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Use of (accurate+quantitative) NMR (and molecular modelling and crystallography)

Use of (accurate+quantitative) NMR (and molecular modelling and crystallography) in glycobiology. Oligosaccharides. Formed by linking monosaccharides via a glycosidic linkage. Each monosaccharide can be a or b at the anomeric carbon (position 1)

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Use of (accurate+quantitative) NMR (and molecular modelling and crystallography)

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  1. Use of (accurate+quantitative) NMR (and molecular modelling and crystallography) in glycobiology

  2. Oligosaccharides Formed by linking monosaccharides via a glycosidic linkage • Each monosaccharide can be a or b at the anomeric carbon (position 1) • Linkages can be made to the 2, 3, 4 or 6 positions (if available) • Several residues can be linked to a single monosaccharide (branching) Much higher degree of complexity than other biopolymers

  3. Features of protein glycosylation • There are several linkage types to proteins, including - • N-linked via Asn (requires an Asn-X-Ser/Thr sequon) • O-linked via Ser or Thr • GPI anchor via peptide C-terminus • Glycosylation is a co-/post-translational modification - • Depends on cell machinery as well as protein sequence • Species, tissue and disease-state specific • A well-defined, reproducible set of oligosaccharides is found at any given site on the protein - • A glycoprotein consists of an ensemble of glycoforms

  4. Thy 1

  5. Oligosaccharide 1D NMR spectrum NeuAc1Gal2Man3GlcNAc4Fuc1

  6. Conformational analysis

  7. Conformations of oligosaccharides Formed by linking monosaccharides via a glycosidic linkage Monosaccharide rings are rigid and have a well-defined conformation independent of environment The conformational analysis of an oligosaccharide reduces to determining the torsion angles about each glycosidic linkage (2 or 3 torsion angles per residue).

  8.  O O O   O  O O Conformations of saccharide linkages - information required to define linkage structure • For each distinct conformer • Average linkage torsion • angles • Fluctuations around the • average position • For the ensemble of conformers • Relative populations of the • different conformers • Rate of transition between • the conformers

  9. Conformations of saccharide linkages - information available • X-ray crystallography - • Most oligosaccharides and glycoproteins either do not crystallise or give no resolvable electron density for the glycan. Glycans that can be seen are incomplete. • Can give average properties of linkages. • Nuclear Magnetic Resonance Spectroscopy - • Experimental structural parameters (inter-nuclear distances and torsion angles) averaged on a msec timescale. • Can be interpreted in terms of a structure if it is assumed that there is a single well-defined conformation. • Molecular Dynamics Simulations - • Theoretical dynamic structures on a nsec timescale. • Can be interpreted in terms of a structure if it is assumed that the theory is correct.

  10. Applications of crystallographic databases of glycosidic linkages • Estimate range of possible allowed conformations • Check experimental glycan structures • correct “incorrect” structures • identify “distorted” structures • Build “average” glycan structures for X-ray refinement • Provide test data for forcefield validation • Model glycoprotein structures and glycan:protein interactions Structural Assessment of Glycosylation Sites (SAGS) database https://sags.biochim.ro/

  11. Crystal structures containing glycosidic linkages - 2002 Crystal structures containing glycosidic linkages - 2009

  12. Crystallographic refinement problems 1dpj 1wbl PDB structure: 6 Bond angle 80 3 sp2 (planar) carbon Biosynthetic structure: 6 3 incorrect monomers or linkages ring or linkage distortion Useful tool: http://www.dkfz-heidelberg.de/spec/glycosciences.de/tools/pdbcare/ Dr M. Crispin – personal communication

  13. Crystallographic glycosidic linkage conformers - Man 1-4 GlcNAc linkage 250 180 C1-O-C4’-C3’ 200 120 150 100 60 50 Frequency C1-O-C4’-C3’ 0 0 250 O5-C1-O-C4’ 200 -60 150 100 -120 50 0 -180 -180 -120 -60 0 60 120 180 -180 -120 -60 0 60 120 180 O5-C1-O-C4’ Torsion angle Major cluster – 56% of structures

  14. Crystallographic glycosidic linkage conformers - 2009

  15. Crystallographic glycosidic linkage conformers - chitobiose From 1999 to 2009: Number of structures increased by 1,800% Percentage of structures within the major cluster decreased from 90% to 70%

  16. Conformational constraints from NOEs • Presence of an NOE - • gives a value for the average 1/r6 inter-proton distance (on a msec timescale). • Absence of an NOE - • gives a minimum value (of3.5 Å) of the inter-proton distance for all significantly populated conformations.

  17. H1 - H2’ distance O 140 H 90 r 40 O H C1-O-C2’-H2’ -10 -60 O -110 NOE intensity  1/r6 -160 -110 -60 -10 40 90 140 H1-C1-O-C2’ 2.75 - 3.0 Å Conformational constraints from NOEs

  18. 1 ROE 0.675 0.5 0.385 0 NOE -1 -1 0.1 1 10 Nuclear Magnetic Resonance Spectroscopy - steady state NOE intensity and mobility Generally assume that c is constant for any given disaccharide. Then, can use an intra residue NOE to calibrate the cross-linkage NOEs. Intensity oc

  19. 5.6 5.4 4.0 3.8 3.6 Nuclear Magnetic Resonance Spectroscopy - effects of local correlation times ROESY NOESY Glc1-2Glc Chemical shift (ppm)

  20. Bo Nuclear Magnetic Resonance Spectroscopy - differing local correlation times The local correlation time for a given proton pair depends on the reorientation of the inter-nuclear vector with respect to the applied magnetic field. Anisotropic tumbling - Inter-nuclear vectors will reorient at different rates depending on their angle with respect to the principle axis. Internal flexibility - Inter-nuclear vectors will reorient at different rates depending on the degree of local flexibility. H H H H H H H H

  21. Nuclear Magnetic Resonance Spectroscopy - NOE intensity, distance and motion • Using the two-spin approximation: • rij is the distance between the two nuclei • B is a constant •  is the precession frequency • J is the spectral density function given by: • ij is the local correlation time for the pair of nuclei Poveda, et al (1997) Carbohydr. Res., 300, 3-10

  22. Nuclear Magnetic Resonance Spectroscopy - measuring local correlation times o = 500 MHz 3 (ROE) (T-ROE) 2 Ratio of build-up rates 1 (NOE) (ROE) 0 (NOE) (T-ROE) -1 0 0.2 0.4 0.6 c (ns) Poveda, et al (1997) Carbohydr. Res., 300, 3-10

  23. Effective local correlation times measured by (NOE)/(T-ROE) ratio Distances calculated from NOESY data assuming a rigid molecule Distances calculated from NOESY data allowing for local correlation times 200 ps 2.13 Å 2.40 Å 2.30 Å 2.30 Å 280 ps 289 ps 2.30 Å 2.24 Å Local correlation times - Glc3ManOMe Glc1-2Glc o = 500 MHz

  24. H2 H1 H3 H1-H2 NOE NOE intensity H1-H3 NOE 0 0 Mixing time Nuclear Magnetic Resonance Spectroscopy - NOE intensity and mixing time • Short mixing time - • Observed NOE intensity is • linear with mixing time •  1/r6 • Long mixing time - • Observed NOE intensity • non-linear with mixing time • includes spin-diffusion • depends on 3-D structure • and correlation time (dynamics)

  25. Bo 1H  13C Partial alignment in a liquid crystal - Residual Dipolar Coupling 1JCH(aligned) = 1JCH(unaligned) + RDC

  26. Bo Partial alignment of oligosaccharides in a liquid crystal H Sij is assumed to be constant for a given monosaccharide residue. H O H H H O The General Degree of Order (GDO) parameter is a measure of molecular motion of a given residue. H H O Tian, et al (2001) J. Am. Chem. Soc., 123, 485-492

  27. GLYCAM force-field parameters Exo-anomeric effect Partial charges AMBER Woods, et al (1995) J. Phys. Chem., 99, 3832-3846

  28. Exo-anomeric effect - Hartree-Fock calculations

  29. Molecular Dynamics of Oligosaccharides • Amber Force Field, parameterised to fit ab initio calculations - • Exo-anomeric effect (torsion angle terms) • Solvation (partial charges) • Obtain starting linkage geometries from calculations on disaccharides • Run unconstrained molecular dynamics simulations in water • Compare results to experimental data - • average data from X-ray crystallography • torsion angle constraints from NMR (NOEs and 3JHH) • back calculate NOE build-up curves

  30. Solution conformation of oligomannose N-linked oligosaccharides Dr Rob Woods Dr Chris Edge Dr Andrei Petrescu, Institute of Biochemistry, Bucharest

  31. Oligomannose glycans Dol Complex glycans Hybrid glycans GlcNAc Man Glc Gal Fuc NeuAc N-glycan biosynthesis ER Golgi

  32. Man Man  Man Man    D3 B 6 3 Man   6 3  4’ Man GlcNAc GlcNAc D2 A 3 2 1 Man Man Man  D1 C 4 Man a1-2 Man linkage Schematic structure of Man9GlcNAc2 1,6 arms 1,3 arm Mana1-2Man linkages occur in oligomannose type N-glycans, polysaccharides such as mannan and GPI anchors.

  33. Crystallographic glycosidic linkage conformers - 2009

  34. Crystallographic glycosidic linkage structures - Man a1-2 Man linkage (2009) 180 70 185 structures H1-C1-O-C2’ C1-O-C2’-H2’ 60 120 50 60 40 0 Population C1-O-C2'-H2' 30 -60 20 -120 10 -180 0 -180 -120 -60 0 60 120 180 -180 -120 -60 0 60 120 180 H1-C1-O-C2' Torsion Angle

  35. 5.4 4.8 4.4 4.0 3.6 Chemical Shift (ppm) Man9GlcNAc2 NOESY traces 4’:C1H 4’:C2H 3:C6H 3:C6H 4’:C3H HDO 3:C5H A:C1H 4’:C3H A:C5H + D2:C5H A:C2H D2:C1H 4’:C2H A:C3H D2:C1H A:C2H D2:C2H A:C1H A:C3H

  36. Man9GlcNAc2 NMR torsion angle map D1-C Man2Man linkage C1H - C1H’ = 2.80 - 3.15 Å C1H - C2H’ = 2.05 - 2.30 Å C1H - C3H’ = 3.1 - 3.7 Å C5H - C1H’ = 2.4 - 2.9 Å C1H - C4H’ > 3.5 Å

  37. Man9GlcNAc2 Molecular Dynamics D1-C Man2Man linkage H1-C1-O-C2’ C1-O-C2’-H2’

  38. f = -60, y = -60 f = -60, y = +60 OH OH HO OH HO O O HO OH OH HO O OH O OH HO O OH OH OH O O H OH H Hydrogen bond Hydrophobic interactions Conformation of the Man a1-2 Man linkage

  39. Man9GlcNAc2 NMR torsion angle map A-4’ ManMan linkage 140 90 • C1H - C2H’ = 2.85 - 3.20 Å • C1H - C3H’ = 2.00 - 2.25 Å • C1H - C4H’ = 2.75 - 3.10 Å • C5H - C2H’ = 2.55 - 2.85 Å • Individual MD • conformations 40 C1-O-C3’-H3’ -10 -60 -110 -110 -60 -10 40 90 140 H1-C1-O-C3’

  40. Man9GlcNAc2 NMR build up curves A-4’ ManMan linkage 10 8 6 NOE Intensity (%) C1H - C3H’ C5H - C2H’ C1H - C4H’ C1H - C2H’ 4 2 0 0 500 1000 1500 2000 Mixing time (ms)

  41. Schematic structure of Man9GlcNAc2 1,6 arms Man Man  Man Man    D3 B 6 3 Man   6 3  4’ Man GlcNAc GlcNAc D2 A 3 2 1 Man Man Man  D1 C 4 1,3 arm

  42. Molecular Dynamics of Man9GlcNAc2 Torsion angle Simulation time (ps)

  43. Molecular Dynamics of Man9GlcNAc2 Overlay of structures (all atoms) from 1000 ps Side view Top view

  44. Flexibility of the tri-glucosylated cap of immature N-linked glycans Mukram Mackeen Drs Andy Almond and Michael Deschamps, Dept. of Biochem., Oxford Dr Terry Butters Dr Antony Fairbanks, Dept. of Chem., Oxford

  45. Dol P P ER processing and recognition of N-linked glycans - protein folding and quality control Nascent peptide OST Glucosidase I Glucosidase II Glucosidase II Glucosyl transferase Recognised by Calnexin and Calreticulin  ER resident chaperones involved in protein folding Golgi ER ERAD

  46. Man Man  Man Man    D3 B 6 3 Man   6 3  4’ Man GlcNAc GlcNAc D2 A 3 2 1 Glc Glc Glc Man Man Man  G1 G2 G3 D1 C 4 Schematic structure of Glc3Man9GlcNAc2 1,6 arms 1,3 arm

  47. Multifunctional roles of GlcxMan9GlcNAc2 Glc3Man Glc 1 Glycan remodelling to produce mature glycans Chaperone assisted folding Calnexin and calreticulin binding OGT substrate Protein folding quality control ERAD recognition Protein N-glycosylation OST substrate binding GlcNAc 1

  48. Crystallographic glycosidic linkage conformers - 2009 Glc3Man linkages: Glca1-2Glc 0 Glca1-3Glc 1 Glca1-3Man 0

  49. -150 -150 -150 -50 -50 -50 +50 +50 +50 +150 +150 +150 Glc3Man NMR torsion angle maps G1-G2 G2-G3 G3-D1 +150 +50  - C1-O-C’x-H’x -50 -150  - H1-C1-O-C’x Solid lines : positive constraints Dotted lines : negative constraints White – H1-H’x-1Grey – H5-H’x-1 Red – H1-H’xPink – H5-H’x Green – H1-H’x+1Yellow – H5-H’x+1

  50. 4.9 D1:1 D1:1 A:1 A:1 5.1 G3:1 G3:1 C:1 C:1 4:1 4:1 5.3 2 – 1H (ppM) 4.9 5.1 5.3 116 115 114 113 112 1 – 13C (ppM) Residual dipolar coupling - Glc3Man7GlcNAc2 Unaligned Aligned

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