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Oxford Glycobiology Institute Use of (accurate+quantitative) NMR (and molecular modelling and crystallography) in glycobiology.

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Presentation on theme: "Oxford Glycobiology Institute Use of (accurate+quantitative) NMR (and molecular modelling and crystallography) in glycobiology."— Presentation transcript:

1 Oxford Glycobiology Institute Use of (accurate+quantitative) NMR (and molecular modelling and crystallography) in glycobiology

2 Oxford Glycobiology Institute Oligosaccharides Formed by linking monosaccharides via a glycosidic linkage  Each monosaccharide can be  or  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 Oxford Glycobiology Institute Features of protein glycosylation There are several linkage types to proteins, including - ð N-linked via Asn (requires an Asn-X-Ser/Thr sequon) ð O-linked via Ser or Thr ð GPI anchor via peptide C-terminus Glycosylation is a co-/post-translational modification - ð 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 - ð A glycoprotein consists of an ensemble of glycoforms

4 Oxford Glycobiology Institute Thy 1

5 Oxford Glycobiology Institute Oligosaccharide 1D NMR spectrum NeuAc 1 Gal 2 Man 3 GlcNAc 4 Fuc 1

6 Oxford Glycobiology Institute Conformational analysis

7 Oxford Glycobiology Institute 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 Oxford Glycobiology Institute O O O O O O      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 Conformations of saccharide linkages - information required to define linkage structure

9 Oxford Glycobiology Institute 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. ðCan give average properties of linkages. Nuclear Magnetic Resonance Spectroscopy - Experimental structural parameters (inter-nuclear distances and torsion angles) averaged on a msec timescale. ðCan 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. ðCan be interpreted in terms of a structure if it is assumed that the theory is correct.

10 Oxford Glycobiology Institute 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 Oxford Glycobiology Institute Crystal structures containing glycosidic linkages - 2002 Crystal structures containing glycosidic linkages - 2009 Type of structureNo. of crystal structures No. of glycans No. of linkages between undistorted glycans No. of incorrect/ distorted linkages Oligosaccharides910110 Glycoproteins with N-linked glycans 2285031091292 Glycoproteins with O-linked glycans 2220 Proteins with glycan ligands 29742001 Glycoproteins with N-linked glycans 110930186352

12 Oxford Glycobiology Institute Crystallographic refinement problems PDB structure: Biosynthetic structure: 1dpj Useful tool: http://www.dkfz-heidelberg.de/spec/glycosciences.de/tools/pdbcare/ ring or linkage distortion Dr M. Crispin – personal communication 3 6 3 6 1wbl Bond angle 80  sp 2 (planar) carbon incorrect monomers or linkages

13 Oxford Glycobiology Institute Crystallographic glycosidic linkage conformers - Man  1-4 GlcNAc linkage O5-C1-O-C4’ 100 -180-120-60060 0 50 150 200 120180 250 0 50 100 150 200 250 C1-O-C4’-C3’ Torsion angle Frequency Major cluster – 56% of structures

14 Oxford Glycobiology Institute Crystallographic glycosidic linkage conformers - 2009 Glycosidic linkageNo. ofDistinct conformersConformer structures  population Fuc  1-3GlcNAc 189 -72.0  6.9-97.7  6.1 --143 Fuc  1-6GlcNAc 281 -75.2  9.3187.3  12.055.7  7.9 49 Gal  1-4GlcNAc 44 -71.4  10.9132.2  7.4 --28 GlcNAc  1-4GlcNAc 2950 -78.7  11.6119.2  14.9 --2122 GlcNAc  1-2Man 122 -77.3  6.8-89.4  4.7 --44 Man  1-4GlcNAc 1202 -86.5  11.4109.4  16.8 --674 Man  1-2Man 185 63.6  4.4-178.6  3.5 --37 69.5  8.8-107.9  14.9 --78 Man  1-3Man 584 73.7  7.1-117.3  18.0 --288 Man  1-6Man 553 62.2  5.186.0  2.6190.1  9.5 17 65.2  10.0174.5  21.6183.3  26.5 72 64.1  10.1184.9  9.460.0  8.1 74

15 Oxford Glycobiology Institute Crystallographic glycosidic linkage conformers - chitobiose Glycosidic linkageNo. ofDistinct conformersConformer structures  population 1999 GlcNAc  1-4GlcNAc 163 -74.4  7.8115.9  15.9 --146 2002 GlcNAc  1-4GlcNAc 398 -75.9  11.6119.0  15.4 --376 2009 GlcNAc  1-4GlcNAc 2950 -78.7  11.6119.2  14.9 --2122 From 1999 to 2009:Number of structures increased by 1,800% Percentage of structures within the major cluster decreased from 90% to 70%

16 Oxford Glycobiology Institute Presence of an NOE - gives a value for the average 1/r 6 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. Conformational constraints from NOEs

17 Oxford Glycobiology Institute -110-60-104090140 -160 -110 -60 -10 40 90 140 H1 - H2’ distance H1-C1-O-C2’ C1-O-C2’-H2’ 2.75 - 3.0 Å Conformational constraints from NOEs NOE intensity  1/r 6 r O O H H O

18 Oxford Glycobiology Institute Nuclear Magnetic Resonance Spectroscopy - steady state NOE intensity and mobility 0 1 0.1110 0.5 NOE 0.675 0.385 ROE Intensity ococ Generally assume that  c is constant for any given disaccharide. Then, can use an intra residue NOE to calibrate the cross- linkage NOEs.

19 Oxford Glycobiology Institute Nuclear Magnetic Resonance Spectroscopy - effects of local correlation times Glc  1-2Glc  5.44.03.83.65.6 Chemical shift (ppm) NOESY ROESY

20 Oxford Glycobiology Institute Nuclear Magnetic Resonance Spectroscopy - differing local correlation times 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. 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. H H H H H H H H BoBo

21 Oxford Glycobiology Institute Nuclear Magnetic Resonance Spectroscopy - NOE intensity, distance and motion Using the two-spin approximation: r ij 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 Oxford Glycobiology Institute Nuclear Magnetic Resonance Spectroscopy - measuring local correlation times Ratio of build-up rates  c (ns)  o = 500 MHz 00.20.40.6 0 1 2 3  (ROE)  (T-ROE)  (NOE)  (T-ROE)  (NOE)  (ROE) Poveda, et al (1997) Carbohydr. Res., 300, 3-10

23 Oxford Glycobiology Institute 2.40 Å 2.24 Å 2.30 Å Distances calculated from NOESY data allowing for local correlation times 200 ps 289 ps 280 ps Effective local correlation times measured by  (NOE)/  (T-ROE) ratio 2.13 Å 2.30 Å Distances calculated from NOESY data assuming a rigid molecule Local correlation times - Glc 3 ManOMe Glc  1-2Glc   o = 500 MHz

24 Oxford Glycobiology Institute Short mixing time - Observed NOE intensity is linear with mixing time  1/r 6 Long mixing time - Observed NOE intensity non-linear with mixing time includes spin-diffusion depends on 3-D structure and correlation time (dynamics) Mixing time NOE intensity 0 0 H1 H2 H3 H1-H2 NOE H1-H3 NOE Nuclear Magnetic Resonance Spectroscopy - NOE intensity and mixing time

25 Oxford Glycobiology Institute 13 C 1H1H BoBo  Partial alignment in a liquid crystal - Residual Dipolar Coupling 1 J CH (aligned) = 1 J CH (unaligned) + RDC

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

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

28 Oxford Glycobiology Institute Exo-anomeric effect - Hartree-Fock calculations

29 Oxford Glycobiology Institute Molecular Dynamics of Oligosaccharides Amber Force Field, parameterised to fit ab initio calculations - ðExo-anomeric effect (torsion angle terms) ðSolvation (partial charges) Obtain starting linkage geometries from calculations on disaccharides Run unconstrained molecular dynamics simulations in water Compare results to experimental data - ðaverage data from X-ray crystallography ðtorsion angle constraints from NMR (NOEs and 3 J HH ) ðback calculate NOE build-up curves

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

31 Oxford Glycobiology Institute Dol ER Golgi N-glycan biosynthesis NeuAcGlcNAcManGlcGalFuc Complex glycans Hybrid glycans Oligomannose glycans

32 Oxford Glycobiology Institute 1,6 arms 1,3 arm Man  Man  Man  Man  Man  GlcNAc  GlcNAc Man  Man  Man  Man   6363 6363 D1C 4 321 4’ A B D2 D3 Man  1-2 Man linkage Schematic structure of Man 9 GlcNAc 2 Man  1-2Man linkages occur in oligomannose type N-glycans, polysaccharides such as mannan and GPI anchors.

33 Oxford Glycobiology Institute Crystallographic glycosidic linkage conformers - 2009 Glycosidic linkageNo. ofDistinct conformersConformer structures  population Fuc  1-3GlcNAc 189 -72.0  6.9-97.7  6.1 --143 Fuc  1-6GlcNAc 281 -75.2  9.3187.3  12.055.7  7.9 49 Gal  1-4GlcNAc 44 -71.4  10.9132.2  7.4 --28 GlcNAc  1-4GlcNAc 2950 -78.7  11.6119.2  14.9 --2122 GlcNAc  1-2Man 122 -77.3  6.8-89.4  4.7 --44 Man  1-4GlcNAc 1202 -86.5  11.4109.4  16.8 --674 Man  1-2Man 185 63.6  4.4-178.6  3.5 --37 69.5  8.8-107.9  14.9 --78 Man  1-3Man 584 73.7  7.1-117.3  18.0 --288 Man  1-6Man 553 62.2  5.186.0  2.6190.1  9.5 17 65.2  10.0174.5  21.6183.3  26.5 72 64.1  10.1184.9  9.460.0  8.1 74

34 Oxford Glycobiology Institute Torsion Angle -180-120-60060120180 0 10 20 30 40 70 H1-C1-O-C2’ C1-O-C2’-H2’ 50 60 Population Crystallographic glycosidic linkage structures - Man  1-2 Man linkage (2009) H1-C1-O-C2' -180-120-60060120180 C1-O-C2'-H2' -180 -120 -60 0 60 120 180 185 structures

35 Oxford Glycobiology Institute Man 9 GlcNAc 2 NOESY traces Chemical Shift (ppm) 5.44.84.44.03.6 HDO 4’:C1H A:C1H D2:C1H A:C2H A:C1H D2:C2H A:C3H 4’:C2H 3:C6H 4’:C3H 3:C6H 3:C5H 4’:C2H A:C2H A:C3H 4’:C3H A:C5H + D2:C5H

36 Oxford Glycobiology Institute 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 Man 9 GlcNAc 2 NMR torsion angle map D1-C Man  Man  linkage

37 Oxford Glycobiology Institute Man 9 GlcNAc 2 Molecular Dynamics D1-C Man  Man  linkage H1-C1-O-C2’ C1-O-C2’-H2’

38 Oxford Glycobiology Institute Hydrogen bond Hydrophobic interactions O H H Conformation of the Man  1-2 Man linkage OH HO O O O OH HO O O O 

39 Oxford Glycobiology Institute -110-109014040-60 H1-C1-O-C3’ 90 -10 -110 40 140 C1-O-C3’-H3’ -60 Man 9 GlcNAc 2 NMR torsion angle map A-4’ Man  Man  linkage 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 Oxford Glycobiology Institute Man 9 GlcNAc 2 NMR build up curves A-4’ Man  Man  linkage C1H - C3H’ C5H - C2H’ C1H - C4H’ C1H - C2H’ 05001000150 0 2000 Mixing time (ms) 0 2 4 6 8 10 NOE Intensity (%)

41 Oxford Glycobiology Institute 1,6 arms 1,3 arm Man  Man  Man  Man  Man  GlcNAc  GlcNAc Man  Man  Man  Man   6363 6363 D1C4 321 4’ A B D2 D3 Schematic structure of Man 9 GlcNAc 2

42 Oxford Glycobiology Institute Simulation time (ps) Torsion angle Molecular Dynamics of Man 9 GlcNAc 2

43 Oxford Glycobiology Institute Molecular Dynamics of Man 9 GlcNAc 2 Overlay of structures (all atoms) from 1000 ps Side viewTop view

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

46 Oxford Glycobiology Institute Schematic structure of Glc 3 Man 9 GlcNAc 2 1,6 arms 1,3 arm Glc  Glc  Glc  Man  Man  Man  Man  Man  GlcNAc  GlcNAc Man  Man  Man  Man   6363 6363 G1G2G3D1C4 321 4’ A B D2 D3

47 Oxford Glycobiology Institute Multifunctional roles of Glc x Man 9 GlcNAc 2 Chaperone assisted folding Calnexin and calreticulin binding OGT substrate Protein N-glycosylation OST substrate binding Protein folding quality control ERAD recognition Glycan remodelling to produce mature glycans Glc 1 GlcNAc 1 Glc 3 Man

48 Oxford Glycobiology Institute Crystallographic glycosidic linkage conformers - 2009 Glycosidic linkageNo. ofDistinct conformersConformer structures  population Fuc  1-3GlcNAc 189 -72.0  6.9-97.7  6.1 --143 Fuc  1-6GlcNAc 281 -75.2  9.3187.3  12.055.7  7.9 49 Gal  1-4GlcNAc 44 -71.4  10.9132.2  7.4 --28 GlcNAc  1-4GlcNAc 2950 -78.7  11.6119.2  14.9 --2122 GlcNAc  1-2Man 122 -77.3  6.8-89.4  4.7 --44 Man  1-4GlcNAc 1202 -86.5  11.4109.4  16.8 --674 Man  1-2Man 185 63.6  4.4-178.6  3.5 --37 69.5  8.8-107.9  14.9 --78 Man  1-3Man 584 73.7  7.1-117.3  18.0 --288 Man  1-6Man 553 62.2  5.186.0  2.6190.1  9.5 17 65.2  10.0174.5  21.6183.3  26.5 72 64.1  10.1184.9  9.460.0  8.1 74 Glc 3 Man linkages: Glc  1-2Glc0 Glc  1-3Glc1 Glc  1-3Man0

49 Oxford Glycobiology Institute Solid lines : positive constraints Dotted lines : negative constraints White – H 1 -H’ x-1 Grey – H 5 -H’ x-1 Red – H 1 -H’ x Pink – H 5 -H’ x Green – H 1 -H’ x+1 Yellow – H 5 -H’ x+1  - C1-O-C’x-H’x Glc 3 Man NMR torsion angle maps G1-G2G2-G3G3-D1  - H1-C1-O-C’x -150-50+50+150-150-50+50+150-150-50+50+150 +50 -50 -150

50 Oxford Glycobiology Institute 116115114113112  1 – 13 C (ppM)  2 – 1 H (ppM) 5.3 5.1 4.9 5.3 5.1 4.9 Unaligned Aligned D1:1 A:1 G3:1 4:1 C:1 D1:1 A:1 G3:1 4:1 C:1 Residual dipolar coupling - Glc 3 Man 7 GlcNAc 2

51 Oxford Glycobiology Institute GDO values for the tri-glucosyl cap Glc 3 ManOMeGlc 3 Man 7 GlcNAc 2 G10.0140.005 G20.0130.005 G30.0300.075 D10.0030.013

52 Oxford Glycobiology Institute Glc 3 ManOMe Molecular Dynamics G1-G2 G2-G3 G3-D1

53 Oxford Glycobiology Institute Proton pair  ij (ps)Distance (Å) X-rayNMRMD G1-G2 : Glc  1–2Glc  linkage G1:H1G1:H22802.34--2.42 G2:H12892.302.18 G2:H22002.452.76 G2:H3-->3.54.35 G2-G3 : Glc  1–3Glc  linkage G2:H1G2:H23382.34--2.41 G3:H2--4.23>3.54.41 G3:H33162.052.192.55 G3:H4--3.74>3.53.32 G3-D1 : Glc  1–3Man  linkage G3:H1G3:H23472.34--2.41 D1:H21723.083.48 D1:H32602.122.30 D1:H4nd~3.13.69 Glc 3 ManOMe – X-ray vs NMR vs MD

54 Oxford Glycobiology Institute D1 G3 G2 G1 Major – 70%Minor – 30% Two conformations of Glc 3 ManOMe

55 Oxford Glycobiology Institute Calnexin Residues involved in the interaction between calreticulin and ERp57 (Asp 344 and Glu 352) Residues involved in glycan binding Crystallographic dimer Schrag, et al (2001) Mol. Cell, 8, 633-644

56 Oxford Glycobiology Institute MinorMajor Calnexin Glc 1 Man 9 GlcNAc 2 binds to Calnexin in the minor conformer Major conformer makes very unfavourable steric interactions with the protein.


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