Structural insights in mammalian sialyltransferases and fucosyltransferases: We have come a long way, but it is still a long way down

Funding Information: The APC was funded by King Abdullah University of Science and Technology. M.C.: L.C. and A.R.S. acknowledge the King Abdullah University of Science and Technology (KAUST) for support. We thank Romina Oliva, University of Parthenope (Naples), for helpful comments. Thanks to the STEMskills Research and Education Lab team members for support.Mammalian cell surfaces are modified with complex arrays of glycans that play major roles in health and disease. Abnormal glycosylation is a hallmark of cancer; terminal sialic acid and fucose in particular have high levels in tumor cells, with positive implications for malignancy. Increased sialylation and fucosylation are due to the upregulation of a set of sialyltransferases (STs) and fucosyltransferases (FUTs), which are potential drug targets in cancer. In the past, several advances in glycostructural biology have been made with the determination of crystal structures of several important STs and FUTs in mammals. Additionally, how the independent evolution of STs and FUTs occurred with a limited set of global folds and the diverse modular ability of catalytic domains toward substrates has been elucidated. This review highlights advances in the understanding of the structural architecture, substrate binding interactions, and catalysis of STs and FUTs in mammals. While this general understanding is emerging, use of this information to design inhibitors of STs and FUTs will be helpful in providing further insights into their role in the manifestation of cancer and developing targeted therapeutics in cancer.publishersversionpublishe

Does Metabolism of (S)-N-[1-(3-Morpholin-4-ylphenyl)ethyl]-3-phenylacrylamide Occur at the Morpholine Ring? Quantum Mechanical and Molecular Dynamics Studies

The mechanism of Cytochrome P450 3A4 mediated metabolism of (S)-N- [1-(3-morpholin-4ylphenyl)ethyl]-3-phenylacrylamide and its difluoro analogue have been investigated by density functional QM calculations aided with molecular mechanics/molecular dynamics simulations. In this article, we mainly focus on the metabolism of the morpholine ring of substrates 1 and 2. The reaction proceeds via a hydrogen atom abstraction from the morpholine ring by Compound I on a doublet potential energy surface. A transition state was observed at an O-H distance of 1.46 Å for 1 while 1.38 Å for 2. Transition state for the rebound mechanism was not observed. The energy barrier for the hydrogen atom abstraction from 1 was found to be 7.01 kcal/mol in gas phase while 19.53 kcal/mol when the protein environment was emulated by COSMO. Similarly the energy barrier for substrate 2 was found to be 11.07 kcal/mol in gas phase while it was reduced to 12.99 kcal/mol in protein environment. Our previous study reported energy barriers for phenyl hydroxylation of 7.4 kcal/mol. Large energy barriers for morpholine hydroxylation indicates that hydroxylation at the phenyl ring may be preferred over morpholine. MD simulations in protein environment indicated that hydrogen atom at C4 position of phenyl ring remains in closer proximity to oxyferryl oxygen of the heme moiety as compared to morpholine hydrogen and hence greater chance to metabolize at phenyl ring

Immunoinformatics-aided rational design of a multi-epitope vaccine targeting feline infectious peritonitis virus

Feline infectious peritonitis (FIP) is a grave and frequently lethal ailment instigated by feline coronavirus (FCoV) in wild and domestic feline species. The spike (S) protein of FCoV assumes a critical function in viral ingress and infection, thereby presenting a promising avenue for the development of a vaccine. In this investigation, an immunoinformatics approach was employed to ascertain immunogenic epitopes within the S-protein of FIP and formulate an innovative vaccine candidate. By subjecting the amino acid sequence of the FIP S-protein to computational scrutiny, MHC-I binding T-cell epitopes were predicted, which were subsequently evaluated for their antigenicity, toxicity, and allergenicity through in silico tools. Our analyses yielded the identification of 11 potential epitopes capable of provoking a robust immune response against FIPV. Additionally, molecular docking analysis demonstrated the ability of these epitopes to bind with feline MHC class I molecules. Through the utilization of suitable linkers, these epitopes, along with adjuvants, were integrated to design a multi-epitope vaccine candidate. Furthermore, the stability of the interaction between the vaccine candidate and feline Toll-like receptor 4 (TLR4) was established via molecular docking and molecular dynamics simulation analyses. This suggests good prospects for future experimental validation to ascertain the efficacy of our vaccine candidate in inducing a protective immune response against FIP

D936Y and Other Mutations in the Fusion Core of the SARS-CoV-2 Spike Protein Heptad Repeat 1: Frequency, Geographical Distribution, and Structural Effect

The crown of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constituted by its spike (S) glycoprotein. S protein mediates the SARS-CoV-2 entry into the host cells. The “fusion core” of the heptad repeat 1 (HR1) on S plays a crucial role in the virus infectivity, as it is part of a key membrane fusion architecture. While SARS-CoV-2 was becoming a global threat, scientists have been accumulating data on the virus at an impressive pace, both in terms of genomic sequences and of three-dimensional structures. On 15 February 2021, from the SARS-CoV-2 genomic sequences in the GISAID resource, we collected 415,673 complete S protein sequences and identified all the mutations occurring in the HR1 fusion core. This is a 21-residue segment, which, in the post-fusion conformation of the protein, gives many strong interactions with the heptad repeat 2, bringing viral and cellular membranes in proximity for fusion. We investigated the frequency and structural effect of novel mutations accumulated over time in such a crucial region for the virus infectivity. Three mutations were quite frequent, occurring in over 0.1% of the total sequences. These were S929T, D936Y, and S949F, all in the N-terminal half of the HR1 fusion core segment and particularly spread in Europe and USA. The most frequent of them, D936Y, was present in 17% of sequences from Finland and 12% of sequences from Sweden. In the post-fusion conformation of the unmutated S protein, D936 is involved in an inter-monomer salt bridge with R1185. We investigated the effect of the D936Y mutation on the pre-fusion and post-fusion state of the protein by using molecular dynamics, showing how it especially affects the latter one

Effects of Water Concentration on the Free Volume of Amino Acid Ionic Liquids Investigated by Molecular Dynamics Simulations

Amino acid ionic liquids (AAILs) are gaining attention because of their potential in CO₂ capture technology. Molecular dynamics simulations of AAILs tetra­methyl­ammonium glycinate ([N₁₁₁₁]­[Gly]), tetra­butyl­ammonium glycinate ([N₄₄₄₄]­[Gly]), and 1,1,1-tri­methyl­hydrazinium glycinate ([aN₁₁₁]­[Gly]) and their corresponding mixtures with water were performed to investigate the effect of water concentration on the cation–anion interactions. The water content significantly influenced the free volume (FV) and fractional free volume (FFV) of the AAILs that varied with the hydrophobic and hydrophilic nature of the ion pairs. Under dry conditions, the FFV increased with increasing cation molecular sizes, indicative of proportional adsorption of any inert gases, such as N₂, as consistent with experimental observations. Furthermore, the polarity of the cation played an important role in FFV and hence the diffusion of the AAILs. Density functional theory calculations suggested that hydrophilic [aN₁₁₁]­[Gly] featured stronger interactions in the presence of water, whereas the hydrophobic IL showed weaker interactions. The carboxylate group of glycinate displayed stronger interactions with water than the cation. The computational study provided qualitative insight into the role of FV of the AAILs on CO₂ and N₂ absorption and suggests that [aN₁₁₁]­[Gly] has CO₂ adsorption capacity in the presence of water superior to that of other studied AAILs

A Saccharomyces cerevisiae assay system to investigate ligand/adipoR1 interactions that lead to cellular Signaling

Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology.Peer Reviewe

Quantum Mechanical and Molecular Dynamics Simulations of Dual-Amino-Acid Ionic Liquids for CO₂ Capture

Global warming is occurring because of emission of greenhouse gases due to human activities. Capture of CO₂ from fossil-fuel industries and absorption of CO₂ for natural gas sweetening are crucial industrial tasks to address the threat from greenhouse gases. Amino acid ionic liquids (AAILs) are used for reversible CO₂ capture. In this study, the effect of CO₂ chemisorption on tetramethylammonium glycinate ([N₁₁₁₁]­[GLY]), tetrabutylammonium glycinate ([N₄₄₄₄]­[GLY]), and 1,1,1-trimethylhydrazinium glycinate ([aN₁₁₁]­[GLY]) were analyzed using density functional theory (DFT) and molecular dynamics (MD) studies. Density functional theory studies predicted different reaction pathways for CO₂ absorption on [GLY]⁻ and [aN₁₁₁]⁺. The activation energy barriers for CO₂ absorption on [GLY]⁻ and [aN₁₁₁]⁺ are 52.43 and 64.40 kJ/mol, respectively. The MD results were useful for mimicking the reaction mechanism for CO₂ absorption on AAILs and its effect on physical properties such as the fractional free volume, diffusion coefficient, and hydrogen bonding. Dry and wet conditions were compared to identify factors contributing to CO₂ solubility and selectivity at room temperature and elevated temperature. Hydrogen bonding between ion pairs was used to understand the increase in viscosity after CO₂ absorption. The MD studies revealed that glycinate and related products after CO₂ absorption contribute the most to the increase in viscosity