biological mediators capable of monitoring changes occurring on the cell surface during fundamental biological processes such as cellular communication, inflammation, development, and differentiation. Their highly conserved structures, exquisite carbohydrate specificity, and ability to modulate a broad spectrum of biological processes have captivated a wide range of scientists from a wide spectrum of disciplines, including biochemistry, biophysics, cell biology, and physiology. However, in spite of enormous efforts to dissect the functions and properties of these glycan-binding proteins, limited information about how structural and biochemical aspects of these proteins can influence biological functions is available. In this review, we aim to integrate structural, biochemical, and functional aspects of this bewildering and ancient family of glycan-binding proteins and discuss their implications in physiologic and pathologic settings.

An integrated computational analysis of the structure, dynamics, and ligand binding interactions of the human galectin network.

Galectins, a family of evolutionarily conserved animal lectins, have been shown to modulate signaling processes leading to inflammation, apoptosis, immunoregulation, and angiogenesis through their ability to interact with poly-N-acetyllactosamine-enriched glycoconjugates. To date 16 human galectin carbohydrate recognition domains have been established by sequence analysis and found to be expressed in several tissues. Given the divergent functions of these lectins, it is of vital importance to understand common and differential features in order to search for specific inhibitors of individual members of the human galectin family. In this work we performed an integrated computational analysis of all individual members of the human galectin family. In the first place, we have built homology-based models for galectin-4 and -12 N-terminus, placental protein 13 (PP13) and PP13-like protein for which no experimental structural information is available. We have then performed classical molecular dynamics simulations of the whole 15 members family in free and ligand-bound states to analyze protein and protein-ligand interaction dynamics. Our results show that all galectins adopt the same fold, and the carbohydrate recognition domains are very similar with structural differences located in specific loops. These differences are reflected in the dynamics characteristics, where mobility differences translate into entropy values which significantly influence their ligand affinity. Thus, ligand selectivity appears to be modulated by subtle differences in the monosaccharide binding sites. Taken together, our results may contribute to the understanding, at a molecular level, of the structural and dynamical determinants that distinguish individual human galectins.

Structural basis for ligand recognition in a mushroom lectin: solvent structure as specificity predictor.

Lectins are able to recognize specific carbohydrate structures through their carbohydrate recognition domain (CRD). The lectin from the mushroom Agaricus bisporus (ABL) has the remarkable ability of selectively recognizing the TF-antigen, composed of Galß1-3GalNAc, Ser/Thr linked to proteins, specifically exposed in neoplastic tissues. Strikingly, the recently solved crystal structure of tetrameric ABL in the presence of TF-antigen and other carbohydrates showed that each monomer has two CRDs, each being able to bind specifically to different monosaccharides that differ only in the configuration of a single hydroxyl, like N-acetyl-d-galactosamine (GalNAc) and N-acetyl-d-glucosamine (GlcNAc). Understanding how lectin CRDs bind and discriminate mono and/or (poly)-saccharides is an important issue in glycobiology, with potential impact in the design of better and selective lectin inhibitors with potential therapeutic properties. In this work, and based on the unusual monosaccharide epimeric specificity of the ABL CRDs, we have performed molecular dynamics simulations of the natural (crystallographic) and inverted (changing GalNAc for GlcNAc and vice-versa) ABL-monosaccharide complexes in order to understand the selective ligand recognition properties of each CRD. We also performed a detailed analysis of the CRD local solvent structure, using previously developed methodology, and related it with the recognition mechanism. Our results provide a detailed picture of each ABL CRD specificity, allowing a better understanding of the carbohydrate selective recognition process in this particular lectin.

Protein dynamics and ligand migration interplay as studied by computer simulation.

Since proteins are dynamic systems in living organisms, the employment of methodologies contemplating this crucial characteristic results fundamental to allow revealing several aspects of their function. In this work, we present results obtained using classical mechanical atomistic simulation tools applied to understand the connection between protein dynamics and ligand migration. Firstly, we will present a review of the different sampling schemes used in the last years to obtain both ligand migration pathways and the thermodynamic information associated with the process. Secondly, we will focus on representative examples in which the schemes previously presented are employed, concerning the following: i) ligand migration, tunnels, and cavities in myoglobin and neuroglobin; ii) ligand migration in truncated hemoglobin members; iii) NO escape and conformational changes in nitrophorins; iv) ligand selectivity in catalase and hydrogenase; and v) larger ligand migration: the P450 and haloalkane dehalogenase cases. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Linking the structure and thermal stability of beta-galactoside-binding protein galectin-1 to ligand binding and dimerization equilibria.

The stability of proteins involves a critical balance of interactions of different orders of magnitude. In this work, we present experimental evidence of an increased thermal stability of galectin-1, a multifunctional beta-galactoside-binding protein, upon binding to the disaccharide lactose. Analysis of structural changes occurring upon binding of lectin to its specific glycans and thermal denaturation of the protein and the complex were analyzed by circular dichroism. On the other hand, we studied dimerization as another factor that may induce structural and thermal stability changes. The results were then complemented with molecular dynamics simulations followed by a detailed computation of thermodynamic properties, including the internal energy, solvation free energy, and conformational entropy. In addition, an energetic profile of the binding and dimerization processes is also presented. Whereas binding and cross-linking of lactose do not alter galectin-1 structure, this interaction leads to substantial changes in the flexibility and internal energy of the protein which confers increased thermal stability to this endogenous lectin. Given that an improved understanding of the physicochemical properties of galectin-glycan lattices may contribute to the dissection of their biological functions and prediction of their therapeutic applications, our study suggests that galectin binding to specific disaccharide ligands may increase the thermal stability of this glycan-binding protein, an effect that could influence its critical biological functions.

Carbohydrate-binding proteins: Dissecting ligand structures through solvent environment occupancy.

Formation of protein ligand complexes is a fundamental phenomenon in biochemistry. During the process, significant solvent reorganization is produced along the contact surface and many water molecules strongly bound to the protein's ligand binding site must be displaced. Both the thermodynamics and kinetics of this process are complex and a clear understanding at the microscopic level has been not achieved so far. Special attention has been paid to the structure of water molecules on carbohydrate recognition sites of various proteins, and many studies support the idea that displacement of these water molecules should have a crucial effect on the binding free energy. Molecular dynamics (MD) simulations in explicit water solvent is a very promising approach for this type of studies. Using MD simulations combined with statistical mechanics analysis, thermodynamic properties of these water molecules can be computed and analyzed in a comparative view. Using this idea, we developed a set of analysis tools to link solvation with ligand binding in a key carbohydrate binding protein, human galectin-1 (hGal-1). Specifically, we defined water sites (WS) in terms of the thermodynamic properties of water molecules strongly bound to protein surfaces. In the present work, we selected a group of proteins whose ligand bound complexes have been already structurally characterized in order to extend the analysis of the role of the surface associated water molecules in the ligand binding and recognition process. The selected proteins are concanavalin-A (Con-A), galectin-3 (Gal-3), cyclophilin-A (Cyp-A), and two modules CBM40 and CBM32 of the multimodular bacterial sialidase. Our results show that the probability of finding water molecules inside the WS, p(v), with respect to the bulk density is directly correlated to the likeliness of finding an hydroxyl group of the ligand in the protein-ligand complex. This information can be used to analyze in detail the solvation structure of the carbohydrate recognition domain (CRD) and its relation to the possible protein ligand complexes and suggests addition of OH-containing functional groups to displace water from high p(v) WS to enhance drugs, specially glycomimetic-drugs, protein affinity, and/or specificity.

Critical role of the solvent environment in galectin-1 binding to the disaccharide lactose.

Galectin-1 (Gal-1), a member of a family of evolutionarily conserved glycan-binding proteins, binds specifically to poly-N-acetyllactosamine-enriched glycoconjugates. Through interactions with these glycoconjugates, this protein modulates inflammatory responses and contributes to tumor progression and immune cell homeostasis. The carbohydrate recognition domain includes the single protein tryptophan (Trp68). UV resonance Raman spectroscopy and molecular dynamic simulation were used to examine the change in the environment of the Trp on ligand binding. The UV Raman spectra and the calculated water radial distribution functions show that, while no large structural changes in the protein follow lactose binding, substantial solvent reorganization occurs. These new insights into the microscopic role of water molecules in Gal-1 binding to its specific carbohydrate ligands provides a better understanding of the physicochemical properties of Gal-1-saccharide interactions, which will be useful for the design of synthetic inhibitors for therapeutic purposes.

Characterization of the galectin-1 carbohydrate recognition domain in terms of solvent occupancy.

Human galectin-1, a galactosil-terminal sugar binding soluble protein, is a potent multifunctional effector that participates in specific protein-carbohydrate and protein-protein interactions. Recent studies revealed that it plays a key role as a modulator of cellular differentiation and immunological response. In this work, we have investigated the solvation properties of the carbohydrate recognition domain of Gal-1 by means of molecular dynamics simulations. Water sites (ws) were identified in terms of radial and angular distribution functions, and properties such as water residence times, interaction energies, and free-energy contributions were evaluated for those sites. Our results allowed us to correlate the thermodynamic properties of the ws and their binding pattern with the N-acetilgalactoside ligand. These results let us further infer that the water molecules located at the ws, which exhibit much more favorable binding, are the ones replaced by -OH groups of the sugar.

Modeling heme proteins using atomistic simulations.

Heme proteins are found in all living organisms, and perform a wide variety of tasks ranging from electron transport, to the oxidation of organic compounds, to the sensing and transport of small molecules. In this work we review the application of classical and quantum-mechanical atomistic simulation tools to the investigation of several relevant issues in heme proteins chemistry: (i) conformational analysis, ligand migration, and solvation effects studied using classical molecular dynamics simulations; (ii) electronic structure and spin state energetics of the active sites explored using quantum-mechanics (QM) methods; (iii) the interaction of heme proteins with small ligands studied through hybrid quantum mechanics-molecular mechanics (QM-MM) techniques; (iv) and finally chemical reactivity and catalysis tackled by a combination of quantum and classical tools.