Temporary electrostatic impairment of DNA recognition: Light-driven DNA binding of peptide dimers

Article abstract of DOI:10.1002/anie.201201627

Appending negatively charged Glu₈ tails to a peptide dimer derived from the GCN4 transcription factor leads to an effective suppression of its DNA binding. The specific DNA recognition can be restored by irradiation with UV light by using a pho

Full text of DOI:10.1002/anie.201201627

Angewandte
Chemie
DOI: 10.1002/anie.201201627
Electrostatic Caging
Temporary Electrostatic Impairment of DNA Recognition: Light-
Driven DNA Binding of Peptide Dimers**
Adriꢀn Jimꢁnez-Balsa, Elena Pazos, Borja Martꢂnez-Albardonedo, Josꢁ L. MascareÇas,* and
M. Eugenio Vꢀzquez*
Dedicated to Professor Miguel ꢃngel Miranda on the occasion of his 60th birthday
Gene expression relies on a myriad of carefully orchestrated
interactions between specialized proteins called transcription
linker suppresses the DNA interaction. Upon irradiation, the
negatively charged appendages are released, and the DNA-
binding activity is thus restored. As reference system for
implementing the strategy we chose the GCN4 transcription
factor, an archetypical bZIP TF that specifically binds to
ATF/CREB (5’-ATGA(c/g)TCAT-3’) or AP1 (5’-ATGA(c)T-
CAT-3’) sites as a leucine zipper-mediated dimer of unin-
terrupted a helices. The N-terminal basic regions feature
many positively charged amino acids that are key for the
DNA recognition (10 Lys or Arg out of 31 residues in the
[1]
factors (TFs) and regulatory DNA sequences. In general,
such interactions are subtly regulated in time and space, so
that many TFs remain inactive until receiving an appropriate
[
2]
activation signal. It is well-established that the DNA
readout by TFs largely relies on interactions between amino
[
3]
acid side chains and the DNA bases and phosphates. Among
these contacts, those involving positively charged basic amino
acids are critical for the thermodynamic stability of their
[
4]
[13]
DNA complexes. We reasoned that the temporary electro-
static deactivation of such contacts might provide for the
development of TF-based systems where DNA binding
activity could be externally controllable, for instance by
light. These systems could be useful methods for transcrip-
basic region; Figure 1, GCN4br). It has been shown that the
leucine zipper itself can be substituted by a number of
dimerizing units without significant loss in the DNA binding
[14]
properties. Therefore, in our first iteration for the design of
the electrostatically impaired DNA-binding peptides, we
selected the minimum sequence of the bZIP basic region
(br) that it is known to retain the DNA binding ability when
[5]
tional control, or for probing spatiotemporal patterns of
[
6]
gene expression in living organisms. It is curious that despite
the well-established use of light-activated compounds in
[15]
engineered as a disulfide dimer. This minimal peptide was
extended at the N-terminus by adding acidic extensions with
four or eight Glu residues linked to the core br sequence
[7,8]
chemical biology,
examples of photocontrolled DNA
binding are certainly scarce. These include reversible switches
based on the photostationary equilibrium of azo-modified
[
5,9]
DNA binders,
binding affinity and/or specificity,
strategies for triggering minor groove binding or intercala-
special chromophores with poor DNA
[10]
and single-use caging
[
11]
tion.
Thus, inspired by the use of negatively charged
elements to modulate cell internalization,
[
12]
we sought to
develop a general strategy for photocontrolling the sequence-
specific DNA binding of TF peptide mimics.
Herein we demonstrate that tethering polyanionic tails to
basic DNA-binding bZIP peptides through a light-sensitive
[*] A. Jimꢀnez-Balsa, Dr. E. Pazos, B. Martꢁnez-Albardonedo,
Prof. J. L. MascareÇas, Prof. M. E. Vꢂzquez
Departamento de Quꢁmica Orgꢂnica y Centro Singular de Inves-
tigaciꢃn en Quꢁmica Biolꢃgica y Materiales Moleculares y Unidad
Asociada al CSIC, Universidade de Santiago de Compostela
15782 Santiago de Compostela (Spain)
E-mail: joseluis.mascarenas@usc.es
eugenio.vazquez@usc.es
Homepage: http://www.usc.es/chembiousc/
[
**] We thank the financial support provided by the Spanish grants
Figure 1. a) Sequence of the GCN4 basic region highlighting the
positively charged residues. Sequences of peptides with acidic tails,
E br and E br. b) Structure of the Aba chromophore (used as internal
SAF2010-20822-C02, CTQ2009-14431/BQU, Consolider Ingenio
2
0
010 CSD2007-00006, and the Xunta de Galicia INCITE09 209
84PR, PGIDIT08CSA-047209PR, and GRC2010/12. A.J.B. and E.P.
4
8
standard), and the ANP photocleavable linker (shown as *). c) Di-
thank the Spanish MINECO and the Xunta for their PhD fellow-
ships.
merization reactions of br, E br, and E br to give the corresponding
4
8
control disulfide br , or benzylic dimers, fbr , f(E br) , and f(E br) .
2
2
4
2
8
2
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201201627.
The acidic tails (Glu or Glu ) are represented by darker gray and the
4 8
photocleavable element as a black circle.
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Angewandte
Communications
through a photolabile 3-amino-3-(2-nitrophenyl)propionic
[
16]
acid (ANP) group. The disulfide control peptide br was
2
constructed by dimerization of the monomers containing a C-
terminal cysteine by using Ellmanꢀs reagent in phosphate
[
17]
buffer. Peptide dimers f(br) , f(E br) , and f(E br) were
2
4
2
8
2
obtained in good yield by direct alkylation of each of the
cystein monomers with 1,4-bis(bromomethyl)benzene.
The DNA binding of fbr , f(E br) , and f(E br) was
2
4
2
8
2
studied by electrophoretic mobility shift assays (EMSA)
[18]
under non-denaturing conditions and using SYBR gold for
DNA staining. Thus, a short ds oligonucleotide containing the
ATF/CREB binding site was incubated with each of the three
peptides at 48C. As a positive binding control, we used the
Figure 3. HPLC traces of the irradiated buffered solutions of
a) f(E br) and b) f(E #br) , showing the improved photolysis in the
8
2
8
2
basic region disulfide dimer br , which in the presence of the
ATF/CREB oligo gave the expected slower migrating band
reversed design (5 to 75%, 0.1% TFA CH CN/H O). c Starting
3 2
2
dimers, a chromatograms after 30 s irradiation. The acidic tail
released upon irradiation of f(E br) is marked with an asterisk. The
8
2
(
Figure 2, lanes 2–4, band b). The fbr2 dimer displayed
major peak in the trace at the right corresponds to the expected
uncaged dimer f(Q#br) . The photoreleased acidic tail containing the
2
reactive nitrosoketone group is not observed in this case, probably
because it decomposes and the degraded products are eluted with the
injection peak; traces of the dimer with one acidic tail were also
observed in the MALDI spectra (see the Supporting Information).
reactive phenylnitroso ketone groups photoreleased at the N-
terminus of the active peptide fragments, we modified the
[21]
design, reversing the orientation of the ANP linker. The
Figure 2. EMSA assays of DNA recognition. Lanes 1–10: target ATF/
new f(E #br) peptide contained a reconfigured ANP linker
8
2
CREB dsDNA (50 nm). Lanes 2–4: 75, 150, 300 nm br ; lanes 5, 6: 150,
2
attached to the side chain of a Glu residue at the N-terminus
of the basic region; cleavage of this inverted ANP would
release the intact native peptide, leaving a natural Gln residue
3
00 nm fbr ; lanes 7, 8: 150, 300 nm f(E br) ; lanes 9, 10: 150,
2
4
2
3
00 nm f(E br) . Band (a) corresponds to the free ds-oligo; the slow-
8
2
[19]
migrating band (b) corresponds to the DNA/peptide complexes.
at the N-terminus (f(Q#br) ; Scheme 1).
ATF/CREB (one strand shown, binding site in italics): 5’-TGGAG AT-
GA cg TCAT CTCGT-3’. Peptide and dsDNA in 5 mm Tris-HCl, pH 6.8,
2
5
9
0 mm NaCl, (48C, 10 min) were added to 18 mm Tris-HCl pH 7.5,
À1
0 mm KCl, 1.8 mm MgCl , 1.8 mm EDTA, 9% glycerol, 0.11 mgmL
2
BSA, 2.25% NP-40 (48C, 10 min) and loaded into the gel.
qualitatively similar binding properties as the positive control,
albeit exhibiting a slightly reduced affinity (Figure 2, lanes 5
and 6). Curiously, the dimer f(E br) also displayed meas-
4
2
urable affinity for the target oligonucleotide, despite the
presence of a significant number of negatives charges (two
Glu tails), as evidenced by the appearance of a retarded band
in the gel similar that observed with the controls (Figure 2,
4
Scheme 1. Structure of the modified photolabile basic regions with the
reversed ANP linker connected through a Glu side chain (f(E #br) ),
and the expected photodissociation product, f(Q#br)2.
8
2
lanes 7 and 8). In contrast, the f(E br) peptide, featuring the
8
2
longer Glu8 acidic tails, was incapable of forming stable
complexes in the electrophoretic gel, and only at high peptide
concentrations it was possible to observe a faint, slower
migrating band (lanes 9 and 10).
The revamped f(E #br) peptide was synthesized follow-
8
2
ing a similar procedure to that described previously for the
Once we had confirmed that the Glu acidic appendages
synthesis of f(E br) . Gratifyingly, we found that photolysis of
8
8
2
significantly impaired the DNA binding, we investigated the
photocleavage of the ANP linker. Unfortunately, HPLC
analysis of the irradiated solution of f(E br) (30 s, l = 300–
f(E #br) was much cleaner than that of f(E br) , as shown by
8 2 8 2
the HPLC analysis of the reaction mixture (Figure 3;
Supporting Information).
8
2
3
75 nm) showed a complex mixture of products that could not
As expected, the inverted peptide f(E #br) qualitatively
8
2
be characterized (Figure 3a) and from which it was not
possible to isolate the expected photocleaved dimer. Addition
of commonly used reagents to capture reactive photolysis
byproducts, such as DTT or hydrazine, did not result in any
significant improvement. Considering that the complex
product mixture could arise from the degradation of highly
reproduces the DNA-binding behavior observed with the
original f(E br) peptide. Thus incubation of f(E #br) with
8
2
8
2
a double stranded oligonucleotide containing the ATF/CREB
target sequence did not show significant retarded bands in the
PAGE experiments (Figure 4a, lanes 2–5). Irradiation of
[
20]
a 50 mm solution of f(E #br)2 in 10 mm Tris-HCl pH 6.8
8
2
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It is well-known that the DNA binding of bZIP dimers is
[23]
coupled to their folding into a-helical conformations.
Therefore we carried out complementary circular dichroism
studies to gain some structural insight into the DNA binding
process. These experiments showed that the caged dimer
f(E #br) is essentially unstructured in the absence of DNA
8
2
(Figure 5a, open circles). Addition of the target ATF/CREB
Figure 4. EMSA analysis of DNA binding by f(E #br) at 48C.
8
2
a) Lanes 1–9: target ATF/CREB oligo (50 nm). Lanes 2–5: 125, 250,
4
6
00, 600 nm f(E #br) , no irradiation; lanes 6–9: 125, 250, 400,
8 2
Figure 5. a) CD spectra of 5 mm f(E #br) in 10 mm Tris-HCl, pH 6.8,
8
2
00 nm f(E #br) , after 30 s irradiation with UV light (10 mm Tris-HCl,
8
2
at 48C (a). c f(E #br) after 30 s irradiation (*); f(E #br) in
8
2
8
2
pH 6.8); lane 10: 600 nm f(E #br) after 30 s irradiation and addition
of a random ds-oligo (rndDNA, 50 nm; see the Supporting Information
for the full sequence). b) DNA binding of f(E #br) ; irradiation in the
presence of the oligonucleotide (10 mm Tris-HCl, pH 6.8). Lanes 1–5:
target ATF/CREB oligo (50 nm). Lane 2: control peptide fbr (300 nm).
8
2
the presence of 5 mm of the target oligo ATF/CREB before (^), and
after 30 s irradiation (*). b) Fluorescence anisotropy titrations at
8
2
5
18 nm of a fluorescein-labeled ATF/CREB (50 nm) with f(E #br) (*),
8 2
with the f(Q#br) control (*), and best fit to a simple 1:1 binding
2
2
model (10 mm Tris-HCl buffer, 10 mm NaCl, pH 6.8 at 108C). c) Fluo-
rescence anisotropy values: oligo ATF/CREB (50 nm) (bar 1); oligo
with 300 nm f(Q#br) (bar 2); same oligo with 300 nm f(E #br)
Lanes 3–5: f(E #br) (300 nm) at increased irradiation times; lanes 6–
8
2
1
0: rndDNA (50 nm); lane 7: control, fbr (300 nm); lanes 8–10:
2
2
8
2
f(E #br) (300 nm) at increasing irradiation times.
8
2
8 2
(bar 3); and with 300 nm f(E #br) after 60 s irradiation (bar 4). The
same scale is used as with the titrations.
buffer for 30 s with a standard gel transilluminator lamp, and
subsequent incubation with the same oligonucleotide,
resulted in the appearance of retarded gel bands, which is
consistent with a specific peptide–DNA complex (Figure 4a,
lanes 6–9). Moreover, these bands were similar to those
observed using the f(Q#br)2 peptide dimer, which was
purposely synthesized de novo as a true uncaged control. As
oligonucleotide induced a significant increase in the negative
CD signal at 222 nm, which is consistent with an increase in
the a-helical content of the peptide that could result from the
high concentrations required to run the CD experiments
(5 mm of peptide and DNA), or from nonspecific interactions
[
22]
[
24]
with the dsDNAs. More importantly, UV irradiation of this
expected, incubation of the irradiated sample of f(E #br)2
f(E #br) /oligonucleotide mixture led to a large increase in
8
8
2
with a random DNA did not result in any new band
the negative ellipticity in the range of what has been observed
when this type of dimers fold upon interacting with their
target DNA sites (Figure 5a).
(
Figure 4a, lane 10). Along with the experiments with the
[23]
symmetrically caged f(E #br) , we synthesized a single-caged
8
2
f(Q#br)(E #br) peptide in which only one of the basic regions
To further confirm these results and rule out any
distortions associated with the gel shift experiments, we
performed fluorescence anisotropy titrations that provided
a more quantitative characterization of the DNA recognition
8
was modified with the acidic tail. Remarkably, this peptide
retained a significant DNA binding affinity, showing in the
EMSA gels as a slightly slower migrating band than that of
[
25]
the complex with the uncaged control f(Q#br) (Supporting
process.
Incubation of a fluorescein-labeled ATF/CREB
2
Information, Figure S17).
oligonucleotide with increasing amounts of f(Q#br) resulted
2
Importantly, the photochemical activation can be carried
out in the presence of the DNA. Therefore, irradiating
a mixture of f(E #br) and the target oligonucleotide allowed
a substantial recovery of the DNA binding, as shown by
EMSA (Figure 4b, lanes 3–5). An irradiation control experi-
ment with a random oligonucleotide confirmed the specificity
of the interaction, as in this case we did not observe the
formation of new retarded bands in the gel (Figure 4b,
lanes 8–10).
in a progressive increase of the anisotropy, which was
consistent with the formation of a DNA complex with
[
26]
higher molecular weight and reduced mobility. Fitting of
8
2
[27]
the resulting isotherm to a 1:1 binding model, allowed to
calculate its dissociation constant (K ꢀ 38 nm). As expected,
D
the caged derivative f(E #br) showed drastically weaker
8
2
binding than the control peptide under the same conditions
(K ꢀ 1 mm; Figure 5b, open circles). Moreover, the uncaging
D
process could be monitored by fluorescence anisotropy. Thus,
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Communications
irradiation of the caged f(E #br) peptide in the presence of
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the fluorescence-labeled ATF/CREB oligonucleotide
resulted in an anisotropy value similar to that obtained
when the target oligonucleotide is incubated with the f-
[
(
Q#br) control peptide (Figure 5c).
2
In conclusion, we have demonstrated that the attachment
15624 – 15629.
of a negatively charged tether to an Arg/Lys-rich bZIP-based
peptide can effectively hamper its DNA recognition ability.
Moreover, connecting this appendage through a photocleav-
able ANP linker allows to restoring the DNA binding upon
simple UV irradiation. The negatively charged patch might
disturb the binding by interfering with electrostatic pairing or
by generating repulsive contacts with the DNA. Typical
photocaging or switching strategies are based on the modi-
fication of specific key residues and usually require linear
synthetic processes as well as detailed structural knowledge of
the interaction. Our electrostatic turn-off strategy, by relying
on simple tethering of highly charged appendages to the
natural recognition elements, should provide a facile, versa-
tile, and general route to temporarily control specific
biological interactions involving highly charged partners,
such as nucleic acids. Furthermore, this method seems
particularly appropriate for modulating interactions involving
multivalent contacts that might tolerate a single caging group.
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Received: February 29, 2012
Published online: && &&, &&&&
Keywords: amino acids · DNA recognition · peptides ·
.
[
photochemistry · protein design
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Angewandte
Chemie
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Angew. Chem. Int. Ed. 2012, 51, 1 – 6
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Angewandte
Communications
Communications
Electrostatic Caging
A. Jimꢁnez-Balsa, E. Pazos,
B. Martꢂnez-Albardonedo,
J. L. MascareÇas,*
M. E. Vꢃzquez*
&&&& — &&&&
Temporary Electrostatic Impairment of
DNA Recognition: Light-Driven DNA
Binding of Peptide Dimers
Appending negatively charged Glu tails
The specific DNA recognition can be
restored by irradiation with UV light by
using a photolabile linker between the
acidic tail and the DNA binding peptide.
8
to a peptide dimer derived from the
GCN4 transcription factor leads to an
effective suppression of its DNA binding.
6
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