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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.

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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 Å2 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 P • A

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

DG°binding = -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

DDG°wt-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.

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

DDG°wt-mutAB = DDG°wt-mutA + DDG°wt-mutB + DDG°i

where DDG°i ≈ 0.

DDG°i has been termed the “interaction energy” (see (Wells

[1990] Biochemistry29, 8509). If DDG°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.

Qasim et al (2003)

Biochemistry42, 6460.

…and the theorists are now beginning to mine this data

to refine their docking programs.

“Good” prediction

“Bad” 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) Biopolymers45, 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. Recognition12, 3.

Jelesarov and Bosshard (1999) J. Molec. Recognition12, 3.

…and when you have DH and DG

(= -RTlnKa), you can calculate DS.

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.