Specific dsDNA recognition by a mimic of the DNA binding domain of the c-Myc/Max transcription factor

Article abstract of DOI:10.1039/c7cc01705g

We here report on the synthesis of the first mimic of the DNA binding domain of the c-Myc/Max-bHLH-ZIP transcription factor able to selectively recognize its cognate E-box sequence 5′-CACGTG-3′ through the major groove of the double-stranded DNA. The designed peptidosteroid conjugate was shown to be effective as DNA binder in the presence of excess competitor DNA.

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Specific dsDNA Recognition by a mimic of the DNA
binding domain of the c-Myc/Max transcription factor
Cite this: DOI: 10.1039/x0xx00000x
a
b
b
Yara Ruiz García , Y. Vladimir Pabon , C. I. Edvard Smith and Annemieke
Madder*a
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
We here report on the synthesis of the first mimic of the DNA rendering the design and synthesis of the mimics of the bHLH-ZIP
1
4,15
protein considerably more complicated (figure 1)
.
binding domain of the c-Myc/Max-bHLH-ZIP transcription
factor able to selectively recognize its cognate E-box sequence
5
’-CACGTG-3’ through the major groove of the double-
stranded DNA. The designed peptidosteroid conjugate was
shown to be effective as DNA binder in the presence of excess
competitor DNA.
In the last decades, developments in the areas of biotechnology
and chemical biology have opened up new prospects in anti-
cancer research. Currently, the inhibition of oncogenes and/or
the reactivation of tumor suppressor genes by transcription
1,2
factors (TFs) are of particular interest . In this context, protein-
DNA interactions have become a principal target of study
through several strategies, as they are a keystone in the regulation
3,4
of gene expression . Amongst other strategies, the design of
synthetic TF models has provided insights into the latter
5
process .
The Myc/Max/Mad network is a family of TFs containing a basic
Helix-Loop-Helix Zipper motif (bHLH-ZIP TF), which is responsible
6
for gene specific transcriptional regulation . These oncoproteins are
Figure
1. Representation of the experimentally-determined
heterodimers composed of proteins from the Myc, Max and Mad
structures of cMyc/Max bHLH-ZIP, PBD -ID: 1NKP (left) and
GCN4 bZIP, PBD -ID: 1YSA (right) Transcription Factors.
7
–9
families . The c-Myc-Max heterodimer binds the double-stranded
DNA (dsDNA) in a sequence-specific manner and its function
consists in the transcription initiation from promoters containing the
hexameric sequence (E-box) 5’-CACGTG-3’ . The dysregulation of
the c-MYC gene causes its overexpression, which is related to the
Due to the relevant role of the c-Myc-Max heterodimer
oncoprotein in cancer development, Kent et al developed the
first approach directed towards the total synthesis of a non-
covalently-linked heterodimeric bHLH-ZIP protein mimic by
convergent chemical ligation of unprotected peptide segments.
Furthermore, various artificial models of homodimeric and
heterodimeric bZIP TFs have been developed by several
groups
a synthetically accessible linker to which the basic regions were
attached. We previously contributed to the area with the
synthesis of simplified bHLH-ZIP TF models which involved the
1
0
14
1
1–13
progression of certain types of cancer
.
The crystal structure of cMyc-Max bHLH-ZIP TF has been studied in
detail. The structural motif of bHLH-ZIP proteins is similar to the
basic Leucine Zipper domain (bZIP) one. Both are characterized by
two domains: (i) a dimerization domain, which is a prerequisite for
DNA binding, and (ii) a DNA recognition domain known as the basic
region that contains the α-helical peptides responsible for the specific
DNA binding. However, the main difference is situated in the
interface between the two domains. In the case of bZIP proteins the α
helices are continuous, while in the case of bHLH-ZIP proteins they
are interrupted by a loop, which does not contribute to DNA binding,
16–27
, through substitution of the dimerization domain by
substitution of the dimerization domain by a steroid-based
moiety (Figure 2).
28
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Figure
2. Illustration of the first mimic of bHLH-ZIP TF by Kent second residue to increase the space between the scaffold and the
(
A
) and our cMyc/Max mimics of the DNA binding domain of peptide as reported in previous models of bZIP TFs. Moreover,
bHLH-ZIP TF (
B
and
C
).
a convergent solution phase approach was applied for the
appendage of the peptides onto the scaffold, as a more efficient
Steroidal building blocks have been extensively studied in drug way to synthesize such peptide conjugates, in comparison with
design due to their amphiphilic nature, bioavailability and the previously developed solid phase-based linear strategy. In
2
9–32
tendency to enhance peptide biostability
. In addition, due to order to dimerize the peptides c-Myc and Max onto the scaffold,
their rigid core, suitable dimensions (in terms of distance an orthogonally functionalized deoxycholic acid scaffold
between the different attachment points on the steroid scaffold) derivative was synthesized to allow the convergent incorporation
for major groove binding increase the helicity of the appended of both basic region peptides (Figure
3). Commercially available
peptides and they can thus be envisaged to substitute the leucine deoxycholic acid ( ) was chosen as starting material as its
1
zipper domain as an anchoring point for the basic region of concave structure allowed the two alcohol functionalities to be
bHLH ZIP TF. While various other scaffolds have been selectively esterified facilitating convergent conjugation of two
3
3–36
37
evaluated as artificial dimerization domains
offers rigidity, proper dimensions for major groove recognition functionality was protected with benzyl bromide to facilitate
as well as the capability of increasing cell-uptake. Indeed, we HPLC monitoring of the conjugation reactions ( ). Furthermore,
have shown with previous models that the steroid moiety within as reported previously, aliphatic linkers have proven useful to
, the steroid different peptide sequences . In a first step, the carboxylic acid
2
2
3
peptide-steroid conjugates substantially increases their cellular ensure DNA binding for this type of TF mimics . These spacers
2
3
uptake . Our previously synthesized artificial peptidosteroid- between the peptides and the scaffold should provide sufficient
based mimic of the DNA binding domain of the c-Myc-Max length and flexibility to allow accommodation of the peptides
oncoprotein suffered from unsuccessful DNA binding due to the into the DNA major groove. Therefore, we chose 4-maleimido-
lack of an α-helix conformation and suitable orientation of the butyric acid and pentynoic acid as linkers to ensure the flexibility
appended peptides. It was shown that the direct conjugation of needed for DNA recognition through the major groove. Selective
the basic region peptides to the steroid scaffold impedes the esterification with pentynoic acid at C3 was carried out using a
correct dimerization of the peptides due to the rigidity and steric catalytic amount of DMAP and EDC·HCl as coupling reagent
2
8
encumbrance imposed by the steroid moiety which could be
(3). Subsequent functionalization of the alcohol at C12 with Boc
derived with the aid of molecular modeling from the high protected gamma-aminobutyric acid was performed with EDC
7
resolution of the protein structure by Burley et al (PDB-ID: and excess of DMAP to allow the reaction to reach completion.
1
NKP).
We now report on the synthesis of an optimized model of the amine into a maleimide resulted in hetero-difunctionalized
DNA binding domain of the cMyc/Max-bHLH ZIP TF able to scaffold ( ). Cu catalyzed azide alkyn cycloaddition click and
Finally, Boc deprotection and transformation of the obtained
6
bind specifically to the non-palindromic target DNA sequence. cysteine-maleimide conjugation strategies were chosen for
We decided to introduce spacers between the dimerization peptide conjugation in view of their generality and orthogonality.
moiety and the appended peptides in addition to using an For this purpose, the c-Myc and Max peptides sequences were
elongated DNA recognition domain. We considered the modified at the C-terminus with cysteine and azido-homoalanine
elongation of the basic region peptides at the C-terminus (Aha) respectively. The Myc/Max basic regions consist of a
corresponding to at least four amino acids or approximately one sequence of 16 amino acids that specifically recognize the E-box
helical turn, which should be added to the 16-17-residue c- DNA sequence 5’- AGCACGTGCT -3’. The monomeric Myc
Myc/Max peptides respectively. A linker was additionally (NVKRRTHNVLERQRRNELKRGC)
foreseen in the final peptide-steroid conjugate in order to provide (ADKRAHHNALERKRRDHIKDGAha)
and
sequences
Max
were
the peptides with the required flexibility needed for insertion into synthesized using Fmoc-/tBu-SPPS on Rink-amide ChemMatrix
to the major groove of the dsDNA. Glycine was coupled as resin.
3
29 | Chem. Commun., 2014, 00, 1-3
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middle (5’-CCATGGCGAGCGTCGCTACTAGCACGTGCT
AGTAGGTGCGCTATCTAAGG-3’).Increasing concentrations
of compound 13 were added, at a constant 5 nM concentration of
dsDNA and different ratios of dsDNA:peptide concentrations
were tested to study binding of compound 13 to the 50-mer E-
box DNA sequence (figure
4 A).
Figure
conjugate 13 to 5 nM of 5’-labeled P-DNA target sequence (50-
. Synthetic scheme towards final peptidosteroid mer) containing the E-box. First lane in all the gels: dsDNA.
conjugate 13. a) DBU, Benzyl bromide, Dry DMF, 50°C, 24h Lanes 2-6 contain peptide concentrations from 0.125, 0.25, 0.5,
78%); b) Pentynoic acid, EDC·HCl, DMAP, dry DCM, rt, and 1.0, up to 2.0 µM of 13. B) EMSA titration in the presence
overnight (60%); c) Boc-Gaba-OH, EDC, DMAP, dry DCM, rt, of
competitor dsDNA sequence 45-mer (5'-
overnight (40%); d) 20% TFA in DCM, 2h (99%); e) Maleic AGCAGAGGGCGTGGGGGAAAAGAAAAAAGATCCACC
anhydride, dry toluene. 80°C, 24h (38%); f) H O/ACN 1:1, GGTCGCCAC-3') at 0.5 µM. C) EMSA titration in the presence
NH HCO pH 6.5, rt, 2h; g) Cu(CH CN) PF , DMSO/H
h (Combined yield for step f and g: 24%).
4. A, B and C) EMSA titration of the peptide-steroid
3
2
Figure
3
(
2
4
3
3
4
6
2
O, rt, of competitor dsDNA sequence 45-mer at 2 µM concentration.
3
It can now be noticed that, at a ratio of 1:50 dsDNA:peptide
The resulting Myc/Max basic regions, functionalized with a complete binding can be observed in the gel, evident by the
cysteine and an azide, were then conjugated to the central steroid presence of an up-shifted band from the dsDNA band (Figure
scaffold resulting in the heterodimeric TF mimic. First, the
conjugation at the C12 position was performed via thiol- peptide:dsDNA ratios (Figure
4
A, lane 3). Again higher-order species are visible at increased
A, lanes (5-6). Additional EMSA
4
maleimide conjugation through 1,4-Michael addition to a experiments were then performed to check specificity of
cysteine-contained peptide resulting in a succinimidyl thioether construct 13 towards the recognition site of the dsDNA.
moiety (12). We decided to perform the conjugation at C12 in Therefore, two competitors were used: 1) a dsDNA sequence
first place as the maleimide functional group could be hindered (45-mer), and 2) a ssDNA sequence (15-mer) both of which are
by the presence of a peptide at C3. Without isolation of the unrelated sequences. Competitor sequences were added at
monopodal intermediate 12, the second conjugation was different concentrations. Whereas binding is clearly less strong
performed via CuAAC at the C3 position of the alkyne- than for our homodimer GCN4 mimics and adding competitor
functionalized scaffold forming a triazole (13, Figure
3
).
DNA influences the binding pattern, our results reveal that
The click conjugation was compatible with the presence of the construct 13 can still bind specifically to the target sequence to a
peptide chain at C12. These and previous results show that the certain extent, even in presence of a high excess of competitor
here applied CuAAC protocol is compatible with different dsDNA (figure
4 B and C). It was however also shown that the
peptide sequences. Both, the succinimidyl thioether and triazole concentration of peptide required for dsDNA binding was very
moieties are generally accepted as stable linkages under high as indicated by the fact that the dsDNA band is still present at
physiological conditions.
2 µM of peptide, which was the concentration required for complete
In order to detect DNA binding affinity of peptide-steroid dsDNA binding without competitor dsDNA. Further EMSA
conjugate 13 an Electrophoretic Mobility Shift Assay (EMSA) experiments were performed using 3 µM (Figure S18) and 12 µM
3
2
using radioactive P was performed (Figure 4). Whereas our (Figure S19) of ssDNA as competitor (5’-CCTTTTCTTTTTTCT-
2
3
previously developed homodimeric GCN4 based mimics , 3’). The used concentrations ensure the same number of competing
when added to a 21-mer dsDNA containing the ATF/CREB negative charges for both types of competitors. Peptide binding was
recognition site, lead to a complete shift in the EMSA analysis, observed even when 1-4 fold excess of the ssDNA was used. As
indicating full binding with only a 10-12 fold excess of peptide, similar results were observed for both types of competitor, this does
the current Myc/Max models bind less good to the 26-mer target support that the peptide is specific for the target sequence, but only at
containing the E-box recognition-binding site in the middle high excess concentration of the peptide (ratio 1:400). This can be
requiring an at least 50-fold excess to detect any binding. explained by the existence of random electrostatic interactions
Additionally, when using larger excesses of peptide, formation between the peptide and the competitors. Although non-specific
of higher order complexes can be observed (see supporting interactions can be observed, the results of the experiment clearly
information). This observation is in line with the low specificity reflect the formation of a specific dsDNA/dimeric peptide
of the native cMyc/Max heterodimer towards the E-Box DNA construct albeit with lower affinity as compared to our previously
sequence, presenting multiple binding sites38–40. Additional reported homodimeric models.
experiments were carried out using a 50-mer dsDNA target
sequence, containing the E-box recognition-binding site in the
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To conclude, we show the possibility of selective functionalization of
steroid-based scaffolds to synthesize heterodimeric peptide-based
conjugates, in principle allowing application of current methodology
to other families of TF, such as bZIP Fos/Jun TF. Moreover, our
design overcomes the difficulties observed when decorating building
blocks by SPPS, since it is based solely on conjugation strategies in
solution phase.
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