Carbohydrate- Protein interac;ons are Cri;cal in Life and Death. Other Cells. Hormones. Viruses. Toxins. Cell. Bacteria

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Shavonne Mosley
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1 ther Cells Carbohydrate- Protein interac;ons are Cri;cal in Life and Death ormones Viruses Toxins Cell Bacteria

2 ow to Model Protein- ligand interac;ons? Protein Protein Protein DNA/RNA Protein Carbohydrate Protein Drug An;body An;gen Enzyme Substrate

3 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! ow 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?

4 ow to generate the ini;al model? igher Accuracy Co- Complex Source X- ray structure of the protein - ligand complex (so called co- complex ) Suitable for 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 Lower Accuracy omology model of the protein with docking of the ligand Guiding protein engineering experiments

5 Amino Acids: igh Chemical Diversity, Low Structural Diversity 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. f the 20 common residues 19 have the general structure shown below: R 3 N + Cα C 2 - R= (Gly), C3 (Ala), etc. The exception is the amino acid proline, whose side chain is bonded to the nitrogen atom to give a cyclic imino acid called proline: C 2 2 C C 2 2 N + Cα C 2 - Because each side chain group attached to Cα is different (except for glycine, in which R=), Cα is asymmetric and, in nature, is always the L-enantiomer. n 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.

6 Carbohydrates: igh Structural Diversity, Low Chemical Diversity β-d-pyranose 1 β α-d-pyranose 1 α 1-3 linkage (β) 1 3 β-d-glucopyranose, β-d-glc NAc 2-N-acetyl-β-D-glucopyranose, β-d-glcnac 1-4 linkage (α) 1 4 β-d-galactopyranose, β-d-gal AcN 5 4 C N -acetyl-α-neuraminic acid, α-neu5ac linkage (β) 1 6 β-d-mannopyranose, β-d-man

7 ligosaccharides: Mul;ple Linkage Posi;ons and Configura;ons α/β α/β Glc-α-(1-4)-Glc (Starch) Glc-β-(1-4)-Glc (Cellulose) The same two amino acids à 1 possible peptide The same two monosaccharides à 20 possible disaccharides AcN C 2-2 AcN -GlcNAc 3 C Avian Flu Receptor uman Flu Receptor α-(2-3)-gal versus α-(2-6)-gal -GlcNAc

8 Electrostatic Interactions (ydrogen-bonds, charge-charge, charge-dipole, dipole-dipole) Dispersive Interactions (Van der Waals attractions and repulsions) Δ < 0 reaction is exothermic, tells us nothing about the spontaneity of the reaction Δ > 0 reaction is endothermic, tells us nothing about the spontaneity of the reaction Examples: And what about xidation of glucose: C C Δ = kjmol -1 Just because a reaction is exothermic (that is because Δ < 0) does not mean that it is spontaneous. Dissolving salt: NaCl(s) + 2 (l) Na + (aq) + Cl - (aq) Δ = 4 kjmol -1 Just because this reaction is endothermic (Δ > 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: Enthalpy (Δ) and Entropy (ΔS) 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

9 Thermodynamics of Ligand- Protein Interac;ons (ΔG) Remember: ΔG reaction = ΔG products ΔG reactants ΔG = Δ - 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 ΔG Binding = ΔG Complex ΔG Ligand ΔG Receptor = (Δ Complex T Δ S Complex ) (Δ Ligand T ΔS Ligand ) (Δ Receptor T ΔS Receptor ) There is a temptation to draw conclusions only from the structure of the complex, but: ΔG Binding MM Energy Complex MM Energy is often just the potential energy from a force field calculation. MM Energy often ignores entropy and desolvation! and is often NT computed as a difference between reactants and products! Bad modeling can t be trusted!

10 ydrogen Bonds Energe;c Contribu;ons to Ligand Binding A molecule which has a weakly acidic proton (, N ) may function as a proton donor (D) in a hydrogen bond with another molecule in which an electronegative atom (, N) is present to act as an acceptor (A). D A A typical hydrogen bond between polar uncharged groups has its maximum stability at an interatomic (A D) separation of Å and may contribute up to approximately 5 kcalmol -1 in the gas phase. ydrogen bonds show a high dependence on the orientation of the donor and acceptor groups, with a tendency for the D A angle to be linear. ydrogen Bond Energy Total Energy (kcal/mol) Total Energy Dipole/Dipole van der Waals R" D A R' r oh Separation (Angstroms) X-ray crystallographic studies of sugar-protein complexes can provide detailed structural information pertaining to hydrogen bonding in the binding site.

11 ydrogen Bonds, Con;nued 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 2 N + N 2 N 2 N 3 + N N + - A. -bonds between a sugar and a protein. B. -bonds between a sugar and water.

12 Effect of Loss of a ydrogen Bond on Binding Energies 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 ENTALPICALLY 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 ΔG Binding (L1) -20 kcal/mol L2 + Receptor Complex2 ΔG Binding (L2) -15 kcal/mol ΔΔG = ΔG Binding (L1) ΔG Binding (L2) = -5 kcal/mol (favoring the binding of L1) ow much would the loss of a single hydrogen bond change the binding affinities? Recall: ΔG = RT ln(k) ΔΔG = RTln(K 1 ) RTln(K 2 ) = RT(ln(K 1 ) ln(k 2 )) = RTln(K 1 /K 2 ) So for the two ligands, the ratio of their binding affinities (at 293 K) is: -5 = RT ln(k 1 /K 2 ) = ln(K 1 /K 2 ) = 0.59 ln(k 1 /K 2 ) Therefore K 1 /K 2 = e 8.47 = 4788, so the net loss of a hydrogen bond decreases affinity ~ 5000 fold. But why isn t counting -bonds a good measure of affinity?

13 The Role of Water in Ligand Binding Thermodynamics 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. + ΔΗ =? 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. owever 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 ENTALPIC 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: K A ~10 3 M -1.

14 Example: Effect of Loss of on Affinity Bacterial surface oligosaccharides from S. flexneri Y with antibody SYAJ6 Vyas, N.K., et al., Biochemistry, : p

15 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 ENTALPIC 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/r 6. 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" ther Enthalpic Contribu;ons to Binding r oh R' 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 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.

16 Wang, J., et al., J. Biol. Chem., (5): p Example: Effect of loss of van der Waals and hydrophobic contacts on affinity Bacterial surface oligosaccharides from V. cholerae with antibodys20-4 Too few favorable interac;ons, or too many unfavorable ones, will hurt binding Affinities of Vibrio cholerae binding to mab S20-4 Me Et Pr ΔG > >0 >0 K A x x

A large contributor to the ENTRPY of binding is from the release of water molecules. This arises from two contributions, desolvation entropy and the hydrophobic effect.

17 A large contributor to the ENTRPY of binding is from the release of water molecules. This arises from two contributions, desolvation entropy and the hydrophobic effect. Desolvation Entropy Effect of Entropy on Binding Energies 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. + ΔΗ =? ΔS > 0 + This release of water results in an increase in the entropy of the system, i.e. ΔS Binding > 0 and so -TΔS Binding < 0. But the desolvation free energy may still be unfavorable (>0) depending on Δ ΔG Desolvation = Δ Desolvation - TΔS Desolvation

18 The ydrophobic Effect 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: ow does the hydrophobic effect differ from van der Waals contacts? ow does it differ from orbital overlap? 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 ENTRPY (ΔS > 0). Moreover, it exposes its hydrophilic face to the solvent and so helps promote good solvent-ligand hydrogen bonding.

19 The rigin of ydrophobicity is Entropic! 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 Δ > 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! Process Δ kj/mol - TΔS kj/mol ΔG kj/mol C 4 in 2 versus C 4 in C C 2 6 in 2 versus C 2 6 in C C 2 4 in 2 versus C 2 4 in C 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.

20 Conforma;onal Entropy 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.

21 Changes in Flexibility Affect Binding Energy Carbohydr. Res., (2005) 340, 1007 PNAS, (2006) 103, 8149

22 Simula;ons Can Quan;ty Each Energy Contribu;on Component Energy Theoretical Contribution Electrostatic Interactions Van der Waals Interactions Total Molecular Mechanical Energy Desolvation Energy Entropy 77.6 Total Binding Energy -4.9 Kadirvelraj, R., et al., PNAS, (21): p

Other Cells. Hormones. Viruses. Toxins. Cell. Bacteria

Other Cells. Hormones. Viruses. Toxins. Cell. Bacteria Other Cells Hormones Viruses Toxins Cell Bacteria 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