Molecular recognition

Transcription

1 Lecture 9 Molecular recognition Antoine van Oijen BCMP201 Spring 2008 Structural principles of binding 4 fundamental functions of proteins: 1) Binding 2) Catalysis 3) Switching 4) Structural All involve binding! Last week: Kinetics and thermodynamics of binding Today: Structural principles of binding 1

2 The nature of protein-protein interactions Binding energy simply sum of energies of - hydrogen bonds - ionic contacts - van der Waals contacts? The nature of protein-protein interactions Chothia and Janin (1975, Nature): Electrostatic, H-bond, vdw interactions are also present in solution; forming them at the binding interface doesn t reduce ΔG 0 2

3 The nature of protein-protein interactions Main determinant of binding energy is hydrophobicity (entropic effect) The nature of protein-protein interactions Binding energy increases with area of interface Maximum ~ 0.1 kj/mol per Å 2 interaction surface 3

4 Specificity High specificity interfaces must be highly complementary Specificity provided by : 1) Ionic complementarity 2) Hydrogen bond complementarity 3) Steric complementarity (van der Waals) Example: a hormone-receptor interface human Growth Hormone - human Growth Hormone receptor 90 4

5 Example: a hormone-receptor interface K D of hgh-hghr interaction = 0.3 nm Using K D = e ("G0 / RT) : (R=8.3 J -1 mol -1 K -1 T=295 K) ΔG 0 = -54 kj/mol (=12.7 kcal/mol) ~ 1300 Å 2 buried surface Alanine scanning mutagenesis Systematically mutate each of the surface residues into Alanine (Clackson et al., Science (1995); 267, 383) 5

6 Example: a hormone-receptor interface Hot spots : W104A and W169A each increase ΔG 0 by >4.5 kcal/mol (~ 19 kj/mol) Each increases K D from 0.3 nm to >0.7 µm (Clackson et al., Science (1995); 267, 383) Example: a hormone-receptor interface Compare interaction surface with cross-section through globular protein: hydrophobic core, hydrophilic exterior Functional epitope Structural epitope (Clackson et al., Science (1995); 267, 383) 6

7 Example: a hormone-receptor interface Complementarity of functional epitopes of GH and GHr (Clackson et al., Science (1995); 267, 383) Lock in key versus induced fit Lock in key Induced fit 7

8 Induced fit 1) Surface side chains can move 2) Surface loops can move 3) Domains can move at hinges Helix-turn-helix motifs 8

9 Lac repressor Lac repressor Folding upon binding to DNA (Kalodimos et al., Science (2004), 305, 386) 9

10 Lac repressor Folding only upon binding to specific DNA Nonspecific complex formed by electrostatic interactions (Kalodimos et al., Science (2004), 305, 386) A mechanism for target location? (Slutsky et al. Biophysical Jrnl. (2004); 87, 4021) 10

11 Antibodies Immunoglobulins / antibodies: Secreted by B cells to bind to antigens Constant domains (C) Variable domains (V) (Figures from: Branden, Tooze; Introduction to Protein Structure) Immunoglobulins Regions in variable domains show hypervariability (complementarity determining regions; CDR) (Figure from: Branden, Tooze; Introduction to Protein Structure) 11

12 Antigen-binding site Antigen-binding site is formed by close association of the hypervariable regions from both heavy and light chains Light chain Heavy chain Antigen-binding site Antigen-binding site is formed by close association of the hypervariable regions from both heavy and light chains Light chain Heavy chain (Figures from: Branden, Tooze; Introduction to Protein Structure) 12

13 Induced fit Induced fit upon binding of an HIV-1 peptide to Fab fragment of IgG Wilson et al., Structure (1993); 1, Domain flexibility Domain flexibility can give rise to dramatic increase in binding affinity: divalent binding virus IgG 13

14 Cooperative association K D Fab 10-6 M; What is K D IgG? virus 1) Calculate ΔG 0 Fab = RTlnK D = -34 kj/mol 2) Multiply by 2 for 2 bonds: -68 kj/mol 3) Plus an entropic factor of ~ -25 kj/mol 4) ΔG 0 IgG = -93 kj/mol K D IgG = 3x10-17 M!! IgG Entropy penalty is only paid once! Immunoglobulin fold in MHC and T-cell receptors Antibody - antigen MHC - peptide T-cell receptor - MHC/peptide High affinity, high specificity High affinity, low specificity Low affinity, high specificity 14

15 Antibody techniques (Lehninger, Principles of Biochemistry) Take-home messages 1) Protein-protein interactions are mainly mediated by hydrophobic effects 2) Surface complementarity contributes to specificity 3) Lock-in-key versus induced fit 15