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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