1-month Practical Course

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1-month Practical Course Genome AnalysisProtein
Structure-Function Relationships Centre for
Integrative Bioinformatics VU (IBIVU) Vrije
Universiteit Amsterdam The Netherlands www.ibivu.
cs.vu.nl heringa_at_cs.vu.nl
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Protein function
Genome/DNA Transcriptome/mRNA Proteome Metabolo
me Physiome
Transcription factors
Ribosomal proteins Chaperonins
Enzymes
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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)
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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)

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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

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How to arrive at a given function

• Divergent evolution homologous proteins
  proteins have same structure and same-ish
  function
• Convergent evolution analogous proteins
  different structure but same function
• Question can homologous proteins change
  structure (and function)?

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Protein function evolution
Chymotrypsin
Active site (combination of ancestral active site
residues)
Putative ancestral barrel structure
Modern 2-barrel structure
Activity 1000-10,000 times enhanced
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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)

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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)

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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
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Example -- Flavodoxin fold
5(??) fold
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Flavodoxin family - TOPS diagrams (Flores et
al., 1994)
These are four variations of the same basic
topology (bottom) Do you see what is inserted as
compared to the basic topology?
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3
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A TOPS diagram is a schematic representation of a
protein fold
alpha-helix
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2
3
4
5
beta-strand
5
1
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Protein structure evolution
Insertion/deletion of structural domains can
easily be done at loop sites
N C
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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).

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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

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Structural domain organisation can be nasty
Pyruvate kinase Phosphotransferase
b barrel regulatory domain a/b barrel catalytic
substrate binding domain a/b nucleotide binding
domain
1 continuous 2 discontinuous domains
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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

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• The DEATH Domain
• 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.

http//www.mshri.on.ca/pawson
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(No Transcript)
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Globin fold ? protein myoglobin PDB 1MBN
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? sandwich ? protein immunoglobulin PDB 7FAB
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TIM barrel ? / ? protein Triose phosphate
IsoMerase PDB 1TIM
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A fold in ? ? protein ribonuclease A PDB 7RSA
The red balls represent waters that are bound
to the protein based on polar contacts
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(No Transcript)
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434 Cro protein complex (phage) PDB 3CRO
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Zinc finger DNA recognition (Drosophila) PDB
2DRP
..YRCKVCSRVY THISNFCRHY VTSH...
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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.  
  
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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
  
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Leucine zipper (yeast) PDB 1YSA
..RA RKLQRMKQLE DKVEE LLSKN YHLENEVARL...