Transcript Protein Structure-Function Relationships - IBIVU

C
E
N
T
R
E
F
O
R
I
N
T
E
G
R
A
T
I
V
E
1-month Practical Course
Genome Analysis
B
I
O
I
N
F
O
R
M
A
T
I
C
S
V
U
Protein Structure-Function
Relationships
Centre for Integrative Bioinformatics VU (IBIVU)
Vrije Universiteit Amsterdam
The Netherlands
www.ibivu.cs.vu.nl
[email protected]
Protein function
Genome/DNA
Transcription
factors
Transcriptome/mRNA
Proteome
Ribosomal
proteins
Chaperonins
Metabolome
Enzymes
Physiome
Protein function
Not all proteins are enzymes:
-crystallin: eye lens protein – needs to stay
stable and transparent for a lifetime (very little
turnover in the eye lens)
Protein function groups
• Catalysis (enzymes)
• Binding – transport (active/passive)
– Protein-DNA/RNA binding (e.g. histones, transcription
factors)
– Protein-protein interactions (e.g. antibody-lysozyme)
– Protein-fatty acid binding (e.g. apolipoproteins)
– Protein – small molecules (drug interaction, structure
decoding)
• Structural component (e.g. -crystallin)
• Regulation
• Transcription regulation
• Signalling
• Immune system
• Motor proteins (actin/myosin)
What can happen to protein
function through evolution
Proteins can have multiple functions (and sometimes
many -- Ig).
Enzyme function is defined by specificity and
activity
Through evolution:
• Function and specificity can stay the same
• Function stays same but specificity changes
• Change to some similar function (e.g. somewhere
else in metabolic system)
• Change to completely new function
How to arrive at a given function
• Divergent evolution – homologous proteins
–proteins have same structure and “sameish” function
• Convergent evolution – analogous proteins
– different structure but same function
• Question: can homologous proteins change
structure (and function)?
Protein function evolution
Active site
Chymotrypsin
(combination
of ancestral
active site
residues)
Putative ancestral
barrel structure
‘Modern’ 2-barrel structure
Activity 1000-10,000 times enhanced
How to evolve
Important distinction:
• Orthologues: homologous proteins in different
species (all deriving from same ancestor)
• Paralogues: homologous proteins in same species
(internal gene duplication)
• In practice: to recognise orthology, bi-directional
best hit is used in conjunction with database
search program (this is called an operational
definition)
How to evolve
By addition of domains (at either end of protein
sequence or at loop sites [see next slides])
Often through gene duplication followed by
divergence
Multi-domain proteins are a result of gene fusion
(multiple genes ending up in a single ORF).
Repetitions of the same domain in a single protein
occur frequently (gene duplication followed by
gene fusion)
Protein structure evolution
Insertion/deletion of secondary structural
elements can ‘easily’ be done at loop sites
These sites are normally
at the surface of a protein
Example -- Flavodoxin fold
Flavodoxin family - TOPS diagrams
These are four
variations of
the same basic
topology
(bottom)
(Flores et al., 1994)
Do you see
what is
inserted as
compared to
the basic
topology?
4
= alpha-helix
= beta-strand
5
4
5
3 2
3
1
1
2
A TOPS diagram is a
schematic
representation of a
protein fold
Protein structure evolution
Insertion/deletion of structural domains can
‘easily’ be done at loop sites
N
C
The basic functional unit of a
protein is the domain
A domain is a:
• Compact, semi-independent unit
(Richardson, 1981).
• Stable unit of a protein structure that can
fold autonomously (Wetlaufer, 1973).
• Recurring functional and evolutionary
module (Bork, 1992).
“Nature is a ‘tinkerer’ and not an inventor” (Jacob, 1977).
Delineating domains is essential for:
•
•
•
•
•
•
•
•
Obtaining high resolution structures (x-ray, NMR)
Sequence analysis
Multiple sequence alignment methods
Prediction algorithms (SS, Class, secondary/tertiary
structure)
Fold recognition and threading
Elucidating the evolution, structure and function of
a protein family (e.g. ‘Rosetta Stone’ method – next
lecture)
Structural/functional genomics
Cross genome comparative analysis
Structural domain organisation can be nasty…
Pyruvate kinase
Phosphotransferase
 barrel regulatory domain
/ barrel catalytic substrate binding
domain
/ nucleotide binding domain
1 continuous + 2 discontinuous domains
Complex protein functions are a
result of multiple domains
• An example is the so-called swivelling domain in
pyruvate phosphate dikinase (Herzberg et al.,
1996), which brings an intermediate enzymatic
product over about 45 Å from the active site of
one domain to that of another.
• This enhances the enzymatic activity: delivery of
intermediate product not by a diffusion process but
by active transport
The DEATH Domain
http://www.mshri.on.ca/pawson
• Present in a variety of Eukaryotic
proteins involved with cell death.
• Six helices enclose a tightly
packed hydrophobic core.
• Some DEATH domains form
homotypic and heterotypic dimers.
Globin fold
 protein
myoglobin
PDB: 1MBN
 sandwich
 protein
immunoglobulin
PDB: 7FAB
TIM barrel
 /  protein
Triose
phosphate
IsoMerase
PDB: 1TIM
A fold in
 + protein
ribonuclease A
PDB: 7RSA
The red balls
represent
waters that
are ‘bound’
to the protein
based on
polar
contacts
434 Cro
protein
complex
(phage)
PDB: 3CRO
Zinc finger
DNA recognition
(Drosophila)
PDB: 2DRP
..YRCKVCSRVY THISNFCRHY VTSH...
Zinc-finger DNA binding protein family
Characteristics of the family:
Function:
The DNA-binding motif is found as part of
transcription regulatory proteins.
Structure:
One of the most abundant DNA-binding motifs.
Proteins may contain more than one finger in a
single chain. For example Transcription Factor
TF3A was the first zinc-finger protein discovered
to contain 9 C2H2 zinc-finger motifs (tandem
repeats). Each motif consists of 2 antiparallel
beta-strands followed by by an alpha-helix. A
single zinc ion is tetrahedrally coordinated by
conserved histidine and cysteine residues,
stabilising the motif.
Zinc-finger DNA binding protein family
Characteristics of the family:
Binding:
Fingers bind to 3 base-pair subsites and specific
contacts are mediated by amino acids in positions 1, 2, 3 and 6 relative to the start of the alpha-helix.
Contacts mainly involve one strand of the DNA.
Where proteins contain multiple fingers, each
finger binds to adjacent subsites within a larger
DNA recognition site thus allowing a relatively
simple motif to specifically bind to a wide range of
DNA sequences.
This means that the number and the type of zinc
fingers dictates the specificity of binding to DNA
Leucine zipper
(yeast)
PDB: 1YSA
..RA RKLQRMKQLE DKVEE LLSKN YHLENEVARL...