4th Year Biochemistry -- Glycobiology option.
Dr M.R. Wormald -Structure and conformation of oligosaccharides
The lecture is based loosely on
Glycoproteins: glycan presentation and protein-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)
⊗ -- 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 “Glycoproteins”, pp.67-86, ed. Montreuil, Vligenthart and Schachter.
•
Computer modeling -Computer modeling of carbohydrates: an introduction
French and Brady (1990) Computer modeling of carbohydrate molecules, 1-19
⊗ Computational carbohydrate chemistry: what theoretical methods can tell us
Woods (1998) Glycoconjugate Journal, 15, 209-216
Oligosaccharide structures: Theory versus experiment
Imberty (1997) Current Opinion In Structural Biology, 7, 617-623
•
X-ray studies --
⊗ 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 glycans: 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
⊗ Primary sequence dependence of conformation in oligomannose oligosaccharides.
Wooten, et al. (1990) European Biophysics Journal, 18, 139-148
⊗ The solution conformation of the Lex group
Wormald, et al. (1991) Biochemical and Biophysical Research Communications, 180, 12141221
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
⊗ The solution NMR structure of glucosylated N-glycans involved in the early stages of
glycoprotein 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 sugars in glycoproteins
Wyss and Wagner (1996) Current Opinion in Biotechnology, 7, 409-16
þ Modulation of protein structure and function by asparagine-linked glycosylation
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
⊗ 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 --
⊗ 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
⊗ 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
⊗ Variations of oligosaccharide-protein interactions in immunoglobulin G determine the sitespecific 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
⊗ 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
OH
O
HO
Structure and Conformation
of Oligosaccharides
HO
O
+
OH
HO
HO
OH
OH
OH
OH
O
HO
O
O
HO
HO
OH
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).
Oxford Glycobiology Institute
Oxford Glycobiology Institute
Possible roles for protein glycosylation
Alter the biophysical properties of proteins ï Membrane attachment via a GPI anchor
ï Changes in solubility
presentation at membranes
protein aggregation / association
protease resistance
Alter protein structure / properties ï Changes in tertiary/quaternary structure/flexibility
ï Restrict or block access to active sites or possible antigenic regions
Provide specific recognition targets -
Conformations of monosaccharide rings
Ring size – determined by ring strain
OH
O
4
Oxford Glycobiology Institute
OH
1
3
2
HO
OH
1
5
4
2
HO
û
3
OH
OH
ü
Ring conformation – determined by steric interactions between substituents
OH
OH
OH
OH
O
HO
ï Interactions with calnexin/calreticulin during protein folding
ï Interactions with lectins
ï Epitopes recognised by the innate/acquired immune system
HO
5
6
OH
O
6
HO
O
OH
HO
OH
ü
OH
û
OH
Oxford Glycobiology Institute
Mouse MHC Class I
Conformations of saccharide linkages
OH
Glycopeptide – presented to T-cell
O
HO
φ ψ
O
HO
OH
ω
O
HO
HO
OH
Asn 86 – glycan is
involved in peptide
loading in the ER
Asn 176
φ – largely determined by the
exo-anomeric effect
O
O
O
H
Asn 256
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ω – gauche preference
determined by solvation
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HO
O
R
ü
ψ – largely determined by nonbonded interactions
OH
H
O
4
1
5
H
H
H
ü
HO
H
1
5
H
OH
H
û
H
O
4
R
ü
HO
H
O
4
1
5
H
HO
H
û
1
Conformations of saccharide linkages
Conformations of saccharide linkages
- Exo
- anomeric effect
O
- information available
X-ray crystallography -
O
H
O
O
Me
H
Me
Relative energy (kJ/mol)
50
40
Nuclear Magnetic Resonance Spectroscopy Experimental structural parameters (inter-nuclear distances and torsion angles)
averaged on a msec timescale.
ï Can be interpreted in terms of a structure if it is assumed that there is a
single well-defined conformation.
30
20
10
0
-180
Most oligosaccharides and glycoproteins either do not crystallise or give no
resolvable electron density for the glycan. Glycans that can be seen are
incomplete.
ï Can give average properties of linkages.
Molecular Dynamics Simulations -120
-60
0
60
120
180 -180
-120
-60
H1-C1-O-C
0
60
120
180
H1-C1-O-C
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Theoretical dynamic structures on a nsec timescale.
ï Can be interpreted in terms of a structure if it is assumed that the theory is
correct.
Oxford Glycobiology Institute
Man α1-2 Man linkage
Conformations of saccharide linkages
- Hydrogen bonding and solvent effects
Manα1-2Man linkages occur in oligomannose type N-glycans,
polysaccharides such as mannan and GPI anchors.
OH
O
HO
O
O
O
H
O
HO
HO
H
H
HO
O
O
O
HO
O
O
H
H
OH
OH
O
OH
O
H
H
O
H
φ
OH
O
ψ
O
H
OH
OH
O
OH
Hydrogen bond
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Conformations of saccharide linkages
- information required to define linkage structure
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Crystallographic glycosidic linkage structuresMan α1
- 2Man linkage
25
180
O
O
O
φ
ψ
O
ω
O
For the ensemble of conformers • Relative populations of the
different conformers
• Rate of transition between the
conformers
H1-C1-O-C2’
20
60
Population
ψ
φ
120
C1-O-C2'-H2'
O
For each distinct conformer • Average linkage torsion angles
• Fluctuations around the
average position
0
-60
-60
0
60
H1-C1-O-C2'
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C1-O-C2’-H2’
10
5
-120
-180
-180 -120
15
120
180
0
-180 -120
-60
0
60
120
180
Torsion Angle
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2
Conformation of the Man α1-2 Man linkage
Conformational constraints from NOEs
H1 - H2’ distance
O
φ = −60, ψ = −60
140
H
90
C1-O-C2’-H2’
r
O
H
HO
-110
-160
O
O
OH
HO
OH
O
OH
-60
NOE intensity ∝ 1/r6
OH
OH
-10
O
OH
O
HO
HO
40
φ = −60, ψ = +60
HO
OH
OH
O
OH
OH
O
O
OH
-110 -60 -10 40
H
90 140
H1-C1-O-C2’
2.75 - 3.0 Å
Oxford Glycobiology Institute
H
Hydrogen bond
Hydrophobic interactions
Oxford Glycobiology Institute
Man9GlcNAc2 NMR torsion angle map
D1-C Manα1→2Manα linkage
Schematic structure of Man9GlcNAc2
180
1,6 arms
120
C1-O-C2'-H2'
Man α1→2 Man α1
D3
60
-60
D2
-180
-180
-120
-60
0
60
120
6
Man α1
3
æ
4’
6
Man β1→4 GlcNAc β1→4 GlcNAc
3
A
Man α1→2 Man α1→2 Man α1
D1
C
3
ä
2
1
4
1,3 arm
C1H - C4H’ > 3.5 Å
-120
æ
Man α1→2 Man α1 ä
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
0
B
180
H1-C1-O-C2'
Oxford Glycobiology Institute
Oxford Glycobiology Institute
Molecular Dynamics of Man9GlcNAc2
Man9GlcNAc2 Molecular Dynamics
D1-C Manα1→2Manα linkage
180
210
90
120
30
180
180
0
120
120
-180
60
0
-60
H1-C1-O-C2’
C1-O-C2’-H2’
-120
-180
0
250
500
750
Simulation Time (ps)
Oxford Glycobiology Institute
1000
Torsion angle
C1-O-C2'-H2'
Torsion Angle
-90
60
0
-60
-120
-180
-180 -120
-60
0
60
H1-C1-O-C2'
120
180
210
-60
D3-B
φ
ψ
ω
B-4'
-150
120
30
180
180
90
90
0
0
-90
-90
D2-A
-180
-60
A-4'
-180
180
180
90
90
90
0
0
0
-90
-90
D1-C
0
450
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180
0
450
0
-90
3-2
0
450
2-1
-180
900
0
450
900
4-3
-180
900
90
0
-90
-180
-90
C-4
-180
900
180
90
180
-180
4'-3
-150
0
450
900
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
Molecular Dynamics of Man9GlcNAc2
- water mediated hydrogen bonds between the arms
28%
46%
21%
3-arm
Conformational properties of the
Glycan-Asn linkage
6(6)-arm
64%
NMR studies on model peptides • H-C-N-H unit is planar and trans in
solution
X-ray crystallography • Cα-Cβ and Cβ-Cγ bonds show the
same range of conformations as
for unsubstituted Asn residues
H
O
O
6(3)-arm
Numbers give the
percentage occupancies
at the sites
OH
C
HO
NH
Ac
H
N
C
O
Core
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Structure of Man9GlcNAc2
Individual linkages are flexible on a sub-nsec timescale, many
showing more than one conformer.
The properties of a linkage depend on the its position in the
molecule.
The overall topology of the molecule is more conserved than
the structures of the individual linkages.
ï correlated motions of the linkages (due to constraints of
the solvent shell ??)
Oxford Glycobiology Institute
Oxford Glycobiology Institute
Crystal and NMR structures of K2G
X-tal structure of
K2G in complex
with MHC Class I
Solution
structure of K2G
from NMR and
modelling
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The presence of the
GlcNAc causes
preferential clustering of
aromatic side chains,
leading to reduced
configurational entropy
of the peptide backbone.
In this case, the
clustering places the
Phe, Tyr and GlcNAc on
the same side of the
peptide as seen in the
complex with MHC.
4
The presentation of the glycan is determined
by interactions with the peptide surface
Domain 1 of CD2
Site 78 of hCG
Oxford Glycobiology Institute
CH2 domain of IgG Fc
- area of the surface with which the glycan interacts
Ribonucleases B glycan dynamics
Two sterically allowed
Asn conformations
Allowing for N-linkage
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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5