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Shawna Thomas Protein Folding Journal Club May 6, 2008

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NOT an equilibrium between the R and T states ... suggest allosteric coupling due to network of interactions connecting A and B ... – PowerPoint PPT presentation

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Title: Shawna Thomas Protein Folding Journal Club May 6, 2008


1
  • Shawna ThomasProtein Folding Journal ClubMay 6,
    2008

2
In a Nutshell
  • Observation allosteric proteins contain higher
    amounts of ID domains
  • Hypothesis ID provides a mechanism for
    allosteric regulation
  • Method study how ID and binding affect
    conformation probabilities in a 2 domain model
  • Result ID optimizes allosteric coupling in the
    model

3
In a Nutshell
  • Observation allosteric proteins contain higher
    amounts of ID domains
  • Hypothesis ID provides a mechanism for
    allosteric regulation
  • Method study how ID and binding affect
    conformation probabilities in a 2 domain model
  • Result ID optimizes allosteric coupling in the
    model

4
Intrinsic Disorder (ID)
  • Intrinsic disorder occurs when large portions of
    a protein remain unstructured under native
    conditions
  • Why conserved?
  • A side effect of in vitro study rather than in
    vivo study
  • For rapid protein degradation when ligand not
    present1
  • To support high-specificity and low-affinity
    binding2,3
  • To support high- specificity to multiple ligand
    targets1-5
  • Provides a mechanism to mediate allosteric
    interactions

1 Wright PE, Dyson HJ (1999) J Mol Biol
293321-331.2 Uversky VN, Oldfield CJ, Dunker AK
(2005) J Mol Recognit 18343-384.3 Liu J, et al.
(2006) Biochemistry 456873-6888.4 Dunker AK, et
al. (2001) J Mol Graphics Model 1926-59.5
Uversky (2006) Protein Sci 11739-756.
5
Allostery
  • Allostery occurs when the binding of ligand A at
    one site (the allosteric site) affects the
    binding of ligand B at another, distant site (the
    active site)

R (relaxed) state
T (tense) state
allosteric site
active site
6
Allosteric Reponse Types
  • Allosteric activation /positive cooperativity
  • Allosteric inhibition /negative cooperativity
  • (Non-cooperative - independent binding of A and B)

7
Allostery Examples
  • Hemoglobin
  • Responsible for oxygen transport
  • Binding of one oxygen increases the binding
    affinity for the second oxygen1

1 Di Cera, E (2004) Thermodynamic Theory of
Site-Specific Binding Processes in Biological
Macromolecules Cambridge University Press, NY.2
www.3dchem.com/molecules.asp?ID213 (image)
8
Allostery Examples
  • Phosphfructokinase (PFK)
  • Tetramer with 4 allosteric sites and 4 active
    sites on the chain interfaces
  • Binding at one of the allosteric sites alters
    binding affinity at the active sites2
  • Relative allosteric contributions differ between
    sites and species2-4

1 Schirmer T, Evans PR (1990) Nature 343140-145.
(PDB 6PFK)2 Ortigosa AD, Kimmel JL, Reinhart GD
(2004) Biochemistry 43577-586.3 Fenton AW,
Paricharttanakul NM, Reinhart GD (2004)
Biochemistry 4314104-14110.4 Paricharttanakul
NM, et al. (2005) Biochemistry 4415280-15286.
9
Traditional Allosteric Models
T state
  • Monod-Wyman-Changeux (MWC) Model1
  • concerted model or symmetry model
  • Equilibrium between the R and T states
  • Ligand presence/binding shifts the equilibrium
  • Conformation change of domains NOT independent
  • Equilibrium change explains allosteric response

R state
1 Monod J, Wyman J, Changeux P (1965) J Mol Biol
1288-118.
10
Traditional Allosteric Models
T state
  • Koshland-Nemethy-Filmer (KNF) Model1
  • sequential model or induced-fit model
  • Conformational change of the domains are
    independent
  • Ligand binding in one domain forces conformation
    change in the other domain
  • NOT an equilibrium between the R and T states
  • Conformation change of one domain explains
    allosteric response

R state
1 Koshland JDE, Nemethy G, Filmer D (1966)
Biochemistry 5365-385.
11
Traditional Allosteric Models
  • General allosteric model1
  • (combining MWC and KNF)

1 Wyman J (1972) Curr Top Cell Regul 6207-223.
12
Allostery and ID
  • Existing models do not use ID directly
  • Cell signaling proteins and transcription factors
    contain disproportionately high levels of ID
    domains1
  • Structure formation often occurs upon ligand
    binding
  • Hypothesis ID provides a mechanism for
    allosteric regulation

1 Liu J, et al. (2006) Biochemistry 456873-6888.
13
In a Nutshell
  • Observation allosteric proteins contain higher
    amounts of ID domains
  • Hypothesis ID provides a mechanism for
    allosteric regulation
  • Method study how ID and binding affect
    conformation probabilities in a 2 domain model
  • Result ID optimizes allosteric coupling in the
    model

14
Two Domain Model
  • Rules/Assumptions
  • Ligand A only binds to domain I
  • Ligand B only binds to domain II
  • Domains may be independently (completely) folded
    or (completely) unfolded
  • Binding occurs only then the domain is completely
    folded

15
Two Domain Model
  • 4 possible states N, 1, 2, and U

Energy free energy of unfolding each domain
energy of breaking interactions between them
(Dgint)
16
Two Domain Model
Q 1 KIIfint KIfint KIKIIfint fint
exp(-Dgint/RT) KI exp(-DGI/RT) KII
exp(-DGII/RT)
17
Effect of Dgint
  • Domain coupling occurs when Dgint ? 0
  • Dgint gt 0
  • Energetically unfavorable to break interaction
  • E.g., complementary hydrophobic surfaces
  • Dgint lt 0
  • Energetically favorable to break interaction
  • E.g., complementary hydrophobic surfaces at low T

18
Exploring Dgint by Perturbation
  • Idea see how domains are coupled by perturbing
    one and observing the effect on the other
  • Perturbation operations
  • Mutation, ionization, or other modification of a
    residue
  • Ligand binding
  • How does binding at 1 site affect the
    probability/ability to bind at another site?

19
Effect of Ligand A Binding
  • Ligand A can only bind to domain I in states 1
    And N

Q 1 KIIfint KIfint KIKIIfint Q ZLig,A
ZLig,AKIIfint KIfint KIKIIfint ZLig,A 1
Ka,AA Ka,A intrinsic association constant of
domain I for ligand A
20
Effect of Ligand A Binding
  • Binding ligand A redistributes the ensemble
    probabilities

PB,Folded (1 KIfint) / (1 KIIfint KIfint
KIKIIfint) PB,Folded (ZLig,A KIfint) /
(ZLig,A ZLig,AKIIfint KIfint KIKIIfint)
21
Effect of Ligand A Binding
  • Effect of ZLig,A depends of DGI, DGII, and Dgint

PB,Folded (ZLig,A KIfint) / (ZLig,A
ZLig,AKIIfint KIfint KIKIIfint)
(DGI -2.3 kcal/mol, DGII -0.7 kcal/mol, Dgint
1.6 kcal/mol, DgLig,A -3.0 kcal/mol)
22
Allosteric Coupling Response
  • The degree to which the probability of binding B
    is affected by perturbing the states that can
    bind A
  • CR (DPB,Folded) / (DlnZLig,A)
  • Measures the sensitivity of site B to
    perturbations to site A (such as ligand binding)

23
In a Nutshell
  • Observation allosteric proteins contain higher
    amounts of ID domains
  • Hypothesis ID provides a mechanism for
    allosteric regulation
  • Method study how ID and binding affect
    conformation probabilities in a 2 domain model
  • Result ID optimizes allosteric coupling in the
    model

24
Where is CR Maximal?
  • All combinations with CR 0.10
  • 2 distinct regions
  • Many values give a high CR
  • Significant Dgint is required (either or -)

25
Where is CR Maximal?
  • For CR 0.07, 0.10, and 0.15
  • 2 regions of maximal CR when one or both domains
    are ID
  • Region 1 negative coupling
  • Region 2 positive coupling

P( )
26
Where is CR Maximal?
  • Different than traditional views that suggest
    allosteric coupling due to network of
    interactions connecting A and B

P( )
27
Extending to 3 Domains
  • For CR gt 0.07
  • CR still maximal when one or many domains are ID

28
Effect of Binding Affinity on CR
  • Maximal CR for low (yellow), moderate (orange),
    and high (red) affinity binding of A
  • Even in rare cases where affinity is low
    (yellow), ID is still expected 50 of the time

29
Effect of Binding Affinity on CR
  • Where equilibrium is poised before adding A
    depends on how much binding energy is available
    to the system
  • Greater binding affinities and/or larger expected
    concentration changes yield more ID

30
Relation to Classical Allostery Models
  • This ID model is most similar to KNF/induced-fit
  • However,
  • Does not impose symmetry like MWC (affinities of
    ligands unrelated)
  • Observed coupling is a consequence of the
    intrinsic stabilities of the domains and their
    interaction, not part of the binding energy like
    KNF

31
Conformational FluctuationsID
  • Conformational fluctuations around the native
    state may play a similar role as ID in allosteric
    coupling
  • Fluctuations can be thermodynamically well
    represented as local order/disorder
    transitions1-3
  • Fluctuations often involve active sites1-3
  • Allosteric coupling energies correlate with
    probes of the regional structural stability4

1 Babu CR, Hilser VJ, Wand AJ (2004) Nat Struct
Mol Biol 11352-357.2 Ferreon JC, Hamburger JB,
Hilser VJ (2004) J Am Chem Soc 12612774-12775.3
Whitten ST, Garcia-Moreno EB, Hilser VJ (2005)
Proc Natl Acad Sci USA 1024282-4287.4 Gekko K,
Obu N, Li J, Lee JC (2004) Biocheistry
433844-3852.
32
Impact on Evolutionary View
  • Model demonstrates that coupling is a result of
    the energetic balance within the protein
  • Allosteric coupling is not brittle
  • Changing the stability in one domain may be
    compensated by changing the other domain or the
    interactions between them
  • Sites may be coupled without requiring a specific
    network of interactions/pathways between the sites

33
In a Nutshell
  • Observation allosteric proteins contain higher
    amounts of ID domains
  • Hypothesis ID provides a mechanism for
    allosteric regulation
  • Method study how ID and binding affect
    conformation probabilities in a 2 domain model
  • Result ID optimizes allosteric coupling in the
    model

34
  • Shawna ThomasProtein Folding Journal ClubMay 6,
    2008
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