Essential Bioinformatics and Biocomputing (LSM2104: Section I) Biological Databases and Bioinformatics Software Prof. Chen Yu Zong Tel: [phone] Email: [e-mail] http://xin.cz3.nus.edu.sg Room 07-24, level 7, SOC1, NUS January 2003

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Advanced Bioinformatics Lecture 3
Protein-protein interaction
ZHU FENG zhufeng_at_cqu.edu.cn http//idrb.cqu.edu.cn
/ Innovative Drug Research Centre in CQU
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Table of Content

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

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Proteinprotein interaction (PPI)
The horseshoe shaped ribonuclease inhibitor
(shown as wireframe) forms a proteinprotein
interaction with the ribonuclease protein. The
contacts (non-covalent interaction) between the
two proteins are shown as colored patches.
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Central importance for processes in cell

• 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.

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Interaction representation
Enzyme Substrate Kinase-ATP complex
inactive-enzyme gt Kinase ADP active enzyme
K
P
ATP
ADP

• One representation

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Interaction representation
Kinase-ATPcomplex
Inactiveenzyme
Activeenzyme
ADP
Another representation
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Generalization of the representation
B
A

C
D
E
F
A biomolecules function can be defined by the
things that it interacts with and the new (or
altered) molecules that result from that
interaction.
Makes it easy to focus on the interaction part
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A simple record
B
A
01. Short label for A 02. Short label for B03.
Molecule type for A 04. Molecule type for B 05.
Database reference for A 06. Database reference
for B07. Where A comes from 08. Where B comes
from 09. Interaction Kinetics 10. Publication
reference
The minimal record has 10 pieces of information
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An example record
B
A
01. EGF 02. EGFR03. Protein 04. Protein 05.
OMIM 131530 06. OMIM 13155007. Homo
sapiens 08. Homo sapiens 09. Equilibrium
dissociation constant (Kd) 130 nM 10. Cancer
Cell 7(4)301-311, 2005
You can view this record in BIND
(http//bind.ca/) with ID 263509
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BIND stores molecular interaction data
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BIND interaction types
Specify method used to confirm the interaction,
what method?
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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.

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Method A Two-hybrid assay
Transcription activation domain (AD)
1.
Transcriptional activator (TA)
3.
2.
Gene
Promoter
DNA-binding domain (BD)
4.
Fields S, et al. Nature. 1989 Jul
20340(6230)245-6.
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Two-hybrid assay
SNF4
1.
B
SNF1
A
3.
2.
UASG
Reporter Gene
4.
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Two-hybrid assay
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Two-hybrid assay
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Some two-hybrid caveats
1.
A
3.
2.
4.
Does the DBD-fusion have activity by itself?
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Some two-hybrid caveats
1.
B
C
A
3.
2.
4.
Is the interaction mediated by some other
protein?
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Some two-hybrid caveats
1.
B
A
3.
2.
4.
Is the interaction bi-directional?
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Method B Affinity purification
This molecule will bind the tag
A
Tag modification(e.g. HA/GST/His)
Protein of interest
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Affinity purification
The cell
A
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Affinity purification
Lots of other untagged proteins
A
B
Naturally binding protein
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Affinity purification
Ruptured membranes
A
B
Cell extract
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Affinity purification
A
B
Untagged proteins go through fastest
(flow-through)
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Affinity purification
A
B
Tagged complexes are slower and come out later
(eluate)
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Some affinity purification caveats
A
Firstly, this is only a representation of
observation You can only tell what proteins are
in the eluate You cant tell how they are
connectedIf 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
A
C
B
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Complex with unknown binding topology
A
A
A
B
C
B
C
B
C
Which of these models is correct? The complex
described by this experimental result is said to
have an unknown topology.
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Complexes with unknown stoichiometry
A
A
B
C
Heres another possibility? The complex described
by this experimental result is also said to have
unknown stoichiometry.
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Spoke and matrix models of PPI
Possible Actual Topology
Spoke
Matrix
Simple, intuitive, more accurate, but can
misrepresent
Theoretical max. no. of interactions, but many FPs
Bader GD, et al. Nat Biotechnol. 2002
20(10)991-7.
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Synthetic genetic interactions in yeast
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Network of the human interactome
Each point represents a protein and each line
between them is an interaction
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Network motifs found in networks
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.
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Yeast interactome project
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Interactome data analysis (1)

• Validation of interactomes 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.

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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 QA!
Biological pathway simulation
2. Computer-aided anti-cancer drug design
3. Disease-causing mutation on drug target
Any questions? Thank you!
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