Upload
salene
View
30
Download
1
Embed Size (px)
DESCRIPTION
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) - PowerPoint PPT Presentation
Citation preview
Oxford Glycobiology Institute
Use of (accurate+quantitative) NMR (and molecular modelling and
crystallography) in glycobiology
Oxford Glycobiology Institute
+O
HOHO
OH
OH
OH OH
OHHO
OHO
O
HOHO
OH
OH
O
OH
OHHO
O
OligosaccharidesFormed 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
Oxford Glycobiology Institute
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
Oxford Glycobiology Institute
Thy 1
Oxford Glycobiology Institute
Oligosaccharide 1D NMR spectrum
9 7 5 3 1C hem ica l S hift (ppm )
NeuAc1Gal2Man3GlcNAc4Fuc1
Oxford Glycobiology Institute
Conformational analysis
Oxford Glycobiology Institute
+O
HOHO
OH
OH
OH OH
OHHO
OHO
O
HOHO
OH
OH
O
OH
OHHO
O
Conformations of oligosaccharidesFormed 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).
Oxford Glycobiology Institute
O
OO
O
OO
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
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.
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.
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) databasehttps://sags.biochim.ro/
Oxford Glycobiology Institute
Crystal structures containing glycosidic linkages - 2002
Crystal structures containing glycosidic linkages - 2009
Type of structure No. of crystal
structures
No. of glycans
No. of linkages between
undistorted glycans
No. of incorrect/distorted linkages
Oligosaccharides 9 10 11 0
Glycoproteins with N-linked glycans
228 503 1091 292
Glycoproteins with O-linked glycans
2 2 2 0
Proteins with glycan ligands
29 74 200 1
Glycoproteins with N-linked glycans
1109 3018 6352
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
sp2 (planar) carbon
incorrect monomers or linkages
Oxford Glycobiology Institute
Crystallographic glycosidic linkage conformers - Man 1-4 GlcNAc linkage
-180 -120 -60 0 60 120 180-180
-120
-60
0
60
120
180
O5-C1-O-C4’
C1-
O-C
4’-C
3’
O5-C1-O-C4’
100
-180 -120 -60 0 600
50
150200
120 180
250
050
100150200250
C1-O-C4’-C3’
Torsion angle
Fre
quen
cy
Major cluster – 56% of structures
Oxford Glycobiology Institute
Crystallographic glycosidic linkageconformers - 2009
Glycosidic linkage No. of Distinct conformers Conformerstructures population
Fuc1-3GlcNAc 189 -72.06.9 -97.76.1 -- 143Fuc1-6GlcNAc 281 -75.29.3 187.312.0 55.77.9 49Gal1-4GlcNAc 44 -71.410.9 132.27.4 -- 28GlcNAc1-4GlcNAc 2950 -78.711.6 119.214.9 -- 2122GlcNAc1-2Man 122 -77.36.8 -89.44.7 -- 44Man1-4GlcNAc 1202 -86.511.4 109.416.8 -- 674Man1-2Man 185 63.64.4 -178.63.5 -- 37
69.58.8 -107.914.9
-- 78
Man1-3Man 584 73.77.1 -117.318.0
-- 288
Man1-6Man 553 62.25.1 86.02.6 190.19.5 1765.210.0 174.521.6 183.326.5 7264.110.1 184.99.4 60.08.1 74
Oxford Glycobiology Institute
Crystallographic glycosidic linkage conformers - chitobiose
Glycosidic linkage No. of Distinct conformers Conformerstructures population
1999GlcNAc1-4GlcNAc 163 -74.47.8 115.915.9 -- 146
2002GlcNAc1-4GlcNAc 398 -75.911.6 119.015.4 -- 376
2009GlcNAc1-4GlcNAc 2950 -78.711.6 119.214.9 -- 2122
From 1999 to 2009: Number of structures increased by 1,800%
Percentage of structures within the major cluster decreased from 90% to 70%
Oxford Glycobiology Institute
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 (of3.5 Å) of the inter-proton distance for all significantly populated conformations.
Conformational constraints from NOEs
Oxford Glycobiology Institute
-110 -60 -10 40 90 140-160
-110
-60
-10
40
90
140
H1 - H2’ distance
H1-C1-O-C2’
C1-
O-C
2’-H
2’
2.75 - 3.0 Å
Conformational constraints from NOEs
NOE intensity 1/r6
r
O
O
H
H
O
Oxford Glycobiology Institute
Nuclear Magnetic Resonance Spectroscopy- steady state NOE intensity and mobility
0
1
-1
0.1 1 10
0.5
-1
NOE
0.675
0.385
ROE
Inte
nsity
oc
Generally assume that c is constant for any given disaccharide.
Then, can use an intra residue NOE to calibrate the cross-linkage NOEs.
Oxford Glycobiology Institute
Nuclear Magnetic Resonance Spectroscopy- effects of local correlation times
Glc1-2Glc5.4 4.0 3.8 3.65.6
Chemical shift (ppm)
NOESY
ROESY
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
Bo
Oxford Glycobiology Institute
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
ij
ij 2ij
J( )1 ( )
ijij 6
ij
AI
r
ij ij ij
ij ij ij
ij ij ij ij
NOE : A B 6J(2 ) J(0)
ROE : A B 3J( ) 2J(0)
1T-ROE : A B 6J(2 ) 3J( ) J(0)2
Poveda, et al (1997) Carbohydr. Res., 300, 3-10
Oxford Glycobiology Institute
Nuclear Magnetic Resonance Spectroscopy- measuring local correlation times
Rat
io o
f bu
ild-u
p ra
tes
c (ns)
o = 500 MHz
0 0.2 0.4 0.6
0
-1
1
2
3(ROE)
(T-ROE)
(NOE)(T-ROE)
(NOE)(ROE)
Poveda, et al (1997) Carbohydr. Res., 300, 3-10
Oxford Glycobiology Institute
2.40 Å
2.24 Å
2.30 Å
Distances calculated from
NOESY data allowing for local correlation times200 ps
289 ps
280 ps
Effective local correlation times
measured by (NOE)/(T-ROE)
ratio2.13 Å
2.30 Å
2.30 Å
Distances calculated from
NOESY data assuming a rigid
molecule
Local correlation times- Glc3ManOMe
Glc1-2Glc o = 500 MHz
Oxford Glycobiology Institute
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)
Mixing time
NO
E in
tens
ity
00
H1 H2
H3
H1-H2 NOE
H1-H3 NOE
Nuclear Magnetic Resonance Spectroscopy- NOE intensity and mixing time
Oxford Glycobiology Institute
13C
1H
Bo
2
3
3 1cosRDC
r
Partial alignment in a liquid crystal- Residual Dipolar Coupling
1JCH(aligned) = 1JCH(unaligned) + RDC
Oxford Glycobiology Institute
cos cosij i jij
RDC S Sij 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.
22
3 ijij
GDO S
Partial alignment of oligosaccharides in a liquid crystal
H
H
HH
HH
H O
O
O
Bo
Tian, et al (2001) J. Am. Chem. Soc., 123, 485-492
Oxford Glycobiology Institute
jibondsH
10ij
ij
12ij
ij
jibondedNon ij
ji
6ij
ij
12ij
ij
)Dihedrals(
n
)Angles(
2eq
Bonds(b)
2eqbTotal
rr
rrr
ncos12
bb
DC
qqBA
V
KKE
GLYCAM force-field parameters
Woods, et al (1995) J. Phys. Chem., 99, 3832-3846
Exo-anomeric effect
Partial charges
AMBER
Oxford Glycobiology InstituteH1-C1-O-C
-180 -120 -60 0 60 120 180
H1-C1-O-C
-180 -120 -60 0 60 120 180
Rel
ativ
e en
ergy
(kJ
/mol
)
0
10
20
30
40
50
Exo-anomeric effect - Hartree-Fock calculations
Oxford Glycobiology Institute
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
Oxford Glycobiology Institute
Solution conformation of oligomannose N-linked
oligosaccharides
Dr Rob WoodsDr Chris Edge
Dr Andrei Petrescu, Institute of Biochemistry, Bucharest
Oxford Glycobiology Institute
Dol
ER
Golgi
N-glycan biosynthesis
NeuAcGlcNAc Man Glc Gal Fuc
Complexglycans
Hybridglycans
Oligomannoseglycans
Oxford Glycobiology Institute
1,6 arms
1,3 arm
Man Man Man
Man
Man GlcNAc GlcNAc
Man Man
Man Man
63
63
D1 C 4
3 2 1
4’A
B
D2
D3
Man 1-2 Man linkage
Schematic structure of Man9GlcNAc2
Man1-2Man linkages occur in oligomannose type N-glycans, polysaccharides such as
mannan and GPI anchors.
Oxford Glycobiology Institute
Crystallographic glycosidic linkageconformers - 2009
Glycosidic linkage No. of Distinct conformers Conformerstructures population
Fuc1-3GlcNAc 189 -72.06.9 -97.76.1 -- 143Fuc1-6GlcNAc 281 -75.29.3 187.312.0 55.77.9 49Gal1-4GlcNAc 44 -71.410.9 132.27.4 -- 28GlcNAc1-4GlcNAc 2950 -78.711.6 119.214.9 -- 2122GlcNAc1-2Man 122 -77.36.8 -89.44.7 -- 44Man1-4GlcNAc 1202 -86.511.4 109.416.8 -- 674Man1-2Man 185 63.64.4 -178.63.5 -- 37
69.58.8 -107.914.9
-- 78
Man1-3Man 584 73.77.1 -117.318.0
-- 288
Man1-6Man 553 62.25.1 86.02.6 190.19.5 1765.210.0 174.521.6 183.326.5 7264.110.1 184.99.4 60.08.1 74
Oxford Glycobiology Institute
Torsion Angle
-180 -120 -60 0 60 120 1800
10
20
30
40
70H1-C1-O-C2’C1-O-C2’-H2’
50
60
Pop
ulat
ion
Crystallographic glycosidic linkage structures -Man 1-2 Man linkage (2009)
H1-C1-O-C2'
-180 -120 -60 0 60 120 180
C1-
O-C
2'-H
2'
-180
-120
-60
0
60
120
180185 structures
Oxford Glycobiology Institute
Man9GlcNAc2 NOESY traces
Chemical Shift (ppm)5.4 4.8 4.4 4.0 3.6
HDO
4’:C1H
A:C1H
D2:C1H
D2:C1H
A:C2H
A:C1HD2:C2HA:C3H
4’:C2H
3:C6H4’:C3H3:C6H
3:C5H
4’:C2HA:C2H
A:C3H
4’:C3HA:C5H +D2:C5H
Oxford Glycobiology InstituteH1-C1-O-C2'
-180 -120 -60 0 60 120 180
C1-
O-C
2'-H
2'
-180
-120
-60
0
60
120
180
C1H - C1H’ = 2.80 - 3.15 ÅC1H - C2H’ = 2.05 - 2.30 ÅC1H - C3H’ = 3.1 - 3.7 ÅC5H - C1H’ = 2.4 - 2.9 Å
C1H - C4H’ > 3.5 Å
Man9GlcNAc2 NMR torsion angle mapD1-C ManMan linkage
Oxford Glycobiology Institute
Simulation Time (ps)
0 250 500 750 1000
Tors
ion
Ang
le
-180
-120
-60
0
60
120
180
H1-C1-O-C2'
-180 -120 -60 0 60 120 180
C1-
O-C
2'-H
2'
-180
-120
-60
0
60
120
180
Man9GlcNAc2 Molecular DynamicsD1-C ManMan linkage
H1-C1-O-C2’C1-O-C2’-H2’
Oxford Glycobiology Institute
Hydrogen bondHydrophobic interactions
O
H
H
Conformation of the Man 1-2 Man linkage
OH
OH
OH
OH
OH
OHHO
HO
O
O
O
OH
OH
OH
OHOH
HO
HO
HO
O
O
O
Oxford Glycobiology Institute
-110
-10 90 14040-60
H1-C1-O-C3’
90
-10
-110
40
140
C1-
O-C
3’-H
3’
-60
Man9GlcNAc2 NMR torsion angle mapA-4’ ManMan linkage
C1H - C2H’ = 2.85 - 3.20 ÅC1H - C3H’ = 2.00 - 2.25 ÅC1H - C4H’ = 2.75 - 3.10 ÅC5H - C2H’ = 2.55 - 2.85 Å
• Individual MD conformations
Oxford Glycobiology Institute
Man9GlcNAc2 NMR build up curvesA-4’ ManMan linkage
C1H - C3H’
C5H - C2H’ C1H - C4H’C1H - C2H’
0 500 1000 1500
2000
Mixing time (ms)
0
2
4
6
8
10
NO
E In
ten
sity
(%
)
Oxford Glycobiology Institute
1,6 arms
1,3 arm
Man Man Man
Man
Man GlcNAc GlcNAc
Man Man
Man Man
63
63
D1 C 4
3 2 1
4’
A
B
D2
D3
Schematic structure of Man9GlcNAc2
Oxford Glycobiology Institute
-150
-60
30
120
210
-180
-90
0
90
180
-180
-90
0
90
180
0 450 900-180
-90
0
90
180
-180
-90
0
90
180
0 450 900-180
-90
0
90
180
0 450 900-180
-90
0
90
180
-150
-60
30
120
210
0 450 900-180
-90
0
90
180
D3-B
D2-A
D1-C C-4 4-3
A-4'
3-2
B-4'
4'-3
0 450 900-180
-90
0
90
180
2-1
Simulation time (ps)
Tor
sion
ang
leMolecular Dynamics of Man9GlcNAc2
Oxford Glycobiology Institute
Molecular Dynamics of Man9GlcNAc2
Overlay of structures (all atoms) from 1000 ps
Side view Top view
Oxford Glycobiology Institute
Flexibility of the tri-glucosylated cap of
immature N-linked glycans
Mukram MackeenDrs Andy Almond and Michael
Deschamps, Dept. of Biochem., Oxford
Dr Terry ButtersDr Antony Fairbanks, Dept. of Chem.,
Oxford
Oxford Glycobiology Institute
Recognised byCalnexin and Calreticulin
ER resident chaperonesinvolved in protein folding
Glucosidase I Glucosidase II Glucosidase II
Glucosyltransferase
ER Golgi
ER processing and recognition of N-linked glycans - protein folding and quality control
OST
P
P
Dol
Nascent peptide
ERAD
Oxford Glycobiology Institute
Schematic structure of Glc3Man9GlcNAc2
1,6 arms
1,3 arm
Glc Glc Glc Man Man Man
Man
Man GlcNAc GlcNAc
Man Man
Man Man
63
63
G1 G2 G3 D1 C 4
3 2 1
4’
A
B
D2
D3
Oxford Glycobiology Institute
Multifunctional roles of GlcxMan9GlcNAc2
Chaperone assisted foldingCalnexin and calreticulin binding
OGT substrate
Protein N-glycosylationOST substrate binding
Protein folding quality controlERAD recognition
Glycan remodelling to produce mature glycans
Glc 1
GlcNAc 1
Glc3Man
Oxford Glycobiology Institute
Crystallographic glycosidic linkage conformers - 2009
Glycosidic linkage No. of Distinct conformers Conformerstructures population
Fuc1-3GlcNAc 189 -72.06.9 -97.76.1 -- 143Fuc1-6GlcNAc 281 -75.29.3 187.312.0 55.77.9 49Gal1-4GlcNAc 44 -71.410.9 132.27.4 -- 28GlcNAc1-4GlcNAc 2950 -78.711.6 119.214.9 -- 2122GlcNAc1-2Man 122 -77.36.8 -89.44.7 -- 44Man1-4GlcNAc 1202 -86.511.4 109.416.8 -- 674Man1-2Man 185 63.64.4 -178.63.5 -- 37
69.58.8 -107.914.9
-- 78
Man1-3Man 584 73.77.1 -117.318.0
-- 288
Man1-6Man 553 62.25.1 86.02.6 190.19.5 1765.210.0 174.521.6 183.326.5 7264.110.1 184.99.4 60.08.1 74
Glc3Man linkages:
Glc1-2Glc 0Glc1-3Glc 1Glc1-3Man 0
Oxford Glycobiology Institute
Solid lines : positive constraintsDotted lines : negative constraints
White – H1-H’x-1 Grey – H5-H’x-1
Red – H1-H’x Pink – H5-H’xGreen – H1-H’x+1 Yellow – H5-H’x+1
-
C1-
O-C
’x-H
’xGlc3Man NMR torsion angle maps
G1-G2 G2-G3 G3-D1
- H1-C1-O-C’x-150 -50 +50 +150-150 -50 +50 +150 -150 -50 +50 +150
+150
+50
-50
-150
Oxford Glycobiology Institute
116 115 114 113 112
1 – 13C (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
4:1C:1
D1:1
A:1
G3:1
4:1C:1
Residual dipolar coupling- Glc3Man7GlcNAc2
Oxford Glycobiology Institute
GDO values for the tri-glucosyl cap
Glc3ManOMe Glc3Man7GlcNAc2
G1 0.014 0.005
G2 0.013 0.005
G3 0.030 0.075
D1 0.003 0.013
Oxford Glycobiology Institute
Glc3ManOMe Molecular Dynamics
G1-G2
G2-G3
G3-D1
Oxford Glycobiology Institute
Proton pair ij (ps) Distance (Å)X-ray NMR MD
G1-G2 : Glc1–2Glc linkageG1:H1 G1:H2 280 2.34 -- 2.42
G2:H1 289 2.30 2.18G2:H2 200 2.45 2.76G2:H3 -- >3.5 4.35
G2-G3 : Glc1–3Glc linkageG2:H1 G2:H2 338 2.34 -- 2.41
G3:H2 -- 4.23 >3.5 4.41G3:H3 316 2.05 2.19 2.55G3:H4 -- 3.74 >3.5 3.32
G3-D1 : Glc1–3Man linkageG3:H1 G3:H2 347 2.34 -- 2.41
D1:H2 172 3.08 3.48D1:H3 260 2.12 2.30D1:H4 nd ~3.1 3.69
Glc3ManOMe – X-ray vs NMR vs MD
Oxford Glycobiology Institute
D1
G3
G2
G1
Major – 70% Minor – 30%
Two conformations of Glc3ManOMe
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
Oxford Glycobiology Institute
MinorMajor
Calnexin
Glc1Man9GlcNAc2 binds to Calnexin in the minor conformer
Major conformer makes very unfavourable steric interactions with the protein.