| 1. |
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| 2. |
- Edgar, Rebecca J., et al.
(author)
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Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides
- 2019
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In: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 15:5, s. 463-
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Journal article (peer-reviewed)abstract
- Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. Here we identify gacH, a gene in the Streptococcus pyogenes group A carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA-secreted phospholipase A(2). Subsequent structural and phylogenetic analysis of the GacH extracellular domain revealed that GacH represents an alternative class of glycerol phosphate transferase. We detected the presence of glycerol phosphate in the GAC, as well as the serotype c carbohydrate from Streptococcus mutans, which depended on the presence of the respective gacH homologs. Finally, nuclear magnetic resonance analysis of GAC confirmed that glycerol phosphate is attached to approximately 25% of the GAC N-acetylglucosamine side-chains at the C6 hydroxyl group. This previously unrecognized structural modification impacts host-pathogen interaction and has implications for vaccine design.
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| 3. |
- Furevi, Axel, 1992-, et al.
(author)
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Complete 1H and 13C NMR chemical shift assignments of mono-to tetrasaccharides as basis for NMR chemical shift predictions of oligo- and polysaccharides using the computer program CASPER
- 2022
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In: Carbohydrate Research. - : Elsevier BV. - 0008-6215 .- 1873-426X. ; 513
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Journal article (peer-reviewed)abstract
- Carbohydrate structure can be elucidated or confirmed by using NMR spectroscopy as the prime technique. Prediction of 1H and 13C NMR chemical shifts by computational approaches makes this assignment process more efficient and the program CASPER can perform this task rapidly. It does so by relying on chemical shift data of mono-, di-, and trisaccharides. In order to improve accuracy and quality of these predictions we have assigned 1H and 13C NMR chemical shifts of 30 monosaccharides, 17 disaccharides, 10 trisaccharides and one tetrasaccharide; in total 58 compounds. Due to different rotamers, ring forms, α- and β-anomeric forms and pD conditions this resulted in 74 1H and 13C NMR chemical shift data sets, all of which were refined using total line-shape analysis for the 1H resonances in order to obtain accurate chemical shifts. Subsequent NMR chemical shift predictions for three sialic acid-containing oligosaccharides, viz., GD1a, a disialyl-LNnT hexasaccharide and a polysialic acid-lactose decasaccharide, and NMR-based structural elucidations of two O-antigen polysaccharides from E. coli O174 were performed by the CASPER program (http://www.casper.organ.su.se/casper/) resulting in very good to excellent agreement between experimental and predicted data thereby demonstrating its utility for carbohydrate compounds that have been chemically or enzymatically synthesized, structurally modified or isolated from nature.
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| 4. |
- Kwon, Jeahoo, et al.
(author)
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Glycan Stability and Flexibility : Thermodynamic and Kinetic Characterization of Nonconventional Hydrogen Bonding in Lewis Antigens
- 2023
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In: Journal of the American Chemical Society. - 0002-7863 .- 1520-5126. ; 145:18, s. 10022-10034
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Journal article (peer-reviewed)abstract
- We provide evidence for CH-based nonconventional hydrogen bonds (H-bonds) for 10 Lewis antigens and two of their rhamnose analogues. We also characterize the thermodynamics and kinetics of the H-bonds in these molecules and present a plausible explanation for the presence of nonconventional H-bonds in Lewis antigens. Using an alternative method to simultaneously fit a series of temperature-dependent fast exchange nuclear magnetic resonance (NMR) spectra, we determined that the H-bonded conformation is favored by ∼1 kcal/mol over the non-H-bonded conformation. Additionally, a comparison of temperature-dependent 13C linewidths in various Lewis antigens and the two rhamnose analogues reveals H-bonds between the carbonyl oxygen of the N-acetyl group of N-acetylglucosamine and the OH2 group of galactose/fucose. The data presented herein provide insight into the contribution of nonconventional H-bonding to molecular structure and could therefore be used for the rational design of therapeutics.
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| 5. |
- Nestor, Gustav, et al.
(author)
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A detailed picture of a protein-carbohydrate hydrogen-bonding network revealed by NMR and MD simulations
- 2021
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In: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 31:4, s. 508-518
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Journal article (peer-reviewed)abstract
- Cyanovirin-N (CV-N) is a cyanobacterial lectin with antiviral activity towards HIV and several other viruses. Here, we identify mannoside hydroxyl protons that are hydrogen bonded to the protein backbone of the CV-N domain B binding site, using NMR spectroscopy. For the two carbohydrate ligands Manα(1→2)ManαOMe and Manα(1→2) Manα(1→6)ManαOMe five hydroxyl protons are involved in hydrogen-bonding networks. Comparison with previous crystallographic results revealed that four of these hydroxyl protons donate hydrogen bonds to protein backbone carbonyl oxygens in solution and in the crystal. Hydrogen bonds were not detected between the side chains of Glu41 and Arg76 with sugar hydroxyls, as previously proposed for CV-N binding of mannosides. Molecular dynamics simulations of the CV-N/Manα(1→2)Manα(1→6)ManαOMe complex confirmed the NMR-determined hydrogen-bonding network. Detailed characterization of CV-N/mannoside complexes provides a better understanding of lectin-carbohydrate interactions and opens up to the use of CV-N and similar lectins as antiviral agents.
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| 6. |
- Riu, Federico, et al.
(author)
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A Lead-Based Fragment Library Screening of the Glycosyltransferase WaaG from Escherichia coli
- 2022
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In: Pharmaceuticals. - : MDPI AG. - 1424-8247. ; 15:2
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Journal article (peer-reviewed)abstract
- Glucosyl transferase I (WaaG) in E. coli catalyzes the transfer of an α-d-glucosyl group to the inner core of the lipopolysaccharide (LPS) and plays an important role in the biogenesis of the outer membrane. If its activity could be inhibited, the integrity of the outer membrane would be compromised and the bacterium would be susceptible to antibiotics that are normally prevented from entering the cell. Herein, three libraries of molecules (A, B and C) were docked in the binding pocket of WaaG, utilizing the docking binding affinity as a filter to select fragment-based compounds for further investigations. From the results of the docking procedure, a selection of compounds was investigated by molecular dynamics (MD) simulations to obtain binding free energy (BFE) and KD values for ligands as an evaluation for the binding to WaaG. Derivatives of 1,3-thiazoles (A7 and A4) from library A and 1,3,4-thiadiazole (B33) from library B displayed a promising profile of BFE, with KD < mM, viz., 0.11, 0.62 and 0.04 mM, respectively. Further root-mean-square-deviation (RMSD), electrostatic/van der Waals contribution to the binding and H-bond interactions displayed a favorable profile for ligands A4 and B33. Mannose and/or heptose-containing disaccharides C1–C4, representing sub-structures of the inner core of the LPS, were also investigated by MD simulations, and compound C42− showed a calculated KD = 0.4 µM. In the presence of UDP-Glc2−, the best-docked pose of disaccharide C42− is proximate to the glucose-binding site of WaaG. A study of the variation in angle and distance was performed on the different portions of WaaG (N-, the C- domains and the hinge region). The Spearman correlation coefficient between the two variables was close to unity, where both variables increase in the same way, suggesting a conformational rearrangement of the protein during the MD simulation, revealing molecular motions of the enzyme that may be part of the catalytic cycle. Selected compounds were also analyzed by Saturation Transfer Difference (STD) NMR experiments. STD effects were notable for the 1,3-thiazole derivatives A4, A8 and A15 with the apo form of the protein as well as in the presence of UDP for A4.
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| 7. |
- Riu, Federico, et al.
(author)
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Antibiotics and Carbohydrate-Containing Drugs Targeting Bacterial Cell Envelopes : An Overview
- 2022
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In: Pharmaceuticals. - : MDPI AG. - 1424-8247. ; 15:8
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Research review (peer-reviewed)abstract
- Certain bacteria constitute a threat to humans due to their ability to escape host defenses as they easily develop drug resistance. Bacteria are classified into gram-positive and gram-negative according to the composition of the cell membrane structure. Gram-negative bacteria have an additional outer membrane (OM) that is not present in their gram-positive counterpart; the latter instead hold a thicker peptidoglycan (PG) layer. This review covers the main structural and functional properties of cell wall polysaccharides (CWPs) and PG. Drugs targeting CWPs are discussed, both noncarbohydrate-related (β-lactams, fosfomycin, and lipopeptides) and carbohydrate-related (glycopeptides and lipoglycopeptides). Bacterial resistance to these drugs continues to evolve, which calls for novel antibacterial approaches to be developed. The use of carbohydrate-based vaccines as a valid strategy to prevent bacterial infections is also addressed.
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| 8. |
- Ruda, Alessandro, 1990-, et al.
(author)
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Glycosidic α-linked mannopyranose disaccharides : an NMR spectroscopy and molecular dynamics simulation study employing additive and Drude polarizable force fields
- 2023
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In: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 25:4, s. 3042-3060
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Journal article (peer-reviewed)abstract
- D-Mannose is a structural component in N-linked glycoproteins from viruses and mammals as well as in polysaccharides from fungi and bacteria. Structural components often consist of D-Manp residues joined via α-(1→2)-, α-(1→3)-, α-(1→4)- or α-(1→6)-linkages. As models for these oligo- and polysaccharides, a series of mannose-containing disaccharides have been investigated with respect to conformation and dynamics. Translational diffusion NMR experiments were performed to deduce rotational correlation times for the molecules, 1D 1H,1H-NOESY and 1D 1H,1H-T-ROESY NMR experiments were carried out to obtain inter-residue proton–proton distances and one-dimensional long-range and 2D J-HMBC experiments were acquired to gain information about conformationally dependent heteronuclear coupling constants across glycosidic linkages. To attain further spectroscopic data, the doubly 13C-isotope labeled α-D-[1,2-13C2]Manp-(1→4)-α-D-Manp-OMe was synthesized thereby facilitating conformational analysis based on 13C,13C coupling constants as interpreted by Karplus-type relationships. Molecular dynamics simulations were carried out for the disaccharides with explicit water as solvent using the additive CHARMM36 and Drude polarizable force fields for carbohydrates, where the latter showed broader population distributions. Both simulations sampled conformational space in such a way that inter-glycosidic proton–proton distances were very well described whereas in some cases deviations were observed between calculated conformationally dependent NMR scalar coupling constants and those determined from experiment, with closely similar root-mean-square differences for the two force fields. However, analyses of dipole moments and radial distribution functions with water of the hydroxyl groups indicate differences in the underlying physical forces dictating the wider conformational sampling with the Drude polarizable versus additive C36 force field and indicate the improved utility of the Drude polarizable model in investigating complex carbohydrates.
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| 9. |
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| 10. |
- Ruda, Alessandro, 1990-, et al.
(author)
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O-Methylation in Carbohydrates : An NMR and MD Simulation Study with Application to Methylcellulose
- 2021
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 125:43, s. 11967-11979
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Journal article (peer-reviewed)abstract
- Methylated carbohydrates are important from both biological and technical perspectives. Specifically, methylcellulose is an interesting cellulose derivative that has applications in foods, materials, cosmetics, and many other fields. While the molecular dynamics simulation technique has the potential for both advancing the fundamental understanding of this polymer and aiding in the development of specific applications, a general drawback is the lack of experimentally validated interaction potentials for the methylated moieties. In the present study, simulations using the GROMOS 56 carbohydrate force field are compared to NMR spin–spin coupling constants related to the conformation of the exocyclic torsion angle ω in d-glucopyranose and derivatives containing a 6-O-methyl substituent and a 13C-isotopologue thereof. A 3JCC Karplus-type relationship is proposed for the C5–C6–O6–CMe torsion angle. Moreover, solvation free energies are compared to experimental data for small model compounds. Alkylation in the form of 6-O-methylation affects exocyclic torsion only marginally. Computed solvation free energies between nonmethylated and methylated molecules were internally consistent, which validates the application of these interaction potentials for more specialized purposes.
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| 11. |
- Ruda, Alessandro, 1990-
(author)
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Tales from the Sweet Side of Life : Structure and dynamics of carbohydrate-based systems
- 2022
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Doctoral thesis (other academic/artistic)abstract
- The field of glycomics has experienced significant developments in the last few decades, revealing a profound and elaborate language behind the intrinsic complexity of the glycans structures. This ‘sugar code’ is at the basis of intercellular interaction and recognition. For this language to be understood, its chemical basis must be unveiled. Two main techniques have played a significant role in this area: NMR spectroscopy and molecular dynamics simulations. In this thesis, via the combined approach of these two techniques, we get an insight of three different aspects of carbohydrate analysis, such as structure elucidation, conformation and dynamics, and interaction mechanisms with specialized proteins. The thesis is divided in three parts. The first part will uncover the biological role of streptococcal bacteria cell wall polysaccharides. Using a variety of different biological, biochemical, and spectroscopic tools, we describe their biosynthesis, structures – including newly discovered substituents – and biological significance. These rhamnose containing polysaccharides are the basis for the bacterial first-line protection against external offense and, at the same time, a Trojan horse for us to eradicate as yet persistent streptococcal pathogens-related diseases. The second part focuses on the conformational analysis of small mannose- and glucose-derivatives using NMR spectroscopy and molecular dynamics simulations. Particularly, the effect of methyl substitution in glucose oligosaccharides is investigated from a structural and thermodynamic point of view. Additionally, polarizable force fields were tested in the conformational analysis of different naturally occurring α-linked mannose disaccharide and compared to previously developed force fields. In the last part, using the same set of techniques, we elucidate the binding mode of small mannose oligosaccharides to the cyanobacterium protein Cyanovirin-N. This interaction is the basis of the virucidal activity of the protein against HIV infections.
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| 12. |
- Rush, Jeffrey S., et al.
(author)
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PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides
- 2021
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Other publication (other academic/artistic)abstract
- The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.
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| 13. |
- Rush, Jeffrey S., et al.
(author)
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PplD is a de-N-acetylase of the cell wall linkage unit of streptococcal rhamnopolysaccharides
- 2022
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
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Journal article (peer-reviewed)abstract
- The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.
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| 14. |
- Söderström, Bill, et al.
(author)
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An OregonGreen488-labelled d-amino acid for visualizing peptidoglycan by super-resolution STED nanoscopy
- 2020
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In: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 166:12, s. 1129-1135
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Journal article (peer-reviewed)abstract
- Fluorescent o amino acids (FDAAs) are molecular probes that are widely used for labelling the peptidoglycan layer of bacteria. When added to growing cells they are incorporated into the stem peptide by a transpeptidase reaction, allowing the timing and localization of peptidoglycan synthesis to be determined by fluorescence microscopy. Herein we describe the chemical synthesis of an Oregon Green 488 labelled FDAA (OGDA). We also demonstrate that OGDA can be efficiently incorporated into the PG of Grampositive and some Gramnegative bacteria, and imaged by superresolution stimulated emission depletion (STED) nanoscopy at a resolution well below 100 nm.
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