Biochemistry 412

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Biochemistry 412 Protein-Protein
Interactions February 22, 2005
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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!
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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
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Formalisms for Characterizing Binding Affinities
For a protein (P), ligand (A), and complex (P
A) P A P A where Ptotal P
P A
ka
kd
The association constant Ka P A/PA
ka/kd
The dissociation constant Kd 1/Ka
PA/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.
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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 DGbinding -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 DDGwt-mut
-RT ln (Kdmut/Kdwt)
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Mapping Antigen-Antibody Interaction Surfaces
(Binding Epitopes) Using Hydrogen-Deuterium
Exchange and NMR Spectroscopy
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Mapping Protein-Protein Interactions Using
Alanine-Scanning Mutagenesis
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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
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Clackson et al (1998) J. Mol. Biol. 277, 1111.
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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
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Cunningham Wells (1993) J. Mol. Biol. 234, 554.
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Cunningham Wells (1993) J. Mol. Biol. 234, 554.
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Clackson et al (1998) J. Mol. Biol. 277, 1111.
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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.
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Structure of the complex to TKY-OM3D P1 Pro
with Streptomyces griseus Protease B
Bateman et al (2001) J. Mol. Biol. 305, 839.
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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 DDGwt-mutAB
DDGwt-mutA DDGwt-mutB DDGi where
DDGi 0. DDGi has been termed the
interaction energy (see (Wells 1990
Biochemistry 29, 8509). If DDGi ? 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.
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Qasim et al (2003) Biochemistry 42, 6460.
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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.
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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, entropie
s, and heat capacities, too.
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Makarov et al (1998) Biopolymers 45, 469.
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Makarov et al (2000) Biophys. J. 76, 2966.
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Makarov et al (2002) Acc. Chem. Res. 35, 376.
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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.
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Jelesarov and Bosshard (1999) J. Molec.
Recognition 12, 3.
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Jelesarov and Bosshard (1999) J. Molec.
Recognition 12, 3.
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Isothermal Titration Calorimetry Yields DH of
Binding
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and when you have DH and DG ( -RTlnKa), you can
calculate DS.
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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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