Nucleoside-Driven Specificity of DNA Methyltransferase.

We have studied the adenosine binding specificities of two bacterial DNA methyltransferases, Taq methyltransferase (M.TaqI), and HhaI methyltransferase (M.HhaI). While they have similar cofactor binding pocket interactions, experimental data showed different specificity for novel S-nucleobase-l-methionine cofactors (SNMs; N=guanosyl, cytidyl, uridyl). Protein dynamics corroborate the experimental data on the cofactor specificities. For M.TaqI the specificity for S-adenosyl-l-methionine (SAM) is governed by the tight binding on the nucleoside part of the cofactor, while for M.HhaI the degree of freedom of the nucleoside chain allows the acceptance of other bases. The experimental data prove catalytically productive methylation by the M.HhaI binding pocket for all the SNMs. Our results suggest a new route for successful design of unnatural SNM analogues for methyltransferases as a tool for cofactor engineering.

Correction to "Substrate Dynamics Contribute to Enzymatic Specificity in Human and Bacterial Methionine Adenosyltransferases".

[This corrects the article DOI: 10.1021/jacsau.1c00464.].

Implications of divergence of methionine adenosyltransferase in archaea.

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosyl methionine from l-methionine and ATP. MAT enzymes are ancient, believed to share a common ancestor, and are highly conserved in all three domains of life. However, the sequences of archaeal MATs show considerable divergence compared with their bacterial and eukaryotic counterparts. Furthermore, the structural significance and functional significance of this sequence divergence are not well understood. In the present study, we employed structural analysis and ancestral sequence reconstruction to investigate archaeal MAT divergence. We observed that the dimer interface containing the active site (which is usually well conserved) diverged considerably between the bacterial/eukaryotic MATs and archaeal MAT. A detailed investigation of the available structures supports the sequence analysis outcome: The protein domains and subdomains of bacterial and eukaryotic MAT are more similar than those of archaea. Finally, we resurrected archaeal MAT ancestors. Interestingly, archaeal MAT ancestors show substrate specificity, which is lost during evolution. This observation supports the hypothesis of a common MAT ancestor for the three domains of life. In conclusion, we have demonstrated that archaeal MAT is an ideal system for studying an enzyme family that evolved differently in one domain compared with others while maintaining the same catalytic activity.

Substrate Dynamics Contribute to Enzymatic Specificity in Human and Bacterial Methionine Adenosyltransferases.

Protein conformational changes can facilitate the binding of noncognate substrates and underlying promiscuous activities. However, the contribution of substrate conformational dynamics to this process is comparatively poorly understood. Here, we analyze human (hMAT2A) and Escherichia coli (eMAT) methionine adenosyltransferases that have identical active sites but different substrate specificity. In the promiscuous hMAT2A, noncognate substrates bind in a stable conformation to allow catalysis. In contrast, noncognate substrates sample stable productive binding modes less frequently in eMAT owing to altered mobility in the enzyme active site. Different cellular concentrations of substrates likely drove the evolutionary divergence of substrate specificity in these orthologues. The observation of catalytic promiscuity in hMAT2A led to the detection of a new human metabolite, methyl thioguanosine, that is produced at elevated levels in a cancer cell line. This work establishes that identical active sites can result in different substrate specificity owing to the effects of substrate and enzyme dynamics.

Rossmann-Fold Methyltransferases: Taking a "ß-Turn" around Their Cofactor, S-Adenosylmethionine.

Methyltransferases (MTases) are superfamilies of enzymes that catalyze the transfer of a methyl group from S-adenosylmethionine (SAM), a nucleoside-based cofactor, to a wide variety of substrates such as DNA, RNA, proteins, small molecules, and lipids. Depending upon their structural features, the MTases can be further classified into different classes; we consider exclusively the largest class of MTases, the Rossmann-fold MTases. It has been shown that the nucleoside cofactor-binding Rossmann enzymes, particularly the nicotinamide adenine dinucleotide (NAD)-, flavin adenine dinucleotide (FAD)-, and SAM-binding MTases enzymes, share common binding motifs that include a Gly-rich loop region that interacts with the cofactor and a highly conserved acidic residue (Asp/Glu) that interacts with the ribose moiety of the cofactor. Here, we observe that the Gly-rich loop region of the Rossmann MTases adapts a specific type II' ß-turn in the proximity of the cofactor (<4 Å), and it appears to be a key feature of these superfamilies. Additionally, we demonstrate that the conservation of this ß-turn could play a critical role in the enzyme-cofactor interaction, thereby shedding new light on the structural conformation of the Gly-rich loop region from Rossmann MTases.

Microarray Analysis of Oligosaccharide-Mediated Multivalent Carbohydrate-Protein Interactions and Their Heterogeneity.

Carbohydrate-protein interactions (CPIs) are involved in a wide range of biological phenomena. Hence, the characterization and presentation of carbohydrate epitopes that closely mimic the natural environment is one of the long-term goals of glycosciences. Inspired by the multivalency, heterogeneity and nature of carbohydrate ligand-mediated interactions, we constructed a combinatorial library of mannose and galactose homo- and hetero-glycodendrons to study CPIs. Microarray analysis of these glycodendrons with a wide range of biologically important plant and animal lectins revealed that oligosaccharide structures and heterogeneity interact with each other to alter binding preferences.

Modeling Glyco-Collagen Conjugates Using a Host-Guest Strategy To Alter Phenotypic Cell Migration and in Vivo Wound Healing.

The constructs and study of combinatorial libraries of structurally defined homologous extracellular matrix (ECM) glycopeptides can significantly accelerate the identification of cell surface markers involved in a variety of physiological and pathological processes. Herein, we present a simple and reliable host-guest approach to design a high-throughput glyco-collagen library to modulate the primary and secondary cell line migration process. 4-Amidoadamantyl-substituted collagen peptides and ß-cyclodextrin appended with mono- or disaccharides were used to construct self-assembled glyco-collagen conjugates (GCCs), which were found to be thermally stable, with triple-helix structures and nanoneedles-like morphologies that altered cell migration processes. We also investigated the glycopeptide's mechanisms of action, which included interactions with integrins and cell signaling kinases. Finally, we report murine wound models to demonstrate the real-time application of GCCs. As a result of our observations, we claim that the host-guest model of ECM glycopeptides offers an effective tool to expedite identification of specific glycopeptides to manipulate cell morphogenesis, cell differentiation metastatic processes, and their biomedical applications.

Exploring the Influence of Shapes and Heterogeneity of Glyco-Gold Nanoparticles on Bacterial Binding for Preventing Infections.

To investigate the effects of the heterogeneity and shape of glyco-nanoprobes on carbohydrate-protein interactions (CPIs), α-d-mannose- and ß-d-galactose-linked homo- and heterogeneous glycodendrons were synthesized and immobilized on spherical and rod-shaped gold nanoparticles (AuNPs). Lectin and bacterial binding studies of these glyco-AuNPs clearly illustrate that multivalency and shape of AuNPs contribute significantly to CPIs than the heterogeneity of glycodendrons. Finally, bacterial infection of HeLa cells was effectively inhibited by the homogeneous glycodendron-conjugated rod-shaped AuNPs relative to their heterogeneous counterparts. Overall, these results provide insight into the role of AuNP shape and multivalency as potential factors to regulate CPIs.

Understanding carbohydrate-protein interactions using homologous supramolecular chiral Ru(ii)-glyconanoclusters.

Multivalent glycodendrimers make promising tools to tackle the basic and translational research in the field of carbohydrate-mediated interactions. Despite advances in glycodendrimers and glycopolymers, the multivalent probes available to date are still far from being ideal biological mimics. This work demonstrates the inherent chirality of glycodendrimers to be one of the promising factors to generate different spatial carbohydrate micro-environments to modulate specific carbohydrate-protein interactions. By exploiting the host-guest strategy, chiral Ru(ii) complexes (Δ and Λ) and mannose capped ß-cyclodextrin (ß-CD), we generated a library of homologous metallo-glycodendrimers (MGDs) with sizes of 50-70 nm. These nanoclusters can enantioselectively bind to specific C-type lectins and displayed selectivity in cellular uptake. We also discovered their potential clathrin-mediated endocytotic pathway in DC-SIGN and SIGNR3-transfected cell lines. Finally, in vivo biodistribution and sequestration of MGDs was determined to understand the role of chirality mediated spatial arrangement in carbohydrate-mediated interactions.

Correction: Supramolecular metalloglycodendrimers selectively modulate lectin binding and delivery of Ru(ii) complexes into mammalian cells.

Correction for 'Supramolecular metalloglycodendrimers selectively modulate lectin binding and delivery of Ru(ii) complexes into mammalian cells' by Harikrishna Bavireddi, et al., Org. Biomol. Chem., 2016, DOI: 10.1039/c6ob01546h.

Supramolecular metalloglycodendrimers selectively modulate lectin binding and delivery of Ru(ii) complexes into mammalian cells.

A host-guest interaction between Ru(ii)-complexes and sugar-capped ß-cyclodextrin was employed to synthesize metalloglycodendrimers. These glycodendrimers demonstrated selective carbohydrate-protein interactions and controlled the delivery of the Ru(ii) complexes into cancer cells, which may facilitate cell-specific apoptosis. Lectin binding assay revealed micromolar range IC50 values with different plant lectins. Cell viability assay and confocal imaging studies of Ru(ii) complexes exhibited cytotoxic activities in cancer cells compared to normal cells with IC50 values close to other literature Ru(ii) complexes. The cell death inducer was found to accumulate favorably to the endoplasmic reticulum (ER) and induced ER stress in cells. The upregulation of CHOP, caspase-3 and caspase-12 disturbed the ER morphology initiating the apoptosis pathway.

Immobilization of multivalent glycoprobes on gold surfaces for sensing proteins and macrophages.

The multivalent display of carbohydrates on the cell surface provides cooperative binding to improve the specific biological events. In addition to multivalency, the spatial arrangement and orientation of sugars with respect to external stimuli also trigger carbohydrate-protein interactions. Herein, we report a non-covalent host-guest strategy to immobilize heptavalent glyco-ß-cyclodextrin on gold-coated glass slides to study multivalent carbohydrate-protein interactions. We have found that the localization of sugar entities on surfaces using ß-cyclodextrin (ß-CD) chemistry increased the avidity of carbohydrate-protein and carbohydrate-macrophage interactions compared to monovalent-ß-CD sugar coated surfaces. This platform is expected to be a promising tool to amplify the avidity of sugar-mediated interactions on surfaces and contribute to the development of next generation bio-medical products.

Effect of Transition Metals on Polysialic Acid Structure and Functions.

Polysialic acid (PSA) is one of the most abundant glycopolymer present in embryonic brain, and it is known to be involved in key roles such as plasticity in the central nervous system, cell adhesion, migration and localization of neurotrophins. However, in adult brain, its expression is quite low. The exception to this is in Alzheimer's disease (AD) brain, where significantly increased levels of polysilylated neural cell adhesion molecule (PSA-NCAM) have been reported. Here, we confirm the role of PSA as a metal chelator, allowing it to decrease cytotoxicity caused by high levels of transition metals, commonly found in AD brain, and as a regulator of cell behavior. UV-visible (UV-vis) and circular dichroism (CD) spectroscopy, atomic force microscopy (AFM), and isothermal titration calorimetry (ITC) techniques were used to investigate the assembly of PSA-metals complexes. These PSA-metal complexes exhibited less toxicity compared to free metal ions, and in particular, the PSA-Cu(2+) complex synergistically promoted neurite outgrowth in PC12 cells.

Exploiting the lactose-GM3 interaction for drug delivery.

Protein-protein and protein-carbohydrate interactions as a means to target the cell surface for therapeutic applications have been extensively investigated. However, carbohydrate-carbohydrate interactions (CCIs) have largely been overlooked. Here, we investigate the concept of CCI-mediated drug delivery. Lactose-functionalized ß-cyclodextrin (L-ß-CD) hosting doxorubicin (Dox) was evaluated for site-specific delivery to cancer cells via interaction with GM3 , a cell-surface carbohydrate. The host-guest complex was evaluated in B16 melanoma cells, which express exceptionally high levels of GM3 , and acute monocytic leukemia (THP-1) and mouse fibroblast (NIH-3T3) cells, which lack GM3 on the cell surface. Doxorubicin (Dox) was delivered more efficiently into B16 cells compared with NIH-3T3 and THP-1 cells. In B16 cells pretreated with sialidase or sodium periodate, thus preventing CCI formation, drug uptake was significantly decreased. Taken together, the results of these studies strongly support CCI-mediated uptake via the GM3 -lactose interaction as the mechanism of controlled drug delivery.

Supramolecular scaffolds on glass slides as sugar based rewritable sensors for bacteria.

We describe here the sugar functionalized ß-cyclodextrin-ferrocene glass slides as fully reversible bacterial biosensors under the influence of external adamantane carboxylic acid. The prototype d-mannose - E. coli ORN 178 and l-fucose - P. aeruginosa interactions serve as a model to illustrate the new approach.