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TRANSCRIPT
Bacteria
Toxins
Viruses
Other Cells
Hormones
Cell
Carbohydrate-‐Protein interac;ons are Cri;cal in Life and Death
How to Model Protein-‐ligand interac;ons?
Protein – Protein Protein – DNA/RNA
Protein – Carbohydrate Protein – Drug
An;body – An;gen Enzyme – Substrate
What do you want to know?
• The 3D structure of the complex! • But why do you need this? • To iden;fy cri;cal interac;ons! • But why?
– To understand the mechanism! • How will you prove it? • Compare to experimental data (such as from muta;ons)!
– To design an inhibitor! • Must be able to compute interac;on energies • What level of accuracy is required?
– To guide protein engineering! • Do you need to know interac;on energies? • What level of accuracy is required?
How to generate the ini;al model?
Co-‐Complex Source Suitable for
X-‐ray structure of the protein -‐ ligand complex (so called “co-‐complex”)
Guiding ligand design Insight into binding mechanism Guiding protein engineering
X-‐ray structure of the protein with docking of the ligand
Insight into binding mechanism Guiding protein engineering experiments
Homology model of the protein with docking of the ligand
Guiding protein engineering experiments
Lower Accuracy
Higher Accuracy
Amino Acids: High Chemical Diversity, Low Structural Diversity
CαH3N+
H
CO2-
R
H2CCH2
CH2
CO2-
H
H2N+ Cα
There are 20 common naturally occurring amino acids termed -amino acids because both the amino- and carboxylic acid groups are connected to the same (α) carbon atom. Of the 20 common residues 19 have the general structure shown below:
The exception is the amino acid proline, whose side chain is bonded to the nitrogen atom to give a cyclic imino acid called proline:
Because each side chain group attached to Cα is different (except for glycine, in which R=H), Cα is asymmetric and, in nature, is always the L-enantiomer.
R=H (Gly), CH3 (Ala), etc.
On the basis of the gross physical properties of the R-groups it is possible to divide the amino acids into classes, namely, hydrophobic, charged, and polar. Further divisions may be made on the basis of the chemical natures of the R-groups.
O
OH
O
OH
O
OH
O
OH
O
OH
HO
HOOH
OH
HOOH
HO
HO
OH
OH OH
OH
OH
OH
O
O
OH
O
OH
OH
O
O
OH
O
OH
OH
O
O
OH
O
OH
αβ1
23
4 5
6
123
4 56
β-D-glucopyranose, β-D-Glc
β-D-pyranose α-D-pyranose
β-D-galactopyranose, β-D-Gal
β-D-mannopyranose, β-D-Man
1-4 linkage (α)
1-3 linkage (β)
1-6 linkage (β)
1
1
1
4
3
6
O
OHHO
HONHAc
OH
2-N -acetyl-β-D-glucopyranose, β-D-GlcNAc
O
CO2-
OH
HOHO
AcHN
OHHO
5−N -acetyl-α-neuraminic acid, α-Neu5Ac
5
1
2
34
67
89
Carbohydrates: High Structural Diversity, Low Chemical Diversity
O
O
HO
HOOH
OHO
O
O
OO
O
α/β
α/β
The same two amino acids à 1 possible peptide The same two monosaccharides à 20 possible disaccharides
O
OH
AcHN
OH
HO CO2-
HO
OHO
OH
O
OH
O-GlcNAc2
3
O
OH
AcHN
OH
HO CO2-
HOO
OH
HO
OH
O-GlcNAc
O2 6
Avian Flu Receptor Human Flu Receptor
α-(2-3)-Gal versus α-(2-6)-Gal
Glc-α-(1-4)-Glc (Starch) Glc-β-(1-4)-Glc (Cellulose)
Oligosaccharides: Mul;ple Linkage Posi;ons and Configura;ons
Electrostatic Interactions (Hydrogen-bonds, charge-charge, charge-dipole, dipole-dipole)
Dispersive Interactions (Van der Waals attractions and repulsions)
ΔH < 0 reaction is exothermic, tells us nothing about the spontaneity of the reaction Δ H > 0 reaction is endothermic, tells us nothing about the spontaneity of the reaction Examples:
Oxidation of glucose: C6H12O6 + 6O2 → 6CO2 + 6H2O ΔH = -2803 kJmol-1
Just because a reaction is exothermic (that is because ΔH < 0) does not mean that it is spontaneous.
And what about
Dissolving salt: NaCl(s) + H2O(l) → Na+(aq) + Cl-(aq) ΔH = 4 kJmol-1
Just because this reaction is endothermic (ΔH > 0) does not mean that it doesn’t happen.
Enthalpy alone is not sufficient to decide whether a reaction will occur. The missing factor is called Entropy or ΔS.
Entropic Contributions:
Solute Related (conformational entropy)
Solvent Related (ligand and receptor desolvation)
ΔS < 0 reaction leads to order, tells us nothing about the spontaneity of the reaction ΔS > 0 reaction leads to disorder, tells us nothing about the spontaneity of the
Enthalpy (ΔH) and Entropy (ΔS)
Remember: ΔGreaction = ΔGproducts – ΔGreactants ΔG = ΔH - T ΔS, The reaction is favourable only when ΔG < 0
Ligand Binding Energy is also computed as if it were a reaction: Ligand + Receptor → Complex ΔGBinding = ΔGComplex – ΔGLigand – ΔGReceptor
= (ΔHComplex – T Δ SComplex) – (ΔHLigand – T ΔSLigand) – (ΔHReceptor – T ΔSReceptor) There is a temptation to draw conclusions only from the structure of the complex, but: ΔGBinding ≠ MM EnergyComplex MM “Energy” is often just the potential energy from a force
field calculation. MM Energy often ignores entropy and desolvation! and is often NOT computed as a difference between reactants and products! Bad modeling can’t be trusted!
Thermodynamics of Ligand-‐Protein Interac;ons (ΔG)
1.25 1.75 2.25 2.75 3.25 3.75 4.25 4.75O...H Separation (Angstroms)
-6
-4
-2
0
2
4
6
8
10
Tota
l Ene
rgy
(kca
l/mol
)
Total EnergyDipole/Dipolevan der Waals
Hydrogen Bond Energy
roh
R"
HO
H
R'
O
Hydrogen Bonds
A molecule which has a weakly acidic proton (O—H, N—H) may function as a proton donor (DH) in a hydrogen bond with another molecule in which an electronegative atom (O, N) is present to act as an acceptor (A).
D—H ···A—
A typical hydrogen bond between polar uncharged groups has its maximum stability at an interatomic (A ···D) separation of 2.7-3.1 Å and may contribute up to approximately 5 kcalmol-1 in the gas phase. Hydrogen bonds show a high dependence on the orientation of the donor and acceptor groups, with a tendency for the D—H ··· A angle to be linear.
X-ray crystallographic studies of sugar-protein complexes can provide detailed structural information pertaining to hydrogen bonding in the binding site.
D—H ··· ··· ··· ··· A
Energe;c Contribu;ons to Ligand Binding
NH2H2N
N
N
H
NH2 OOHO
HO OHOH
OH HO
NH3+
H
+
O
O
-+
H
O
H
H
O
H
H
O
H
OHO
HO OHOH
OH
H
O
H
H
O
H
HO
H
The hydrophilic nature of sugars arises from the presence of hydroxyl groups attached typically to 5 out of 6 of the carbon atoms of the sugar:
The polyhydroxylated structure of a sugar has often been cited in support of the importance of hydrogen bonding in the interaction between the sugar and either a receptor or with solvent.
For example, in the case of arabinose binding protein, the arabinopyranose is involved in approximately 54 hydrogen bonds either with the protein, or with coordinated water molecules.
A B
A. H-bonds between a sugar and a protein. B. H-bonds between a sugar and water.
Hydrogen Bonds, Con;nued
The presence of hydrogen bonding is essential to the binding of a sugar to a protein. If there are not at least as many hydrogen bonds in the complex as there are between the sugar and the solvent, the binding will not be ENTHALPICALLY favored.
If each hydrogen bond stabilizes the interaction by 5 kcal/mol, the loss of a single hydrogen bond would severely diminish the binding affinity.
Consider two ligands: one makes 4 hydrogen bonds to the receptor, the other makes 3 hydrogen bonds.
L1 + Receptor → Complex1 ΔGBinding (L1) ≅ -20 kcal/mol
L2 + Receptor → Complex2 ΔGBinding (L2) ≅ -15 kcal/mol
ΔΔG = ΔGBinding (L1) – ΔGBinding (L2) = -5 kcal/mol (favoring the binding of L1)
How much would the loss of a single hydrogen bond change the binding affinities?
Recall: ΔG = –RT ln(K)
ΔΔG = –RTln(K1) – RTln(K2) = –RT(ln(K1) – ln(K2)) = –RTln(K1/K2)
So for the two ligands, the ratio of their binding affinities (at 293 K) is:
-5 = –RT ln(K1/K2) = –0.00198·398ln(K1/K2) = –0.59 ln(K1/K2)
Therefore K1/K2 = e8.47 = 4788, so the net loss of a hydrogen bond decreases affinity ~ 5000 fold. But why isn’t counting H-bonds a good measure of affinity?
Effect of Loss of a Hydrogen Bond on Binding Energies
Since many sugars all have the same number of hydroxyl groups and differ only in the configuration of the hydroxyl groups, they all can exhibit very similar hydrogen bonding patterns if they can physically fit into the receptor site.
H O
HH
OH
O
OHOH
HOHO
OH
HOH
HO
H
H
OH
OH
H
OH
OH
++
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH
ΔΗ = ?
O
OHOH
HOHO
OH
OHOH
ΔS > 0
Each hydroxyl group in a sugar may act as both a proton acceptor and a proton donor in hydrogen bonds.
In solution it is possible for two water molecules to orient themselves along each sugar hydroxyl group lone-pair axis and so an optimum hydrogen bonding network is present. However in the complex it may not be possible to orient the protein side chains as optimally.
Since for every hydrogen bond the sugar forms with the protein, it must break at least one with the water, thus the net ENTHALPIC gain from hydrogen bonding may be relatively small.
Consequently, while hydrogen bonding is essential to the binding of the sugar, it is not sufficient to generate very tight binding, or to discriminate between different sugars. This may explain why monosaccharide-protein interactions are often very weak: KA~103 M-1.
The Role of Water in Ligand Binding Thermodynamics
Bacterial surface oligosaccharides from S. flexneri Y with antibody SYAJ6
Vyas, N.K., et al., Biochemistry, 2002. 41: p. 13575-13586.
Example: Effect of Loss of OH on Affinity
Van der Waals Interactions (instantaneous dipole - induced dipole)
As any pair of atoms approach each other a weak attraction develops that is called a van der Waals interaction.
In order to provide a noticeable ENTHALPIC benefit the atoms must be no further apart than the sum of their van der Waals radii (typically less than ~ 4 Å). VdW energies decrease with distance as a function of 1/r6. In a ligand protein complex there may be many such interactions, and although each one provides very little energy, their sum may be significant.
R"
HO
H
R'
O
roh
Because of the extreme sensitivity of the energies of van der Waals contacts to interatomic distance, a slight change in ligand shape or binding orientation can greatly alter the number of van der Waals contacts. Thus, ligand specificity depends very highly on van der Waals contacts.
Other Enthalpic Contribu;ons to Binding
The maximum energetic contribution from vdw interaction is small (only about 0.2 kcal/mol) per interacting atom, but can add up to a significant contribution
OH OMe OEt OPr H
ΔG >0 -7.6 -6.2 >0 >0
KA ---- 3.9 x 105 3.6 x 104 ---- ----
Bacterial surface oligosaccharides from V. cholerae with antibodyS20-4
Wang, J., et al., J. Biol. Chem., 1998. 273(5): p. 2777-2783.
Affinities of Vibrio cholerae binding to mAb S20-4
Too few favorable interac;ons, or too many unfavorable ones, will hurt binding
Example: Effect of loss of van der Waals and hydrophobic contacts on affinity
A large contributor to the ENTROPY of binding is from the release of water molecules. This arises from two contributions, desolvation entropy and the hydrophobic effect.
Desolvation Entropy
As already seen, when the sugar binds to the protein, it displaces water molecules that were previously present in the binding site. It also must release water molecules that were directly coordinated to the sugar itself.
H O
HH
OH
O
OHOH
HOHO
OH
HOH
HO
H
H
OH
OH
H
OH
OH
++
H
OH
H
OH
H
OH
H
OH
H
OH
H
OH
ΔΗ = ?
O
OHOH
HOHO
OH
OHOH
ΔS > 0
This release of water results in an increase in the entropy of the system, i.e. ΔSBinding > 0 and so -TΔSBinding < 0.
But the desolvation free energy may still be unfavorable (>0) depending on ΔH
ΔGDesolvation = ΔHDesolvation - TΔSDesolvation
Effect of Entropy on Binding Energies
While it is obvious that a sugar is highly hydrophilic (typically being soluble only in water), sugars are also capable of hydrophobic interactions.
In the crystal structures of bound sugar-protein complexes it has frequently been observed that aromatic residues, such as Tyr, Trp and Phe, are present in the binding site. Moreover, these residues appear to stack against the “lower” face of the sugar:
Aromatic residues on the surface of the protein are not able to hydrogen bond effectively with the solvent and so they force the nearby water into non-ideal orientations.
When the sugar places its hydrophobic face against the aromatic residues, it releases the waters from their non-ideal orientation. This results in a gain in ENTROPY (ΔS > 0). Moreover, it exposes its hydrophilic face to the solvent and so helps promote good solvent-ligand hydrogen bonding.
How does the hydrophobic effect differ from van der Waals contacts? How does it differ from orbital overlap?
The Hydrophobic “Effect”
Process ΔH
kJ/mol - TΔS
kJ/mol ΔG
kJ/mol CH4 in H2O versus CH4 in C6H6 11.7 -22.6 -10.9
C2H6 in H2O versus C2H6 in C6H6 9.2 -25.1 -15.9
C2H4 in H2O versus C2H4 in C6H6 6.7 -18.8 -12.1
The solubility of a molecule in water depends on a balance between the energy needed to create a cavity in the water and the energy gained by the resulting interactions.
Thermodynamic data indicate that it is not enthalpy, but rather entropy that drives the non-polar molecules to avoid water.
if ΔH > 0 then this implies that energy must be added to get the reaction to occur
if ΔS > 0 (- TΔS < 0) then this implies that the reaction favours disorder. In all cases ΔG is negative indicating that hydrocarbons will spontaneously separate from water. It is enthalpically more favorable for small hydrocarbons to dissolve in water than in large non-polar solvents!
Entropy is a measure of disorder in a system. It decreases with increasing order. If -T ΔS is negative as in the above table then ΔS must be positive.
Rationalization: Because the non-polar group can not hydrogen bond to water, the water molecules at the surface of the non-polar molecule have fewer ways in which to hydrogen bond to each other. That means they have less freedom, or that they must reorient themselves into a more ordered structure at the surface of the cavity. This causes the entropy to decrease. So the preference for a hydrophobic group to avoid water is because otherwise it would force the water into entropically unfavorable orientations.
The Origin of Hydrophobicity is Entropic!
Entropy may also change as a function of the properties of the ligand or the protein. In flexible ligands, particularly oligosaccharides and polysaccharides, binding reduces the flexibility (entropy) of the ligand, which disfavors binding.
Thus, certain regions of the ligand (or protein) may introduce unfavorable entropies upon binding.
Conforma;onal Entropy
Carbohydr . Res., (2005) 340, 1007 PNAS , (2006) 103, 8149
Changes in Flexibility Affect Binding Energy
Component Energy Theoretical Contribution
Electrostatic Interactions -167.5
Van der Waals Interactions -126.9
Total Molecular Mechanical Energy -294.4
Desolvation Energy 211.9
Entropy 77.6
Total Binding Energy -4.9
Kadirvelraj, R., et al., PNAS, 2006. 103(21): p. 8149-8154.
Simula;ons Can Quan;ty Each Energy Contribu;on