4th Year -- option.

Dr M.R. Wormald -- Structure and conformation of oligosaccharides

The lecture is based loosely on

Glycoproteins: presentation and -fold stability. M.R. Wormald and R.A. Dwek (1999) Structure, 7, R155-R160. and

Conformational studies of oligosaccharides and glycopeptides: Complementarity of NMR, X-ray crystallography, and molecular modelling. M.R. Wormald, A.J. Petrescu, Y.-L. Pao, A. Glithero, T. Elliott and R.A. Dwek (2002) Chemical Reviews, published on-line, due out March 2002. and the references therein. These can be obtained from computers within the Department at http://www.bioch.ox.ac.uk/glycob/publications.html, references 324 and 432.

The following are selected other references

þ -- recommended and easy (at least as easy as they come) Ä -- recommended but a bit more advanced

Oligosaccharide conformation/dynamics :-

· General --

þ Polysaccharide shapes Rees (1977)

þ Three dimensional structure of oligosaccharides explored by NMR and computer simulations Homans (1995) in “”, pp.67-86, ed. Montreuil, Vligenthart and Schachter.

· Computer modeling --

Computer modeling of : an introduction French and Brady (1990) Computer modeling of molecules, 1-19

Ä Computational : what theoretical methods can tell us Woods (1998) Journal, 15, 209-216

Oligosaccharide structures: Theory versus experiment Imberty (1997) Current Opinion In Structural Biology, 7, 617-623

· X-ray studies --

Ä A statistical analysis of N- and O-glycan linkage conformations from crystallographic data. Petrescu, et al. (1999) Glycobiology, 9, 343-352.

· NMR studies --

Tertiary structure of N-linked oligosaccharides. Homans, et al. (1987) Biochemistry, 26, 6553-6560.

Solution conformation of the branch points of N-linked : synthetic model compounds for tri-antennary and tetra-antennary glycans Cumming, et al. (1987) Biochemistry, 26, 6655-6663

Uncertainties in structural determination of oligosaccharide conformation using measurements of nuclear Overhauser effects. Wooten, et al. (1990) Carbohydrate Research, 203, 13-17

Ä Primary sequence dependence of conformation in oligomannose oligosaccharides. Wooten, et al. (1990) European Biophysics Journal, 18, 139-148

Ä The solution conformation of the Lex group Wormald, et al. (1991) Biochemical and Biophysical Research Communications, 180, 1214- 1221

The systematic use of negative nuclear Overhauser constraints in the determination of oligosaccharide conformations: application to sialyl-Lewis X. Wormald and Edge (1993) Carbohydrate Research, 246, 337-344

Ä The solution NMR structure of glucosylated N-glycans involved in the early stages of biosynthesis and folding Petrescu, et al. (1997) The EMBO Journal, 16, 4302-4310

The high degree of internal flexibility observed for an oligomannose oligosaccharide is not reproduced in the overall topology of the molecule. Woods, et al. (1998) Eur. J. Biochem., 258, 372-386.

· Others --

Differential flexibility in three branches of an N-linked triantennary glycopeptide Wu, et al. (1991) Proceedings of the National Academy of Sciences, USA, 88, 9355-9359

Glycopeptide/Glycoprotein Structure :-

þ The structural role of in glycoproteins Wyss and Wagner (1996) Current Opinion in Biotechnology, 7, 409-16

þ Modulation of protein structure and function by asparagine-linked O'Connor and Imperiali (1996) Chemistry and Biology, 3, 803-12

· Computer modelling --

þ Protein surface oligosaccharides and protein function Woods, et al. (1994) Nature Structural Biology, 1, 499-501

· X-ray studies --

Crystallographic refinement and atomic models of human Fc fragment and its complex with Fragment B of Protein A from Staphylococcus aureus at 2.9 and 2.8 A resolution Deisenhofer (1981) Biochemistry, 20, 2361-2370

Ä The three-dimensional structure of the carbohydrate within the Fc fragment of immunoglobulin G. Sutton and Phillips (1983) Biochemical Society Transactions, 11, 130-132

Human leukocyte and porcine pancreatic elastase: X-ray crystal structures, mechanism, substrate specificity and mechanism-based inhibitors. Bode, et al. (1989) Biochemistry, 28, 1951-1963

Structure of a legume lectin with an ordered N-linked carbohydrate in complex with lactose Shaanan, et al. (1991) Science, 254, 862-866

· NMR studies --

Ä Monitoring the carbohydrate component of the Fc fragment of human IgG by 13C nuclear magnetic resonance spectroscopy. Rosen, et al. (1979) Molecular Immunology, 16, 435-436

Ä Effects of glycosylation on the conformation and dynamics of O-linked glycoproteins: carbon-13 NMR studies of ovine submaxillary mucin Gerken, et al. (1989) Biochemistry, 28, 5536-5543

The conformational effects of N-glycosylation on the tailpiece from serum IgM. Wormald, et al. (1991) European Journal of Biochemistry, 198, 131-139

Effects of glycosylation on protein conformation and amide proton exchange rates in RNase B. Joao, et al. (1992) FEBS Letters, 307, 343-346

þ The conformational effects of N-linked glycosylation Edge, et al. (1993) Biochemical Society Transactions, 21, 452-455

Effects of glycosylation on protein structure and dynamics in ribonuclease B and some of its individual glycoforms Joao and Dwek (1993) European Journal of Biochemistry, 218, 239-244

1H NMR studies on an Asn-linked glycopeptide Davis, et al. (1994) Journal of Biological Chemistry, 269, 3331-3338

Ä Variations of oligosaccharide-protein interactions in immunoglobulin G determine the site- specific glycosylation profiles and modulate the dynamic motion of the Fc oligosaccharides Wormald, et al. (1997) Biochemistry, 36, 1370-1380

Conformation and function of the N-linked glycan in the adhesion domain of human CD2. Wyss, et al. (1995) Science, 269, 1273-1278.

· Other techniques –

Internal Movements in Immunoglobulin Molecules. Nezlin (1990) Advances in Immunology, 48, 1-40

Ä The dynamics of glycan-protein interactions in immunoglobulins. Results of spin label studies. Sykulev and Nezlin (1990) Glycoconjugate Journal, 7, 163-182

Conformations of oligosaccharides Formed by linking monosaccharides via a glycosidic linkage

OH O H O O HO + HO OH HO HO Structure and Conformation OH OH OH OH O HO O of Oligosaccharides O HO OH HO OH

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).

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Possible roles for protein glycosylation Conformations of monosaccharide rings Alter the biophysical properties of - Ring size – determined by ring strain ï Membrane attachment via a GPI anchor OH ï Changes in solubility HO 6 O OH 5 HO 5 1 6 presentation at membranes 4 O 4 2 1 OH HO 3 protein aggregation / association 3 OH HO 2 protease resistance û OH ü OH Alter protein structure / properties - ï Changes in tertiary/quaternary structure/flexibility Ring conformation – determined by steric interactions between substituents

ï Restrict or block access to active sites or possible antigenic regions OH OH OH O OH Provide specific recognition targets - HO O ï Interactions with calnexin/calreticulin during protein folding HO OH ï Interactions with lectins OH OH ï Epitopes recognised by the innate/acquired immune system ü OH û

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Mouse MHC Class I Conformations of saccharide linkages OH Glycopeptide – presented to T-cell O f HO y O w HO O OH HO HO OH

f – largely determined by the O O Asn 86 – glycan is exo-anomeric effect involved in peptide Asn 176 O O loading in the ER R R y – largely determined by non- ü û bonded interactions

OH H H H H H 4 O 4 O O HO HO HO 4 w – gauche preference 1 1 1 5 5 determined by solvation 5 Asn 256 H H H OH HO H H H H Oxford Glycobiology Institute Oxford Glycobiology Institute ü ü û

1 Conformations of saccharide linkages Conformations of saccharide linkages - Exo- anomeric effect - information available O O X-ray crystallography - H O Most oligosaccharides and glycoproteins either do not crystallise or give no Me resolvable electron density for the glycan. Glycans that can be seen are O H Me incomplete. 50 ï Can give average properties of linkages. 40 Nuclear Magnetic Resonance Spectroscopy -

30 Experimental structural parameters (inter-nuclear distances and torsion angles) averaged on a msec timescale. 20 ï Can be interpreted in terms of a structure if it is assumed that there is a single well-defined conformation. 10

Relative energy (kJ/mol) Molecular Dynamics Simulations - 0 Theoretical dynamic structures on a nsec timescale. -180 -120 -60 0 60 120 180 -180 -120 -60 0 60 120 180 ï Can be interpreted in terms of a structure if it is assumed that the theory is H1-C1-O-C H1-C1-O-C correct.

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Conformations of saccharide linkages Man a1-2 Man linkage - Hydrogen bonding and solvent effects Mana1-2Man linkages occur in oligomannose type N-glycans, OH polysaccharides such as mannan and GPI anchors. HO O

O OH OH O O OH O H HO O O O OH HO O HO H H O f O O OH O H O y H H OH HO H O H H O OH

Oxford Glycobiology Institute Hydrogen bond Oxford Glycobiology Institute

Conformations of saccharide linkages Crystallographic glycosidic linkage structures - - information required to define linkage structure Man a1- 2 Man linkage

180 25 H1-C1-O-C2’ For each distinct conformer - 120 y 20 O f • Average linkage torsion angles O 60

O H2'

• Fluctuations around the - 15

average position C2' 0 -

O C1-O-C2’-H2’

- 10 O f y -60 Population

For the ensemble of conformers - C1 O w • Relative populations of the 5 O -120 different conformers • Rate of transition between the -180 0 conformers -180 -120 -60 0 60 120 180 -180 -120 -60 0 60 120 180 H1-C1-O-C2' Torsion Angle Oxford Glycobiology Institute Oxford Glycobiology Institute

2 Conformational constraints from NOEs Conformation of the Man a1-2 Man linkage H1 - H2’ distance f = -60, y = -60 f = -60, y = +60 OH O 140 OH HO H OH 90 HO O HO O r OH HO OH H2’ 40 O O H - OH O

C2’ -10 HO O - OH

O OH - -60 OH OH C1 O -110 O O H OH H NOE intensity µ 1/r6 -160 -110 -60 -10 40 90 140 H1-C1-O-C2’ Hydrogen bond Oxford Glycobiology Institute 2.75 - 3.0 Å Oxford Glycobiology Institute Hydrophobic interactions

Man GlcNAc NMR torsion angle map 9 2 Schematic structure of Man GlcNAc D1-C Mana1®2Mana linkage 9 2 180 1,6 arms 120 Man a1®2 Man a1 æ D3 B 6 60 Man a1 H2' 3 æ - ä C1H - C1H’ = 2.80 - 3.15 Å Man a1®2 Man a1 4’ 6 Man b1®4 GlcNAc b1®4 GlcNAc C2' 0 D2 A - C1H - C2H’ = 2.05 - 2.30 Å 3 3 2 1 O

- ä C1H - C3H’ = 3.1 - 3.7 Å Man a1®2 Man a1®2 Man a1 -60 C1 C5H - C1H’ = 2.4 - 2.9 Å D1 C 4

-120 C1H - C4H’ > 3.5 Å 1,3 arm

-180 -180 -120 -60 0 60 120 180 H1-C1-O-C2' Oxford Glycobiology Institute Oxford Glycobiology Institute

Man9GlcNAc2 Molecular Dynamics Molecular Dynamics of Man9GlcNAc2 D1-C Mana1®2Mana linkage 180 210 90 120 f 180 180 0 30 210 -90 -60 y D3-B B-4' 120 120 120 -180 -150 30 180 180 w 60 60 -60 H2' - 90 90 4'-3 -150 180 180

C2' 0 0 0 - 0 90 90

O -90 -90 - D2-A A-4' 0 0 -180 -180 -60 C1 -60 -90 -90 Torsion Angle

Torsion angle 3-2 2-1 180 180 180 -180 -180 -120 H1-C1-O-C2’ -120 0 450 900 0 450 900 90 90 90 C1-O-C2’-H2’ 0 0 0 -180 -180 -90 -90 -90 0 250 500 750 1000 -180 -120 -60 0 60 120 180 D1-C C-4 4-3 -180 -180 -180 Simulation Time (ps) H1-C1-O-C2' 0 450 900 0 450 900 0 450 900 Oxford Glycobiology Institute Oxford Glycobiology Institute Simulation time (ps)

3 Molecular Dynamics of Man9GlcNAc2 Factors determining bio-polymer structure Overlay of structures (all atoms) from 1000 ps Proteins - Secondary structure – molecular orbital effects and sequential hydrogen bonds. Tertiary structure – non-sequential attractive forces and solvent interactions. DNA – Dimerisation – cross-strand hydrogen bonds. Tertiary structure – sequential attractive forces and solvent interactions.

Oligosaccharides - Linkage conformation – molecular orbital effects, steric repulsion and solvent interactions. Tertiary structure – non-sequential repulsive forces and solvent interactions. Side view Top view Oxford Glycobiology Institute Oxford Glycobiology Institute

Conformational properties of the Molecular Dynamics of Man9GlcNAc2 - water mediated hydrogen bonds between the arms Glycan-Asn linkage 28% 46%

21% NMR studies on model peptides - 3-arm 6(6)-arm • H-C-N-H unit is planar and trans in OH solution 64% H O O 6(3)-arm N HO C C X-ray crystallography - NH H O • Ca-Cb and Cb-Cg bonds show the Ac Numbers give the same range of conformations as percentage occupancies for unsubstituted Asn residues at the sites Core Oxford Glycobiology Institute Oxford Glycobiology Institute

Crystal and NMR structures of K2G Structure of Man9GlcNAc2 The presence of the GlcNAc causes Individual linkages are flexible on a sub-nsec timescale, many preferential clustering of showing more than one conformer. X-tal structure of aromatic side chains, K2G in complex leading to reduced The properties of a linkage depend on the its position in the with MHC Class I configurational entropy molecule. of the peptide backbone. The overall topology of the molecule is more conserved than Solution In this case, the the structures of the individual linkages. structure of K2G clustering places the ï correlated motions of the linkages (due to constraints of from NMR and modelling Phe, Tyr and GlcNAc on the solvent shell ??) the same side of the peptide as seen in the complex with MHC. Oxford Glycobiology Institute Oxford Glycobiology Institute

4 The presentation of the glycan is determined by interactions with the peptide surface

Domain 1 of CD2 Site 78 of hCG CH2 domain of IgG Fc Oxford Glycobiology Institute - area of the surface with which the glycan interacts

Ribonucleases B glycan dynamics

Two sterically allowed Allowing for N-linkage Asn conformations flexibility

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Ribonucleases A and B H/D-exchange

Ribonuclease A – aglycosyl Ribonuclease B – single glycosylation site

Glycosylation effects the backbone flexibility in all regions of the protein

H/D exchange reduced in ribonuclease B compared to A

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