Biochemistry 412 Protein-Protein Interactions February 22, 2005 Macromolecular
Biochemistry 412 Protein-Protein Interactions February 22, 2005
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! Special Features of Protein-Protein Interfaces Critical for macromolecular recognition Typically, ca. 500 - 1500 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 Formalisms for Characterizing Binding Affinities For a protein (P), ligand (A), and complex (P A) P+A
ka kd PA
where [P]total = [P] + [P A] The association constant: Ka = [P A]/[P][A] = ka/kd The dissociation constant: Kd = 1/Ka = [P][A]/[P A] please note that Kd has units of concentration, and so when Kd = [A]
then [P] = [P A], and thus Kd is equal to the concentration of the ligand A at the point of half-maximal binding. 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 Gbinding = -RT ln ([A]/Kd) 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 Gwt-mut = -RT ln (Kdmut/Kdwt) Mapping Antigen-Antibody Interaction Surfaces (Binding Epitopes)
Using Hydrogen-Deuterium Exchange and NMR Spectroscopy Mapping Protein-Protein Interactions Using Alanine-Scanning Mutagenesis
If amino acids had personalities, alanine would not be the life of the party! - George Rose Johns Hopkins Univ.
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Auguste Rodin The Kiss 1886 (100 Kb); Bronze, 87 x 51 x 55 cm; Musee
Rodin, Paris Clackson et al (1998) J. Mol. Biol. 277, 1111. Most mutations that markedly affect the binding affinity
(Ka) do so by affecting the off-rate (kd or koff). In general, mutational effects on the on-rate (ka or kon) 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
Cunningham & Wells (1993) J. Mol. Biol. 234, 554. Cunningham & Wells (1993) J. Mol. Biol. 234, 554. Clackson et al (1998) J. Mol. Biol. 277, 1111.
Reference Molecule: 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. X-ray structures were performed on a subset
of the mutant complexes. Structure of the complex to TKY-OM3D P1 Pro with Streptomyces griseus Protease B
Bateman et al (2001) J. Mol. Biol. 305, 839. 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 Gwt-mutAB = Gwt-mutA + Gwt-mutB + Gi where Gi 0. Gi has been termed the interaction energy (see (Wells
 Biochemistry 29, 8509). If Gi 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. Qasim et al (2003) Biochemistry 42, 6460.
and the theorists are now beginning to mine this data to refine their docking programs. Bad prediction
Good prediction Lorber et al (2002) Protein Sci. 11, 1393. If you want to be hard core and really
understand protein-protein interactions, you need to know more than just the free energies of association. You (ultimately) will need to know something about enthalpies, entropies, and heat capacities, too.
Makarov et al (1998) Biopolymers 45, 469. Makarov et al (2000) Biophys. J. 76, 2966.
Makarov et al (2002) Acc. Chem. Res. 35, 376. When two proteins form a complex, solvent must be displaced from the interfacial regions and the conformational freedom (configurational
entropy) of the main chain and side chain atoms will change also. Jelesarov and Bosshard (1999) J. Molec. Recognition 12, 3.
Jelesarov and Bosshard (1999) J. Molec. Recognition 12, 3. Isothermal Titration Calorimetry Yields H of Binding and when you have H and G
(= -RTlnKa), you can calculate S. Some examples of experimentally-measured thermodynamic quantities for interacting proteins, measured using isothermal titration calorimetry:
Note: isothermal titration calorimetry also directly yields n, the stoichiometry of binding. Weber and Salemme (2003) Curr. Opin. Struct. Biol. 13, 115.
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