PROTEIN-PROTEIN INTERACTIONS

Proteinprotein interactions (PPIs) refer to intentional physical contacts established between

two or more proteins as a result of biochemical events and/or electrostatic forces.
In fact, proteins are vital macromolecules, at both cellular and systemic levels, but they rarely act
alone. Diverse essential molecular processes within a cell are carried out by molecular machines
that are built from a large number of protein components organized by their PPIs. Indeed, these
interactions are at the core of the entire interactomics system of any living cell and so,
unsurprisingly, aberrant PPIs are on the basis of multiple diseases, such as CreutzfeldJacob, Alzheimer's disease, and cancer.
PPIs have been studied from different perspectives: biochemistry, quantum chemistry, molecular
dynamics, signal transduction, among others. All this information enables the creation of large
protein interaction networks similar to metabolic orgenetic/epigenetic networks that
empower the current knowledge on biochemical cascades and disease pathogenesis, as well as
provide putative new therapeutic targets.
Factors that regulate protein-protein interactions:

Protein concentration, which in turn are affected by expression levels and degradation
rates;

Protein affinity for proteins or other binding ligands;

Ligands concentrations (substrates, ions, etc.);

Presence of other proteins, nucleic acids, and ions;

Electric fields around proteins.

Occurrence of covalent modifications;

YEAST TWO-HYBRID PRINCIPLE

The development and gradual improvements of the yeast two-hybrid system since the early 90s
revolutionized the way protein interactions could be detected.

Yeast two-hybrid is based on the reconstitution of a functional transcription factor (TF) when
two proteins or polypeptides of interest interact. This takes place in genetically modified yeast
strains, in which the transcription of a reporter gene leads to a specific phenotype, usually growth
on a selective medium or change in the color of the yeast colonies. The most popular reporter
genes are HIS3 to select yeast on a medium lacking histidine, and LacZ to screen yeast in a
colorimetric assay.

Two fusions (hybrids) are constructed between each protein of interest and either the DNA
Binding Domain (DBD) or the Activation Domain (AD) of the TF. The protein fused to the DBD
is referred to as the bait, and the protein fused to the AD as the prey.
Upon interaction between the bait and the prey, the DBD and AD are brought in close proximity
and a functional TF is reconstituted upstream of the reporter gene. The most popular fusions use
the DBD and AD of the yeast TF Gal4. The bacterial protein LexA is also frequently used as a
DBD in combination with Gal4 AD.
As a genetic technique, yeast two-hybrid offers a sensitive and cost-effective mean to test the
direct interaction between two targeted proteins, or to use ones favorite protein as a bait to
screen libraries of proteins fragments prepared from the desired cell types, tissues or entire
organisms. The identity of the interacting partners is then obtained by sequencing the
corresponding plasmids in the selected yeast colonies. Collections of full-length proteins
(ORFeomes) are also becoming available for several species, but they do not cover the entire
proteome yet.
Yeast two-hybrid was rapidly adopted by the scientific community and screens in various species
and research fields already led to dozens of thousands of publications. It remains the method of

choice when it comes to discover novel protein interactions, as reflected by the recently
published literature.
Variations of the yeast two-hybrid were developed to conduct screens in the presence of a cofactor, or an enzyme required for a given post-translational modification of the protein partners.
Other versions allow to screen integral membrane proteins and the technique was adapted to
detect protein-protein interactions in mammalian cells. Finally, yeast n-hybrid protocols were
also devised to screen for novel DNA-protein, RNA-protein and small molecule-protein
interactions.
Yeast two-hybrid screening
This system was firstly described in 1989 by Fields and Song using Saccharomyces cerevisiae as
biological model. Yeast two hybrid allows the identification of pairwise PPIs (binary method) in
vivo, indicating non-specific tendencies towards sticky interactions.
Yeast cells are transfected with two plasmids: the bait (protein of interest fused with the DNAbinding domain of a yeast transcription factor, like Gal4), and the prey (a library of cDNA
fragments linked to the activation domain of the transcription factor. Transcription of reporter
genes does not occur unless bait and prey interact with each other and form a functional
transcription factor. Thus, the interaction between proteins can be inferred by the presence of the
products resultant of the reporter gene expression.
Despite its usefulness, the yeast two-hybrid system has limitations: specificity is relatively low;
uses yeast as main host system, which can be a problem when studying other biological models;
the number of PPIs identified is usually low because some transient PPIs are lost during
purification steps; and, understates membrane proteins, for example. Limitations have been
overcoming by the emergence of yeast two-hybrid variants, such as the membrane yeast twohybrid (MYTH) and the split-ubiquitin system, which are not limited to interactions that occur
in the nucleus; and, the bacterial two-hybrid system, performed in bacteria.

References
1. Fields, S. and Song, O. A novel genetic system to detect protein-protein interactions
(1989) Nature 340, 245-246
2. Naba, A., Reverdy, C., Louvard, D. and Arpin, M. Spatial recruitment and activation of
the Fes kinase by ezrin promotes HGF-induced cell scattering (2008) EMBO J. 27(1), 3850.
3. Stagljar, I., Korostensky, C., Johnsson, N. and te Heesen, S. A genetic system based on
split-ubiquitin for the analysis of interactions between membrane proteins in vivo (1998)
PNAS 95(9), 5187-5192.
4. Eyckerman, S. , Verhee, A., der Heyden, J.V., Lemmens, I., Ostade, X.V.,
Vandekerckhove, J. and Tavernier, J. Design and application of a cytokine-receptor-based
interaction trap (2001) Nat Cell Biol. 3(12), 1114-1119.
5. Li, J.J. and Herskowitz, I. Isolation of ORC6, a component of the yeast origin recognition
complex by a one-hybrid system (1993) Science 262(5141), 1870-1874.
6. Putz, U., Skehel, P. and Kuhl, D. A tri-hybrid system for the analysis and detection of
RNA--protein interactions (1996) Nucleic Acids Res 24, 4838-4840.
7. Licitra, E.J. and Liu, J.O. A three-hybrid system for detecting small ligand-protein
receptor interactions (1996) PNAS 93(23), 12817-12821