From the single proteins to protein

complexes

Protein-Protein interactions (PPIs)

 Protein-Protein interaction (PPI)
Questions:
i) Which are the interacting structures and the interaction areas
in proteins ?
ii) Which aminoacids are involved ?
iii) How the interacting areas determine the strength and the
stability of the interaction ?

Objectives:
• definining PPIs
• being able to identify PPIs and to
• devise strategies to study protein complexes
 Definition of PPIs

Commonly they are understood as physical contacts with molecular docking between
proteins that occur in a cell or in a living organism in vivo. As discussed previously [5,6],
the issue of whether two proteins share a ‘‘functional contact’’ is quite distinct from the
question of whether the same two proteins interact directly with each other. Any protein
in the ribosome or in the basal transcriptional apparatus shares a functional contact with
the other proteins in the complex, but certainly not all the proteins in the particular
complex interact. Indubitably, the existence of many other types of functional links
between biomolecular entities (genes, proteins, metabolites, etc.) in living organisms
should not be confused with protein physical interactions. Investigating these functional
links requires different experimental techniques designed to find such specific types of
relationships, for example, double mutant synthetic lethality to find genetic interactions
[7] or transcriptome expression profiling to find gene co-expression [8]. Identification of
other types of protein interactions (protein–DNA, protein–RNA, protein–cofactor, or
protein–ligand) is also important for a comprehensive study of the interactome, but again
these types of data should not be mixed or confused with PPI data. The physical contact
considered in PPIs should be specific, not just all proteins that bump into each other by
chance. It also should exclude interactions that a protein experiences when it is being
made, folded, quality checked, or degraded.
 Proteins in the complex interact
directly only in pairs;
we see that protein circle and
protein triangle do not interact
directly
But…
Only when the 3 proteins are
associated, the function is
activated, therefore the 3 share a
functional contact

Only when the 3 proteins are

associated, the function is
activated, therefore the 3
share a functional contact
and… all
 Definition of PPIs

For example, all proteins at one point ‘‘touch’’ the ribosome, many touch chaperones, and
most make contact with the degradation machinery. In many experimental assays, such
generic interactions are rightfully filtered out. Therefore, the definition of PPI has to
consider (1st) the interaction interface should be intentional and not accidental, i.e., the
result of specific selected biomolecular events/forces; and (2nd) the interaction interface
should be non-generic, i.e., evolved for a specific purpose distinct from totally generic
functions such as protein production, degradation, and others. That PPIs imply physical
contact between proteins does not mean that such contacts are static or permanent. The
cell machinery undergoes continuous turnover and reassembly. Some protein assemblies
are stable because they constitute macromolecular protein complexes and cellular
machines, for example ATP synthase (eight different proteins in mammals) or cytochrome
oxidase (13 proteins in mammals). These proteins included in complexes are called
‘‘subunits’’. Other protein assemblies are only built to carry out transient actions, for
example, the activation of gene expression by the binding of transcription factors and
activators on the DNA promoter region of a gene. Another essential element for defining
PPIs is the biological context. Not all possible interactions will occur in any cell at any time.
Instead, interactions depend on cell type, cell cycle phase and state, developmental stage,
environmental conditions, protein modifications (e.g., phosphorylation), presence of
cofactors, and presence of other binding partners.
Protein-Protein interaction (PPI)
How many kinds of Protein-Protein Interactions
consortia in Nature?
How many kinds of Protein-Protein Interactions
consortia in Nature?
The protein complex
 The protein complex

Crystal structure of a complex displaying the hot regions between two M chains of the human muscle L-lactate
dehydrogenase (PDB ID: 1i10). Two interacting chains are shown in yellow and cyan. The hot spot residues (red) are
shown in ball and stick representation. There are two hot regions in this interface of the homodimer. The figure
illustrates that hot spots are in contact with each other and form a network of interactions forming hot regions. The
bottom hot region is composed of residues Ser183, Val205, Val179, Gly178 from Chain C and Val269 and Ile293 from
Chain A of the complex.
Observations: the contact points are relatively small in comparison to the area exposed by each protein partner.
How many kinds of Protein-Protein Interactions
consortia in Nature?
Interface
Interface
Interface
Propensity of aminoacids for the location at the
interface of a PPI

No clear AA propensity for the interface

Frequency of occurrence of AA at the contact
points
Frequency of occurrence of AA at the contact
points
 Propensity of aminoacids for the interaction surface:
is that enough ?
The frequency for an aminoacid to be found at the contact point between two
protein partner shows preferences for hydrophobic residues.
Aromatic residues that can form pi-pi interactions with a likewise aromatic
residues present on the protein partner are also quite frequent, indeed the pi-pi
is a way to stabilize the complex.
Cysteine is quite frequent.
Infrequent residues are also evidenced by the research.
Note that it was observed that the prediction of aminoacid frequency to define
hot spots can be not sufficient to accurately identify a contact area.

The question is: is there another characteristic that we can use to define which
areas of a protein might be involved in PPIs?
Accessible surface area (ASA)
Accessible surface area (ASA)
Accessible surface area (ASA)
Accessible surface area (ASA)
Accessible surface area (ASA)

Protein-protein interactions occurs at the interface.

PP Interface definition and the identification of the
interface are based on the concept of solvent
accessible surface area (ASA).

The ASA describes the extent to which a protein can

form contacts with water (Fig. A).
The ASAs of the dimers (Fig. B) are normally
calculated using an implementation of the Lee and
Richards (1971) algorithm developed by Hubbard
(1992).
With a probe sphere of radius 1.4 Å the accessible
surface was defined as the surface mapped out by
the centre of the probe as if it was rolled around the
van der Waals surface of the protein.
Accessible surface area (ASA)
Accessible surface area (ASA)
Buried surface area (BSA) in PP complexes
Buried surface area (BSA) in PP complexes
How structure defines affinity in protein-protein
interactions.
Dependence of Kd on various single biophysical
features.
(A) Change in the accessible interface surface
area (ASA); (B) ΔASA normalized to the total
interface area; (C) percent of non-polar change
in the accessible surface area; (D) the total
number of interfacial H bonds, (E) the number
of intermolecular interfacial H bonds, (F) the
number of intra-molecular H bonds; (G) Van der
Waals energy; (H) volume of cavities; (I) number
of hotspots; (J) electrostatic columbic energy.
Each point represents one PDB file in the
database and the line corresponds to a linear fit
to all data points in the database.
How structure defines affinity in protein-protein
interactions.

(A) Amino acid propensities to

be in an interface compared
to protein surface

(B) Amino acid propensities for

high-affinity (black) and
low-affinity (grey)
complexes.
 Surface buried area and affinity

(A) Scatter plot showing the relationship between the logarithm (base 10) of dissociation constant (log Kd) and the buried surface
area in Å2 of 113 heterodimers. The blue line is the linear least-squares regression fit of the data with slope corresponds to a free
energy change per unit area of 1.6 cal mol−1 Å−2 (Spearman's correlation ρ = −0.53, P value = 2.5E-9; R2 = 0.25), indicating that as
buried surface area increases, the affinity increases (i.e. Kd decreases). Each point represents a co-crystal structure of a
heterodimer. Protein-peptide complexes, which contain a component polypeptide chain with less than 20 amino acids, are denoted
by magenta crosses while all other protein-protein complexes are black circles. Horizontal red lines indicate 1 mM, 1 μM, and 1 nM
dissociation constants for calibration of the log Kd values. The cyan lines are the 90% predicted interval for each new predicted value
(or ‘predicted interval’). For comparison, we also computed the slopes for the protein-peptide and protein-protein complexes. They
correspond to a free energy change per unit area of 1.6 cal mol-1 Å−2 (Spearman s correlation ρ = -0.39, P value = 0.0039; R2 = 0.15)
for the protein-peptide complexes and 0.91 cal mol-1 é-2 (ρ = −0.24, P value = 0.062; R2 = 0.058) for the protein-protein complexes.
 Surface buried area and affinity

(B) Scatter plot showing the relationship between the free energy change per Å−2, or ‘surface
energy density’, and the buried surface area in Å2 of 113 heterodimers. It shows two regimes:
below 2000 Å2, energy density decreases with increasing area; above 2000 Å2, a plateau is
observed. As in 1A, each point represents a co-crystal structure of a heterodimer, protein-peptide
complexes (as defined previously) are denoted by magenta crosses and all other protein-protein
complexes are black circles.