Protein-Protein Interactions I

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1 Biochemistry 412 Protein-Protein Interactions I March 11, 2008

2 Macromolecular Recognition by Proteins Protein folding is a process governed by intramolecular recognition. Protein-protein association is an intermolecular process. Note: the biophysical principles are the same!

3 Special Features of Protein-Protein Interfaces Critical for macromolecular recognition Typically, ca Å 2 of surface buried upon complex formation by two globular proteins Epitopes on protein surface thus may have a hybrid character, compatible with both a solvent-exposed ( free ) state and a buried, solvent-inaccessible ( bound ) state Energetics of binding primarily determined by a few critical residues Flexibility of surface loops may be quite important for promoting adaptive binding and for allowing high specificity interactions without overly-tight binding (via free state entropy effects) Most contacts between two proteins at the interface involve amino acid side chains, although there are some backbone interactions

4 Formalisms for Characterizing Binding Affinities For a protein (P), ligand (A), and complex (P A) P + A k a k d P A where [P] total = [P] + [P A] The association constant: K a = [P A]/[P][A] = k a /k d The dissociation constant: K d = 1/K a = [P][A]/[P A] please note that K d has units of concentration, and so when K d = [A] then [P] = [P A], and thus K d is equal to the concentration of the ligand A at the point of half-maximal binding.

5 At a given ligand concentration [A] the free energy of binding, in terms of the difference in free energies between the free and the bound states, can be described as ΔG binding = -RT ln ([A]/K d ) It is also often useful to describe the difference in binding affinity between a wild type protein and a mutant of the same protein, which is an intrinsic property independent of the ligand concentration. In that case we can express this as ΔΔG wt-mut = -RT ln (K d mut /K d wt )

6 Mapping Protein-Protein Interactions Using Alanine-Scanning Mutagenesis

7 If amino acids had personalities, alanine would not be the life of the party! - George Rose Johns Hopkins Univ.

8 Auguste Rodin The Kiss 1886 (100 Kb); Bronze, 87 x 51 x 55 cm; Musee Rodin, Paris

15 Clackson et al (1998) J. Mol. Biol. 277, 1111.

16 Most mutations that markedly affect the binding affinity (K a ) do so by affecting the off-rate (k d or k off ). In general, mutational effects on the on-rate (k a or k on ) are limited to the following circumstances: Long-range electrostatic effects (steering) Folding mutations masquerading as affinity mutations (i. e., mutations that shift the folding equilibrium to the non-native [and non-binding] state) Inadvertent creation of alternative binding modes that compete with the correct binding mode

17 Cunningham & Wells (1993) J. Mol. Biol. 234, 554.

18 Cunningham & Wells (1993) J. Mol. Biol. 234, 554.

19 Alanine Shaving (a. k. a., Mohawk Mutagenesis ) Clackson et al (1998) J. Mol. Biol. 277, 1111.

22 Quiz: When we say we are comparing the mutant with wt, what does wt mean?

23 (just kidding!)

Seriously though, the reference molecule here is: Turkey Ovomucoid Third Domain (a Serine Protease Inhibitor) All nineteen possible amino acid substitutions were made for each of the residues shown

24 Seriously though, the reference molecule here is: Turkey Ovomucoid Third Domain (a Serine Protease Inhibitor) All nineteen possible amino acid substitutions were made for each of the residues shown in blue (total = 190). For each inhibitor, binding constants were measured precisely for each of six different serine proteases (>1000 separate measurements!). X-ray structures were performed on a subset of the mutant complexes.

25 3D View of Turkey Ovomucoid Third Domain in Complex with a Cognate Protease Li et al (2005) Proteins: Struct. Funct. Bioinformat. 58, 661.

26 Partial view of the structure of the complex of TKY-OM3D P1 Pro with Streptomyces griseus Protease B Bateman et al (2001) J. Mol. Biol. 305, 839.

27 The Principle of Additivity Consider the double mutant, AB, composed of mutation A and mutation B. In general (but not always -- see below), the binding free energy perturbations caused by single mutations are additive, in other words ΔΔG wt-mutab = ΔΔG wt-muta + ΔΔG wt-mutb + ΔΔG i where ΔΔG i 0. ΔΔG i has been termed the interaction energy (see (Wells [1990] Biochemistry 29, 8509). If ΔΔG i 0, then mutations A and B are said to be nonadditive and it can therefore be inferred that the two residues at which these mutations occur must physically interact, directly or indirectly, in the native structure. Note: this has important implications regarding how evolution shapes proteins.

28 Example of a perfectly* additive pair of mutations in avian ovomucoids *Note: within experimental error Qasim et al (2003) Biochemistry 42, 6460.

29 Predicted vs. observed binding free energies for all possible members of the Kazal (ovomucoid) inhibitor family Laskowski et al (2003) Curr. Opin. Struct. Biol. 13, 130.

30 Residue clustering at a protein-protein interface Reichmann et al (2005) Proc. Natl. Acad. Sci. USA 102, 57.

31 Protein-protein interface clusters and the free energy additivity of mutations Mutations within clusters are generally nonadditive (ΔΔG i 0) Mutations between clusters are generally additive (ΔΔG i 0) Reichmann et al (2007) Curr. Opin. Struct. Biol. 17, 67.

32 Theorists are now beginning to mine the OM3D data to refine their docking programs. Bad prediction Good prediction Lorber et al (2002) Protein Sci. 11, 1393.

Biophysical characterization of proteinprotein

Biophysical characterization of proteinprotein interactions Rob Meijers EMBL Hamburg EMBO Global Exchange Lecture Course Hyderabad 2012 Bottom up look at protein-protein interactions Role of hydrogen bonds