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

Oxford Glycobiology Institute

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

crystallography) in glycobiology


+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


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


Thy 1


Oligosaccharide 1D NMR spectrum

9 7 5 3 1C hem ica l S hift (ppm )

NeuAc1Gal2Man3GlcNAc4Fuc1


Conformational analysis


+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).


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


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.


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/


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


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


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


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


Crystallographic glycosidic linkage conformers - chitobiose


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%


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


-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


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.


Nuclear Magnetic Resonance Spectroscopy- effects of local correlation times

Glc1-2Glc5.4 4.0 3.8 3.65.6

Chemical shift (ppm)

NOESY

ROESY


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


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


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


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


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


13C

1H

Bo

2

3

3 1cosRDC

r

Partial alignment in a liquid crystal- Residual Dipolar Coupling

1JCH(aligned) = 1JCH(unaligned) + RDC


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


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


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


Solution conformation of oligomannose N-linked

oligosaccharides

Dr Rob WoodsDr Chris Edge

Dr Andrei Petrescu, Institute of Biochemistry, Bucharest


Dol

ER

Golgi

N-glycan biosynthesis

NeuAcGlcNAc Man Glc Gal Fuc

Complexglycans

Hybridglycans

Oligomannoseglycans


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.


Crystallographic glycosidic linkageconformers - 2009



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


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


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


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’


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


-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


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

(%

)


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


-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


Molecular Dynamics of Man9GlcNAc2

Overlay of structures (all atoms) from 1000 ps

Side view Top view


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


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


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


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


Crystallographic glycosidic linkage conformers - 2009



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


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


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


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


Glc3ManOMe Molecular Dynamics

G1-G2

G2-G3

G3-D1


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


D1

G3

G2

G1

Major – 70% Minor – 30%

Two conformations of Glc3ManOMe


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


MinorMajor

Calnexin

Glc1Man9GlcNAc2 binds to Calnexin in the minor conformer

Major conformer makes very unfavourable steric interactions with the protein.

Documents

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