Protein protein interactions
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Protein-Protein Interactions. Genome. Transcriptome. Proteome. Interactome. Atomic NMR spectroscopy X-ray crystallography: co-crystallisation Electron microscopy Molecular modelling: docking  Protein Data Bank. Experimental methods to identify PPI’s. Biochemical/biophysical binary

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Protein-Protein Interactions

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Protein protein interactions

Protein-Protein Interactions


Protein protein interactions

Genome

Transcriptome

Proteome

Interactome


Experimental methods to identify ppi s

Atomic

NMR spectroscopy

X-ray crystallography: co-crystallisation

Electron microscopy

Molecular modelling: docking

 Protein Data Bank

Experimental methods to identify PPI’s


Protein protein interactions

Biochemical/biophysical

binary

yeast two-hybrid (interactions are identified by the transcription of reporter gene)

surface plasmon resonance (SPR) f.e. BIAcore

etc.

multiprotein complexes

co-immunoprecipitation

tagged genes, affinity chromatography

…. followed by mass spectroscopy

 Various databases

Experimental methods to identify PPI’s


Computational methods genomics

correlated mRNA expression (microarray): mostly permanent complexes

homologues known to operate sequentially in metabolic pathways, conserved operons (prokaryotes)

related phylogenetic profiles / correlated evolution

gene fusion: homologues that are fused into a single gene in different organism (Rosetta Stone method)

 Some databases

Computational methods (Genomics)


Computational methods

homologues of proteins known to interact based on experimental data (e.g. 3D structure)

subcellular localisation

Medline abstract search

if biomolecules are cited in the same abstract, they are likely to be functionally related or to physically interact

Computational methods


Protein protein interactions

 Proteomics

 Functional genomics


Ppi paradigms

Localisation: co-expression, subcellular, compartmentalisation

Affinity and concentration

Binary or multimeric (cooperativity)

PPI paradigms


Pdb structural characterisation of different ppi s

PDBStructural characterisation of different PPI’s


Types of pp interactions

Types of PP-interactions

lifetime of

assembly

examples

folding/structure

Arc repressor

homodimer

flavoprotein

heterodimer

permanent

obligate

2-state

intracellular

signalling

enzyme-inhibitor

antibody-antigen

transient/

permanent

non-obligate

3-state


Protein protein interactions

Rho-GAP

(non-obligate)

Arc repressor

(obligate)

Thrombin-inhibitor

(non-obligate)


Datasets from pdb

Crystal contacts of monomers (non-biological)

Homo/heterodimers

Non-obligate/obligate

Transient/permanent

Datasets from PDB


Wheat germ agglutinin

Wright (1990) J. Mol. Biol.215, 635. [PDB-code: 9WGA]

Wheat Germ Agglutinin


Wheat germ agglutinin1

Wright (1990) J. Mol. Biol.215, 635. [PDB-code: 9WGA]

Wheat Germ Agglutinin


Pdb file

Protein Data Bank (PDB):

PDB file


Crystal symmetry

Crystal symmetry


Monomer dimer tetramer

Monomer, dimer, tetramer …?


Crystal properties

Number of chains in Asymmetric Unit (ASU)

for data set of homo-multimers

Crystal properties

Number of chains in ASU

multimer type


Inferring quaternary structure

macromolecular assemblies from PDB

sparse PDB annotation of multimers

Protein Quaternary Structure (PQS) server

http://pqs.ebi.ac.uk (Kim Henrick)

crystal contacts  interfaces

score interfaces:

DASA, solvation free energy of folding, number of salt bridges and di-sulphide bonds

add atom-pair frequencies, statistical pair potential

Inferring Quaternary Structure


Protein protein interactions

cut-off at

-70

cut-off at

856 Å2

Ponstingl et al. (2000) Proteins 41, 47-57


Performance

<Counts for homo-multimers> / <counts for hetero-multimers>

Performance

Contingency table for oligomer classification

classified as

reference


Problems improvements

contact scores - not binding energies

long-range interactions (electrostatics), …

ligand binding in protein-protein interface

different physiological/crystallisation conditions

protein concentration, pH, …

Oligomeric equilibrium

Improve confidence by including sequence conservation

Problems & improvements


Datasets from pdb1

Crystal contacts of monomers (non-biological

Homo/heterodimers

Non-obligate/obligate

Transient/permanent

Datasets from PDB


Interface properties

Accessible Surface Area (ASA)

Planarity

Protrusion

Hydrophobicity

Percentage polarity

Interface properties


Transient interactions

Dataset I: ‘weak’ transient homodimers

Both oligomeric forms populated at physiological conditions; dynamic equilibrium

Dataset II: functionally validated transient heterodimers

Intracellular signalling complexes

Transient interactions


Protein protein interactions

‘weak’ transient interactions have small and flat interfaces:

DASA < 1000 Å2, planarity < 3.5Å


Protein protein interactions

transient homodimer

miscellaneous homodimer

structurally obligate homodimer

60

50

y

t

i

r

a

l

o

p

40

e

g

a

t

n

e

30

c

r

e

p

20

10

0

1000

2000

3000

4000

5000

6000

contact area (A)

2


Protein protein interactions

obligate heterodimer

co-expressed

co-localised

intracellular signalling

other heterodimer

} compartimentalised

antibody-antigen

enzyme-inhibitor

extracellular receptor-ligand

60

50

y

t

i

r

a

l

40

o

p

e

g

a

t

n

30

e

c

r

e

p

20

structural rearrangements likely to occur upon complexation

10

0

1000

2000

3000

4000

5000

6000

2

contact area (A)


Interface properties diverse pp interactions

Continuum between a monomer and stable homodimer: weak, transient homodimers can be distinguished from more permanent or obligate homodimers: DASA < 1000 Å2, planarity < 3.5Å, polarity > 25%

The above correlation is less distinct for non-obligate, non-colocalised heterodimers

Co-expressed/co-localised, obligate complexes are generally more hydrophobic than non-obligate complexes (co-translation and co-folding)

PP complexes with interfaces larger than about 1000 Å2 are likely to undergo structural rearrangements upon association (e.g. co-folding homodimers, ‘strong’ transient complex)

Interface properties diverse PP-interactions


Protein protein interactions

effector X

further oligomerisation/

cooperativity

competition

effector X

X = D protein concentration, i.e. level of gene

expression/secretion, degradation, temporary

storage, diffusion (viscocity, steric environment)

X = D pH, D temperature, D ionic strength

X = molecular (cooperative/allosteric) binding i.e. D concentration of metabolite, protein or ions

( e.g. ATP, Ca2+) or covalent modification through enzymatic activity ( e.g. PO4-)

strong transient complexes

weak transient complexes

large interface

small interfaces

CONTINUUM

large D conformation

no/minor D conformation


Homology of ppi s

Homology of PPI’s


Protein protein interactions

Structural data

PPI ?

Homologous sequences (paralogues and orthologues)

residue level

PPI ?

PPI ?

PPI

Experimental data

PPI ?

Homologous sequences (paralogues and orthologues)

protein level

PPI ?

PPI ?


Defining the ppi interface

Defining the PPI interface

Outer Zone

Exposed

accessible in monomer and does not lose asa on complexation

asa in dimer <

asa in monomer

Central Zone

accessible in monomer and inaccessible in dimer

Core

inaccessible in monomer

Central Zone

Outer Zone

Total interface

“Accessible” = relative accessible surface

area of > 5%

Total Interface

Exposed

Surface


Alignments and conservation

Alignments and conservation

Use PSI-Blast alignment

PSI-Blast

NR database

Inclusion E value = 10E-40

Max iterations = 20

1 = highly conserved

Assess significance

of contact conservation

Score conservation

for each position in the alignment

0 = unconserved


Protein protein interactions

Residue conservation, consider:

physical and chemical properties of amino acids

frequency distribution of amino acids observed

-> use PAM matrix similarity score

normalise against redundancy in the alignment


Interface conservation central zone

Interface conservation: Central zone

Significantly conserved

Not significantly conserved


Interface conservation total interface

Interface conservation: Total interface

Significantly conserved

Not significantly conserved


Contact conservation

Strong tendency for biological contacts to be more conserved

Contact conservation

Conserved

Variable


Protein protein interactions

Tertiary structure – function

Quaternary structure – function OR happenstance?


Sequence conservation of weak transient interactions in homologous family

sequence conservation of ‘weak’ transient interactions in homologous family

protein

no of

mean conservation

mean conservation

ratio mean conservation

sequences

score surface

score interface

interface / surface

cro repressor

4

0.78

0.88

1.20

dimerization and SH3-like

14

0.40

0.50

1.32

domain of tox repressor

51

0.60

0.77

1.47

dimerization domain RHR NFkB

IL-8

54

0.61

0.55

1.08

MCP-1

56

0.62

0.62

1.19

a

10

0.99

1.00

1.01

SDF1

SCF

29

0.84

0.92

1.19

CNTF

10

0.76

0.78

0.98

insulin

28

0.81

0.89

1.20

cytochrome c’, type I

1

-

-

-

cytochrome c’, type II

1

-

-

-

b

44

0.57

0.52

0.97

-lactoglobulin

Ecad12

243

0.47

0.44

1.30

LC8

24

0.73

0.85

1.28

lysin

25

0.71

0.81

1.23

galectin I

60

0.45

0.18

0.39

average

0.68

0.71

1.11

other obligate homodimers (Valdar et al.)

0.63

0.79

1.30


Protein protein interactions

human MCP-1 dimer

human interleukin-8 dimer

different interfaces


Protein protein interactions

Lost rest of slides

data management: databases:

total 50-60, specific to species, biological process or experimental methods

BIND, DIP, MINT, etc.

data management: data integration:

figures from Bork review

IntAct database (http//:www.ebi.ac.uk/intact)

OEPS ...


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