Lecture 3 Glycan Binding Proteins Principles of Glycan Recognition

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Lecture 3Glycan Binding ProteinsPrinciples of
Glycan Recognition
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The Glycome is Rich
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GBPs to Translate the Glycome
Soluble Glycan-Binding Proteins
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Two Classes of GBPs - Lectins and GAG Binding
Proteins
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Mannose Binding Protein (C-type lectin)

• Mannose binding protein is trimeric and each
  subunit binds to mannose
• Notice that binding occurs in shallow pockets
  near the surface

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Cholera Toxin

• Cholera toxin exists as a AB5 complex, each B
  subunit binds to a single molecule of GM1
• The glycan binds to shallow pockets near the
  surface

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Flu Hemagglutinin
B
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?-mannose in the CRD of FimH
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?1-4Galactosyltransferase
Van der Waals surface diagram around the
trisaccharide GlcNAc?1-2Man?1-6Man
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Link Module - Hyaluronan Binding
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Flavivirus Adhesin
Heparan sulfate interacts with protein across a
groove lined with positive amino acids (Lys Arg)
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Cholera Toxin

• Cholera toxin exists as a AB5 complex, each B
  subunit binds to a single molecule of GM1
• The glycan binds to shallow pockets near the
  surface
• Kd of monomer for GM1 40 nM, very unusual
  (usually µM to mM)

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Cholera Toxin

• Multivalency raises affinity of interaction to
  40 pM

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• Only 3 sugars are bound
• Selectivity is high, since binding occurs with
  great precision

• Electrostatics
• H-bonding
• van der waals interactions
• Tyr, Phe, or alkyl side chains
• Water and divalent cations can act as bridging
  groups that increase selectivity

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Cholera Toxin

• Multivalency raises affinity of interaction to
  40 pM
• Notice that many lectins are multimeric

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• High Avidity can also result from coopretive
  interactions at independent sites
• Whats the advantages/disadvantages of
  multivalency?

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What is meant by affinity?
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Equilibrium-Dialysis
G
L
Set up dialysis and allow equilibrium to occur
(days). Measure G in both chambers
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Leq
Keq LGeq / LeqGeq
Geq
LGeq
Leq Linit - LGeq
LGeq Gtotal in left compartment - Gright
Written this way Keq Ka (units M-1)
1/ Ka Kd (units M)
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ITC titration profile (top panel) of SBA (a
lectin) titrated with Tn-PSM (a mucin). The lower
panel shows a fit of the binding data
Dam, T. K. et al. J. Biol. Chem.
200728228256-28263
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peptide repeats
2300 glycans
23 glycans
11-12 glycans
Dam, T. K. et al. J. Biol. Chem.
200728228256-28263
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From Table 1. Hemagglutination inhibition data
for SBA Ligand Kd K(rel) GalNAc?1-O-Ser
1000 1 38/40-mer Tn-PSM 5.6 180 81-mer
Tn-PSM 0.15 7000 Tn-PSM 0.0003 3,000,000
From Table 2. Thermodynamic binding parameters
for SBA Kd K(rel) ?G ?H T?S n µM
(kcal/mol) GalNAc?1-O-Ser 170 1 5.2
7.9 2.7 1.0 38/40-mer Tn-PSM 1.4 120 8.0
32.2 24.2 0.2 81-mer Tn-PSM 0.06 2,800 9.8
56.1 46.3 0.12 Tn-PSM 0.0002 850,000 13.1
4310 4297 0.0018
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Questions to Ponder

• 1. What is a lectin? What is a CRD?
• 2. Why are sulfated GAG binding proteins
  distinguished from lectins?
• 3. What determines the binding affinity of a
  glycan for a GBP?
• 4. What are the advantages and disadvantages of
  achieving high affinity through multivalency?
• 5. How does the density of carbohydrate ligands
  affect binding of a GBP? Is this relevant in
  vivo?
• 6. Would you predict the existence of GBPs that
  bind to the tips of GAG chains? What would be
  the advantages and disadvantages of this binding
  mode?

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FGF-FGFR Heparin