Advanced BioinformaticsLecture 3: Protein-protein interaction

ZHU FENGzhufeng@cqu.edu.cn

http://idrb.cqu.edu.cn/Innovative Drug Research Centre in CQU

创新药物研究与生物信息学实验室

1. Protein-protein interaction

2. Interaction representations

3. Method A: Two-hybrid assay

4. Method B: Affinity purification

5. Spoke and matrix models of PPI

Table of Content

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The horseshoe shaped ribonuclease inhibitor (shown as wireframe) forms a protein–protein interaction with the ribonuclease protein. The contacts (non-covalent interaction) between the two proteins are shown as colored patches.

Protein–protein interaction (PPI)

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Central importance for processes in cell

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Signal transduction: signals from the exterior of a cell are

mediated inside by PPI of the signaling molecules.

Protein transportation: from cytoplasm to nucleus or vice

versa in the case of the nuclear pore importins.

Protein modification: a protein kinase will add a phosphate to

a target protein.

Chain interaction: proteins with SH2 domains only bind to

other proteins when they are phosphorylated on the amino acid

tyrosine while specifically recognize acetylated lysines.

Enzyme + Substrate

Kinase-ATP complex + inactive-enzyme ==> Kinase + ADP + active enzyme

K

ATP ADP

P

One representation

Interaction representation

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Interaction representation

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Kinase-ATPcomplex

Activeenzyme

Inactiveenzyme

ADP

Another representation

B

C

A

D E F…

Generalization of the representation

A biomolecule’s function can be defined by the things that it interacts with and the new (or altered) molecules that result from that interaction.

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Makes it easy to focus on the interaction part

BA

A simple record

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The minimal record has 10 pieces of information

01. Short label for A 02. Short label for B03. Molecule type for A 04. Molecule type for B05. Database reference for A 06. Database reference for B07. Where A comes from 08. Where B comes from09. Interaction Kinetics10. Publication reference

BA

An example record

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You can view this record in BIND (http://bind.ca/) with ID: 263509

01. EGF 02. EGFR03. Protein 04. Protein05. OMIM: 131530 06. OMIM: 13155007. Homo sapiens 08. Homo sapiens09. Equilibrium dissociation constant (Kd) = 130 nM10. Cancer Cell 7(4):301-311, 2005

BIND stores molecular interaction data

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Specify method used to confirm the interaction, what method?

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BIND interaction types

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Methods for detecting interactions

Many interactions in BIND originates from high-throughput experiments designed to detect interactions between proteins

The most common methods are

– Two-hybrid assay– Affinity purification

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Methods comparison

Yeast two hybrid screens allow for interactions between proteins that are never expressed in the same time and place, lowering the specificity, but better indicate non-specific tendencies towards sticky interactions

Affinity purification better indicates functional in vivo protein-protein interactions.

Method A: Two-hybrid assay

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1.

2. 3.

4.

Fields S, et al. Nature. 1989 Jul 20;340(6230):245-6.

DNA-binding domain (BD)

Transcription activation domain (AD)

Transcriptional activator (TA)

Promoter

Gene

1.

2. 3.

4.

A

B

UASG

SNF1

SNF4

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Two-hybrid assay

Reporter Gene

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Two-hybrid assay

Two-hybrid assay

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1.

2. 3.

4.

A

Does the DBD-fusion have activity by itself?

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Some two-hybrid caveats

1.

2. 3.

4.

A

B

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Some two-hybrid caveats

C

Is the ‘interaction’ mediated by some other protein?

1.

2. 3.

4.

A

B

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Some two-hybrid caveats

Is the ‘interaction’ bi-directional?

A

Protein of interest

Tag modification(e.g. HA/GST/His)

This molecule will bind the ‘tag’

Method B: Affinity purification

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AThe cell

Affinity purification

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Affinity purification

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Lots of other untagged proteins

B

A

Naturally binding protein

Affinity purification

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Ruptured membranes

B

A

Cell extract

B

A

Untagged proteins go through fastest (flow-through)

Affinity purification

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B

A

Affinity purification

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Tagged complexes are slower and come out later (eluate)

B

A Firstly, this is only a representation of observation

You can only tell what proteins are in the eluate

You can’t tell how they are connected

If there is only one other protein present (B), then

its likely that A and B are directly interacting

But, what if I told you that two other proteins (B

and C) were present along with A …B

AC

Some affinity purification caveats

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B

A

Which of these models is correct?The complex described by this experimental result is said to have an unknown topology.

C B

A

C B

A

C

Complex with unknown binding topology

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B

A

C

A

Complexes with unknown stoichiometry

Here’s another possibility?The complex described by this experimental result is also said to have unknown stoichiometry.

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Spoke MatrixPossible ActualTopology

Simple, intuitive, more accurate, but can misrepresent

Theoretical max. no. of interactions, but many FPs

Spoke and matrix models of PPI

Bader GD, et al. Nat Biotechnol. 2002 20(10):991-7.

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Cell PolarityCell Wall Maintenance Cell StructureMitosisChromosome StructureDNA Synthesis DNA RepairUnknownOthers

Synthetic genetic interactions in yeast

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Network of the human interactome

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Each point represents a protein and each line between them is an interaction

Network motifs found in networks

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The feed-forward loop, bi-fan and biparallel are over-represented, whereas feedback loop is under-represented in gene regulatory networks and neuronal connectivity networks.

Yeast interactome project

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Interactome data analysis (1)

Validation of interactome’s coverage and quality

Interactomes are never complete, given the limitations

of experimental methods. For instance, it has been estimated

that typical Y2H screens detect only 25% or so of all interactions

in an interactome.

The coverage of an interactome can be assessed by

comparing it to benchmarks of well-known

interactions that have been found and validated by

independent assays.

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Interactome data analysis (2)

Protein function prediction

Assumption: uncharacterized proteins have similar

functions as their interacting proteins. For example, YbeB

with unknown function was found to interact with ribosomal

proteins and later shown to be involved in translation.

Although such predictions may be based on single

interactions, usually several interactions are found.

Thus, the whole network of interactions can be used to

predict protein functions, given that certain functions

are usually enriched among the interactors.36

Interactome data analysis (3)

Perturbations and disease

The topology of an interactome makes it possible to

predict how a network reacts to the perturbation (e.g.

removal) of nodes (proteins) or edges (interactions).

Mutations of genes (and thus their proteins) can cause

perturbations of networks and thus disease.

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Interactome data analysis (4)

Network structure and modules

The distribution of properties among the proteins of an

interactome has revealed functional modules within a

network that indicate specialized subnetworks.

Such modules can be purely functional, as in a

signaling pathway, or structural, as in a protein

complex. In fact, it is a formidable task to identify

protein complexes in an interactome, given that

typically no affinities are known.

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Projects Q&A!

1. Biological pathway simulation

2. Computer-aided anti-cancer drug design

3. Disease-causing mutation on drug target

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Any questions? Thank you!