¿Podemos diseñar sistemas de expresión que respondan a señales ambientales predeterminadas?...

¿Podemos diseñar sistemas de expresión que respondan a

señales ambientales predeterminadas?

Reguladores a la carta

The transcriptional regulator XylR

A (receptor)

B interdomain

ATP binding

C (activation) D HTH

DNA binding in Pu y Ps

COOHNH2

211 233

Recognition and binding of aromatic effectors

ATP hydrolysisMultimerizationContacts with 54

R

R

XylR active

B

D

A

C

XylR inactive

Intramolecular repressionSpecific A-C interactions

Binding to A domainRelease of repression

Activation of XylR in response to inducers

R

B

D

A

C

Regulators á la carte: can we change at will the effector specificity of XylR?

The B domain of XylR: a coiled coil?

A C D

B/Q linker

1 211 472233 514 554

CN

566

208 236

(XylR16-1) DP HHHR (XylR3H)

IVDE RYELQTQ VANLRNR LKQYDGQ YYGIG

Native inducer

XylR

BA

C

OH4

phenolF

CH3

7

4-FBA

CH3

Cl

8

4-ClBA

CH3

11

1-methyl

13

biphenyl

CH3-(CH2)6-CH3

14

octane

CH3

CH3

m-xileno

2

5

benzene

NH2

6

aniline

CH3

NO2

9

4-NT

10

naphthalene

CH3

12

2-methyl

CH3

Cl

3-Cl tol

3

Suboptimal inducers

Non-inducers

0

5

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14

BC

MAD16-1, XylR16-1

Inducer compounds

0

5

10MAD1, XylR

BA

cti

vity

Pu

(-

Ga

l, x

10

3)

Effects of 16-1 mutation in the B domain of XylR

A

A

B

D

A

C

Can we change the effectorspecificity of XylR?

R

R

Non naturalinducers

XylRactive

Generation of regulators á la carte

D

AB

C

XylRinactive

Structural prediction

The A domain of XylR

Genetic approach: generation of diversity

1 211

CH3

NO2

D

AB

C

XylR

CH3

NO2

CH3

NO2

toluene m-xylene

2-NT 3-NT 4-NT biphenyl

p-xylene

CH3 CH3

CH3

CH3

CH3

Native inducers

New inducers

Reshaping the effector pocket of the A domain

A

CH3

CH3

generation

of diversity

R

R

1. Preparation of target DNA2. Digestion DNase I (30-300 bp)

3. PCR without oligonucleotides

4. PCR with oligonucleotides

Generation of diversity through mutation-prone shuffling of homologous A domains

XylR A domainDmpR A domain TbuT A domain

Family of similarDNA sequences

Genetic screening

Elimination ofNon productivecombinations

Selection ofnew activecombinations

Shuffled A domains

Pool of A domainvariants

Shuffling of A domains of XylR-like activators

XylR A domainDmpR A domain TbuT A domain

Family of similarDNA sequences

The genetic tricks

• Positive selection

• Negative selection

• Visual screening

• Phenotypical characterisation

Po npt (km)

Po sacB

Po luxAB

Pu lacZ

Cloning vector

pCon918

A C DPr

NdeI SnaBI

ligation E. coli XL1 colony poolP. putida KT2440

Po-Km/Po-sacB

Growthon plates

Conjugation

M9 succ Km + new effector

A domain Shuffling libraryNdeI/SnaBI

Sucrose

Plasmid extraction

P. putida KT2440 Po-luxAB Light emission

Genetic screening/selection

No Km

3-nt decanal test assayLB+3NT

LB+3NT

no inducer

no inducer

C-

C-

XylR 1

XylR 2

XylR 3

XylR 4

XylR 5

XylRwt

no inducer m-xylene phenol benzene

XylR 1

XylR 2

XylR 3

XylR 4

XylR 5

XylRwt

2-nt 3-nt 4-nt biphenyl

Visualization of effector-specificity changes

Sequence analysis of hybrid regulators

XylR1

XylR5

XylR DmpR

1 V124A 161-166 220

XylR2

F65L

XylR3

46-50

XylR4

L184I

161-166

In vivo activity of hybrid regulators m

-xile

nobe

ncen

o0

2

4

6

8

2 3 4 5 6 7 8 91XylR wt

no in

d-

feno

l

2-N

T3-

NT

4-N

Tbi

feni

lo 0

2

4

6

8

XylR12 3 4 5 6 7 8 91

no in

d-

feno

l

2-N

T3-

NT

4-N

Tbi

feni

lo 0

2

4

6

8

XylR22 3 4 5 6 7 8 91

no in

d-m

-xil

feno

lbe

nc2-

NT

3-N

T4-

NT

bife

nilo

0

2

4

6

8

XylR32 3 4 5 6 7 8 91

no in

d-m

-xil

feno

lbe

nc2-

NT

3-N

T4-

NT

bife

nilo

no in

d-m

-xil

feno

lbe

nc2-

NT

3-N

T4-

NT

bife

nilo0

2

4

6

8

XylR42 3 4 5 6 7 8 91

0

2

4

6

8

XylR52 3 4 5 6 7 8 91

no in

d-m

-xil

feno

lbe

nc2-

NT

3-N

T4-

NT

bife

nilo

m-x

ilbe

nc

m-x

ilbe

nc

Pro

mote

r act

ivit

yr

Pu

(b

-Gal, x

10

3)

(Apparent) affinity assays

0

2

4

6

8

110-4 10-3 10-2 10-1

XylR wt

XylR2XylR1

0

2

4

6

8

XylR2XylR1

3-MBA (mM) 3-NT (mM)

Pro

mote

r act

ivit

y P

u (

b-G

al, x

10

3)

XylR wt

110-4 10-3 10-2 10-1

Inhibition by 3-NT

0

1

2

3

4

5

6

0 0.5 1 1.5 2 2.5 3

3-NT (mM)

3-MBA 1 mM

XylR wt

Pro

mote

r act

ivit

y P

u (

b-G

al, x

10

3)

Structural prediction for the A domain of XylR

Grupo de Diseño de Proteínas-CNB

Contact surfaces protein/effector

Mapping structural changes in the model

XylR2

F65L

XylR3

46-50

XylR5

161-166

XylR2

F65L

XylR3

46-50

XylR5

161-166

Mapping structural changes in the model

XylR2

F65L

XylR3

46-50

XylR5

161-166

Mapping structural changes in the model

XylR1

XylR4

V124A

L184I

Mapping structural changes in the model

XylR1

XylR4

V124A

L184I

Mapping structural changes in the model

XylR wt

XylR5XylR4

XylR1

XylR3

XylR2

Loops involved in the effector pocket

Conclusions

• Mutants XylR1 to XylR5 bear changes that unlock the ability of XylR to respond to many non-natural effectors

• The changes involve not only the shape of the effector pocket, but also the structural transmission caused by inducer binding

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