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8/8/2019 Presentation Bis
1/20
Surface Sites for Engineering
Allosteric Control in Proteins
Jeeyeon Lee, et al., Science 2008
Francesco Di Giacomo
Shuang Ma
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Background
PAGE 117-1-2011
Instead of local interactions, protein
function also depends on nonlocal,
long-range communication between
amino acids.
K. Henzler-Wildman Nature 450, 964 (2007)
Expectation: The above features are universal rather than idiosyncratic
in protein family
Examples: information transmission;
allosteric regulation in enzyme catalysis;
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Technique
PAGE 2
17-1-2011
Statistical coupling analysis (SCA)
Basic principle: Quantifies how much the amino acid distribution at
some position ichanges upon a perturbation of the amino acid
distribution at another position j
The resulting statistical coupling energyGstat indicates
the degree of evolutionary dependence between the
residues, with higher
Gstat
corresponding to increaseddependence
!(x
x
i
statPG
2)(lnWhere P(x,i) Denotes the
probability of finding amino
acid x at position i
Provide a general tool for computational prediction
of conserved allosteric surface by finding networks
of amino acid correlated with the active site
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Allosteric surface can control protein function
PAGE 317-1-2011A. I. Shulman, C. Larson, D. J.Mangelsdorf, R. Ranganathan, Cell 116, 417 (2004).
The existence of correlated amino acids
networks that connect the active site with
distant secondary site
predictions of allosteric surfaces at whichbinding of regulatory molecules (or
covalent modifications) might control
protein function.
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How to utilize this finding
PAGE 417-1-2011
What if two proteins were joined at surface sites such that
their statistically correlated networks were juxtaposed and
could form functional interactions?
Couple the activity of protein by
linking the connection sites to
their respective active sites
through allosteric mechanisms
intrinsic to each protein
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Examples in natural systems
PAGE 517-1-2011Schematic diagram from SCA computation
Mutagenesis studies
confirm that theseinteractions contribute to
allosteric signaling
regulator
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ExperimentsChoosing input module
PAGE 617-1-2011
Light-sensing domain from plant phototropin
[Avena sativa LOV2 (15)], a member of the
Per/Arnt/Sim (PAS) family of signaling modules
Conformational changeInitiation
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PAGE 717-1-2011
ExperimentsChoosing output module
E.colidihydrofolate reductase (EcDHFR):
catalyze the folate metabolism in all organisms
Catalyzes the stereospecific reduction
of 7,8-dihydrofolate H2F to H4F, using
NADPH as a cofactor
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PAGE 817-1-2011
ExperimentsCoupling of LOV2 and DHFR
Core LOV2 domain is inserted via its N- and C-terminal
helical extensions into DHFR at two different surface sites:
Site A and Site B.
A Site (F-G loop) B Site (C-E loop)
Allosteric surface
correlated with the
active site
Main experiment
object
Uncorrelated with
active site
Serve as a control for
potential nonspecific
coupling
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PAGE 917-1-2011
ExperimentsTest independent domain activity
All LOV2-DHFR chimeras were well expressed and
rescued growth in the DHFR auxotropic E. coli
strain under minimal media conditions (WT stand
for wild type E.coli, -ve stand for empty vector)
insertion of the LOV2-J domain did not
abolish DHFR activity in any instance
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PAGE 1017-1-2011
ExperimentsTest independent domain activity
B
(Control)
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PAGE 1117-1-2011
ExperimentsTest independent domain activity
Peak at 447nm, consistent
with the locked dark state
Shift to 390nm due to formation
of covalent thiol-FMN adduct
Nearly identical to isolated domain
Basic intrinsic features of the PAS
domain and DHFR are structurally
and functionally intact in the
chimeras
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PAGE 1217-1-2011
ExperimentsTest chimeras activity (1)
Strategy: comparing khyd (hydride transfer rate)
of dark and light states
Site B no light dependent
changes in enzyme activity
A120: Light dependent
A120-C450S lock the
molecule at dark state
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PAGE 1317-1-2011
ExperimentsTest chimeras activity (2)
Curve shape identical
Relaxation rate match well
Indicating the light dependent
enzymatic activity in A120 is due to
the establishment of alostericcommunication between LOV2 and
DHFR
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PAGE 1417-1-2011
ExperimentsTest chimeras activity (3)
Strategy: Measured the light dependence of the H4F off-rate and of the
equilibrium dissociation constant for NADPH
A factor of 3 decrease in the A120 chimera
Small but significant
light dependence
No light dependence
Previous work shows:
dynamics of F-G loop
specifically controls khydand product release
Engineered interdomain allostery
in A120 chimera likely works
through light-dependent
modulation of F-G loop
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Conclusion
PAGE 1517-1-2011
1. Modular allosteric networks in each protein can
be brought together to initiate the formation of
new allosteric control.
2. Specific surface locations might act as
evolutionarily conserved hotspots for allosteric
control.
3. Linkage of conserved networks of amino acidinteractions might represent a statistically
preferential strategy for the evolution of
allosteric signaling in multidomain proteins.
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Significance
PAGE 1617-1-2011
1. The work presents a initial step towards a general
scheme for the creation of allosteric multidomain
system
2. By combining computational prediction of
potential allosteric surface sites with physics-
based interface design as well as experimentalscreening, it is possible to design high-
performance allosteric system.
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Discussion
The insertion of an allosteric regulator module
greatly decrease the activity of output module
PAGE 1717-1-2011
wild-type Act-A120chimera
khyd 220 s1 0.4 s1
What is the purpose to synthetized dark-locked
chimera to get the conclusion?
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Input module SCA (extra slide)
PAGE 1817-1-2011
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Output module SCA (extra slide)
PAGE 1917-1-2011