A chemical approach for the synthesis of the DNA-binding domain of the oncoprotein MYC

Article abstract of DOI:10.1039/c9ob01209e

We describe the first chemical synthesis of a functional mutant of the DNA binding domain of the oncoprotein MYC, using two alternative strategies which involve either one or two Native Chemical Ligations (NCLs). Both routes allowed the efficient synthesis of a miniprotein which is capable of heterodimerizing with MAX, and replicate the DNA binding of the native protein. The versatility of the reported synthetic approach enabled the straightforward preparation of MYC and Omomyc analogues, as well as fluorescently labeled derivatives.

Full text of DOI:10.1039/c9ob01209e

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Takeharu Haino et al.
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A chemical approach for the synthesis of the DNA-binding domain
of the oncoprotein MYC
Renata Calo-Lapido,a Cristina Penas,a Adrián Jiménez-Balsa,a M. Eugenio Vázquez,*,a and José L.
Received 00th January 20xx,
Accepted 00th January 20xx
Mascareñas*,a
DOI: 10.1039/x0xx00000x
www.rsc.org/
We describe the first chemical synthesis of a functional mutant of Herein, we report the total synthesis of
a 82-residue
the DNA binding domain of the oncoprotein MYC, using two miniprotein featuring the DNA interacting domain of natural
alternative strategies which involve either one or two Native MYC, with two specific mutations, Asn500→Ala and Asn515→Gln,
Chemical Ligations (NCLs). Both routes allowed the efficient which do not affect its DNA binding but facilitate the synthesis.8
synthesis of a miniprotein which is capable of heterodimerizing The assembly of such long peptide was carried in a convergent
with MAX, and replicate the DNA binding of the native protein. The way by using Native Chemical Ligations (NCL). In its standard
versatility of the reported synthetic approach enabled the format, NCL involves the reaction between a C-terminal
straightforward preparation of MYC and Omomyc analogues, as peptide-thioester and and anN-terminal cysteinyl peptide.9
well as fluorescently labeled derivatives.
Since MYC lacks cysteine residues in its sequence, we chose Xaa-
Ala as ligation sites because Ala residues can be readily obtained
by desulfurization of the Cys required for the NCL.10
The oncogenic MYC transcription factor is a key signaling hub
that integrates multiple cellular pathways,1 and is deregulated
and overexpressed in many tumors.2 As a member of the basic
helix–loop–helix-leucine zipper (bHLH-Zip) family of
transcription factors,3 MYC effects are mediated by its
association with other proteins of the same family, and in
particular with MAX. The resulting MYC/MAX heterodimers are
capable of binding selectively to E-box sites (CACGTG) of gene
promoters. Despite its pharmacological interest, targeting MYC
and affecting its dimerization properties is far from trivial.4,5 In
this context, recent work by the group of L. Soucek has shown
that a protein analogue, omomyc, interferes with the activity of
MYC and induces growth arrest.6
Preliminary assays of the solid phase assembly of the
N-terminal peptide fragments revealed the formation of a small
proportion of undesired products resulting from spontaneous
deamination of asparagine residues Asn500 and Asn515 11
.
Considering that these residues are not involved in DNA
contacts of the natural protein, or in the formation of the
MYC/MAX heterodimer, we decided to replace Asn500 by Ala,
and Asn515 by Gln, so that our final target would be a double
MYC mutant [AQ]MYC (Figure 1). We designed two
disconnection schemes using either one or two NCL reactions
(Figure 1b). The first case requires the synthesis of two long
peptides corresponding to Ala500–Lys536 (37 residues) and
Ala537–Leu581 (45 residues) fragments (2Fr-NCL, Figure 1b, left),
while the second approach involved three shorter peptide
A
major drawback for exploring the biological and
therapeutic properties of these proteins derives from the lack
of efficient methods for their production. Recombinant
methods are slow,7 and more importantly, they do not easily
allow the introduction of non-natural elements or purposely
designed modifications, such as the introduction of small
fluorophores. In this context, alternative chemical approaches
are desirable, and they provide practical and versatile syntheses
of this protein and designed derivatives.
segments, Ala500–Phe522
, ,
Ala523–Thr547 and Ala548–Leu581
,
featuring 23, 25 and 34 residues, respectively (3Fr-NCL, Figure
1b, right). The three-fragment approach is more convergent,
and hence more appropriate to obtain N-labeled [AQ]MYC
derivatives. Moreover, we anticipated that the double Cys
intermediate, [AQ,C523,C548]MYC, obtained in the 3-Fr-NCL
strategy, might be exploited to implement redox- controlled
DNA binding.
Peptides were assembled using the Nbz method,12 which
involves the postsynthetic generation of a C-terminal N-acyl
urea leaving group (N-acyl-benzimidazolinone, Nbz) by on-resin
modification of a precursor 3,4-diaminobenzoic acid (Dbz) with
p-nitrophenyl chloroformate (ESI).
a. Departamento de Química Orgánica and Centro Singular de Investigación en
Química Biolóxica e Materiais Moleculares (CIQUS). Universidade de Santiago de
Compostela. 15782 Santiago de Compostela, Spain.
Electronic Supplementary Information (ESI) available: Peptide synthesis, purification
and characterization, DNA binding experiments. See DOI: 10.1039/x0xx00000x
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Figure 1. a) Sequence and structural elements of the DNA binding domain of MYC. b) Alternative disconnection schemes for synthesizing the Asn500→Ala and Asn515→Gln
MYC mutant ([AQ]MYC) from two (2Fr-NCL, left) or three peptide fragments (3Fr-NCL, right), showing the positions selected for the introduction of the reactive Cys (or Thz)
residues. NCL conditions: 6 M Gdn HCl, 200 mM phosphate buffer (pH 7.5), 100 mM 4-mercaptophenylacetic acid (MPAA), 40 mM tris(2-carboxyethyl)phosphine hydrochloride
(TCEP·HCl), 4.5 h; Desulfurization: 6 M Gdn HCl, 200 mM phosphate buffer (pH 7.0), 500 mM TCEP (tris(2-carboxyethyl) phosphine), EtSH, VA-044 (2-2’-azobis[2-(2-imidazolin-2-
yl) propane]dihydrochloride) 1 M, 37º C, 5 h.
After cleavage and deprotection, the Nbz group can be milligram scale to give the desired miniprotein [AQ]MYC, which
readily exchanged with thiol additives at neutral pH to produce presented a good water solubility.
the peptide thioesters required for the NCL (Figure 1b). Briefly,
With [AQ]MYC at hand, we studied its DNA binding
the two-fragment approach allowed the assembly of [AQ]MYC properties in comparison with the wild-type DNA binding
using a single NCL by coupling peptide segments A500K536–Nbz domain of MYC (wtMYC).15 In agreement with earlier reports,
and C537L581 in the presence of 4-mercaptophenyl acetic acid MAX is capable of binding as homodimer to the E-box site,
(MPAA) as arylthiol catalyst. The reaction was essentially although with a low association constant (Figure 2a and b, lanes
complete after 4.5 h (54 %, 15 mg scale). The desulfurated 2 and 3). In contrast, wtMYC does not appear to dimerize and
target peptide ([AQ]MYC) was readily obtained [AQ,C537]MYC bind to the DNA (Figure 2a, lanes 4 and 5). As expected,
using standard radical-based methods.13 Assembly of [AQ]MYC incubation of MAX with increasing concentrations of wtMYC,
following the three-fragment approach (3Fr-NCL) was carried resulted in the appearance of a slightly less retarded band
out in a similar way. In this case, one of the peptide fragments (Figure 2a, lanes 6-9) that is consistent with the formation of the
used in the first NCL step features the Cys in the form of DNA complex of the MAX/wtMYC heterodimer.16 Importantly,
thiazolidine (Thz) to avoid oligomerization and unwanted self- our synthetic MYC analog, [AQ]MYC, reproduces the MAX
couplings (Thz523T547, Figure 1).14 Following the first NCL, the recognition and DNA binding of wtMYC, although with some
Thz was removed by adding hydroxylamine to the reaction differences: [AQ]MYC shows a slightly more tendency to
mixture and adjusting the pH to 4, and the resulting peptide homodimerize (Figure 2b, lane 5), and the heterodimer of
(C523Q581) was isolated by HPLC (over 20 % for this NCL). The [AQ]MYC with MAX binds the target E-DNA at lower
second NCL afforded the expected product (in this case concentrations than that of wtMYC (Figure 2a and 2b, compare
containing two Cys residues in positions 548 and 523), which lanes 7). Control EMSA experiments with mutated DNAs
was isolated by reverse phase HPLC (Figure S18). After demonstrated that the [AQ]MYC/MAX heterodimer binds the
desulfurization and purification, we obtained [AQ]MYC (over 4 E-DNA with high selectivity (Figure S30). Circular Dichroism
% global yield). It must be highlighted that both synthetic studies were also consistent with the expected binding/folding
approaches (2Fr-NCL and 3Fr-NCL) can be carried out at a processes (Figure S39).17
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[AQ]MYC(SH) and TMR-[AQ]MYC, featuring View ATrMticlRe On(li5ne-
Carboxytetramethylrhodamine) fluorophore at the N-terminal
positions (see the ESI).
a
DOI: 10.1039/C9OB01209E
Moreover, following the same three-fragment approach, we
synthesized two Omomyc analogues: (TMR-[AQS]OMOMYC
and the bis-cysteine derivative (TMR-[AQS]OMOMYC(SH) (ESI).
19
Preliminary uptake studies revealed that the cysteine-
containing miniproteins are more efficiently internalized (over
4 times in A549 and 7-8 times in HeLa) than the desulfurized
analogues (Figure 4, top vs bottom micrographies and Figure
S37).20,21
Figure 2. Non-denaturing EMSA showing the binding of MAX, the expressed wtMYC, and
the synthetic [AQ]MYC to a double stranded oligo containing the target E-box sequence
(E-DNA, 5´-CTGTAGGC CACGTG ACCGGATG-3´, only one chain shown). a) Lanes 1-3: 0,
500, and 1000 nM MAX; lanes 5-6: 500 and 1000 nM wtMYC; lanes 7-10: 250, 500, and
750 nM wtMYC with constant 500 nM MAX. b) same as in gel a, but with the synthetic
[AQ]MYC instead of wtMYC. All lanes contain 50 nM E-DNA in 20 mM Tris-HCl pH 7.5, 90
mM KCl, 1.8 mM MgCl2, 1.8 mM EDTA, 9% glycerol, 0.11 mg/mL BSA, 2.25% NP-40 (20
ºC). The gels were visualized by staining with SYBRTM Gold.
These experiments confirm that the synthetic product
[AQ]MYC heterodimerizes with MAX to bind the E-box site. One
of the advantages of the synthetic approach is that, in addition
to the final product [AQ]MYC, we also obtained the synthetic
intermediate [AQ,C523,C548]MYC, featuring Cys at the ligation
sites. Inspection of the X-ray structure of the cMYC/cMAX/DNA
complex reveals that the positions corresponding to these
cysteine residues are not involved in DNA binding (Figure 3a),
and therefore we predicted that this mutant might show similar
DNA binding properties than [AQ]MYC. We also reasoned that
engaging these cysteines in an intramolecular disulfide bond
should abolish the DNA binding. For simplicity, from now on we
will refer to the double Cys mutant, [AQ,C523,C548]MYC, as
[AQ]MYC(SH). Treatment of [AQ]MYC(SH) with DTNB (5,5’-
dithiobis-(2-nitrobenzoic acid), in 100 mM Tris⋅HCl buffer (pH
7.5) for 1 h,18 led to the corresponding oxidized peptide
[AQ]MYC(S-S), featuring a disulfide crosslink between residues
Cys523 and Cys548 (isolated and identified by ESI-MS, see the ESI).
Addition of increasing amounts of [AQ]MYC(SH) to a
mixture of MAX and E-DNA resulted in two slow-migrating
bands, the first, predominant at low [AQ]MYC(SH)
concentrations, must correspond to the homodimeric MAX2/E-
DNA complex, (Figure 3a, lanes 1-4, band a); the second band,
observed at higher concentrations of [AQ]MYC(SH), is
consistent with the heterodimeric [AQ]MYC(SH)/MAX/E-DNA
complex (Figure 3a, band b). As expected, oxidation provides a
disulfide that is unable to cooperate with MAX and bind DNA,
and therefore we only observe the band arising from the MAX
homodimer (Figure 3b, lane 1). However, in situ addition of a
reducing agent (TCEP) regenerates the parent reduced peptide
[AQ]MYC(SH), and the heterodimeric DNA binding (Figure 4b,
lane 2). These preliminary results propose a strategy to develop
externally activatable MYC surrogates. Using the three-
fragment NCL we also synthesized the labeled analogs TMR-
Figure 3. Top: X-ray structure of the complex MYC-MAX/DNA, highlighting the positions
of the cysteines in [AQ]MYC(SH) (shown in orange). Middle: Schematic representation
of the reduction/activation process: The crosslinked [AQ]MYC(S-S) adopts
a
conformation incompatible with MAX binding. Bottom: a) EMSA analysis of the DNA
binding of [AQ]MYC(SH) and MAX to E-DNA: Lanes 1-4: 50 nM E-DNA; lanes 2-4: 500 nM
MAX and 250, 500, and 1000 nM of [AQ]MYC(SH); b) Redox switching: 50 nM E-DNA,
lane 1: 500 nM MAX and 1000 nM [AQ]MYC(S-S); lane 2: after addition of TECP. All lanes
contain 50 nM DNA in 20 mM Tris-HCl pH 7.5, 90 mM KCl, 1.8 mM MgCl2, 1.8 mM EDTA,
9 % glycerol, 0.11 mg/mL BSA, 2.25 % NP-40 (20 ºC). Dashed line indicates that the top
and bottom of the gel with the bands was mounted in the final image.
Additionally, the Omomyc analogue is also better uptaken
than TMR-[AQ]MYC; and tends to accumulate in the
perinuclear space (Figure S36). Our results confirm that small
variations in the sequence of MYC can have very important
consequences in solubility, aggregation and internalization
properties.
In summary, we have reported the first synthesis of a
functional, DNA-binding analog of the bHLHzip domain of MYC
using chemical methods. The flexibility of the methodology
allowed a practical synthesis of a redox-activatable, and
fluorescently tagged derivatives not only of MYC but also of its
therapeutically-relevant analog Omomyc. These derivatives
could not be assembled using expression approaches. Cell
uptake studies revealed that all the miniproteins are
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8
1
Residues numbered according to the X-ray structure of the
MYC-MAX heterodimer bound to the DNA E-box (PDB ID:
1NKP). S. K. Nair and S. K. Burley, Cell, 2003, 112, 193–205.
Figure 4. Overlay of both red channel and DIC fluorescence micrographies of A549 cells
incubated for 4 h with 250 nM of the synthetic peptides in DMEM (without FBS). Cells
were then washed twice with PSB solution and three more times with DMEM. Pictures
were taken at 60X and λexc = 561 nm for the red channel, in a confocal microscope.
9
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Conflicts of interest
10 L. Z. Yan and P. E. Dawson, J. Am. Chem. Soc., 2001, 123, 526–
533.
There are no conflicts to declare.
11 T. Geiger and S. Clarke, J. Biol. Chem., 1987, 262, 785–794.
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15 This protein was obtained through bacterial expression of the
pGEX Myc c92 plasmid. Sequence: GSPSSDTEEN VKRRTHNVLE
RQRRNELKR SFFALRDQI PELENNEKA PKVVILKKA TAYILSVQA
EEQKLISEE DLLRKRREQ LKHKLEQLR NSCA; wild-type MAX:
GIQMSDNDDI EVESDADKRA HHNALERKRR DHIKDSFHSL
RDSVPSLQGE KASRAQILDK ATEYIQYMRR KNHTHQQDID
Acknowledgements
Financial support from the MINECO (CTQ2015-70698-R and
SAF2016-76689-R, orfeo-cinqa CTQ2016-81797-REDC), Xunta
de Galicia (2015-CP082, ED431C 2017/19, ED431B 2018/04,
Centro singular de investigación de Galicia accreditation 2016–
2019, ED431G/09) and the European Union (European Regional
Development Fund - ERDF), Fundación AECC (IDEAS197VAZQ),
and the European Research Council (Advanced Grant No.
340055) are acknowledged. R.C.L. thanks Xunta de Galicia and
European Social Fund for her fellowship. A.J.B. thanks Xunta de
Galicia for his Post-doctoral fellowship. We are grateful to Prof.
Dr. Bernhard Lüscher, who generously shared with us the
pBluescript p21 Max, pGEX Myc c92, pGEX-2T-Myc C176, and
pGEX 3X plasmids for expression of the MYC and MAX protein
DLKRQNALL
EQQVRALEK
ARSSAQLQTN
YPSSDNSLYT
NAKGSTISAF DGGSDSSSES EPEEPQSRK KLRMEAS.
16 C. Muhle-Goll, M. Nilges and A. Pastore, Biochemistry, 1995,
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18 J. Mosquera, A. Jiménez-Balsa, V. I. Dodero, M. E. Vázquez and
J.L. Mascareñas, Nat. Commun., 2013, 4, 1–8.
19 These derivatives contain key mutations present in omomyc
with respect to MYC, as well as the A and Q mutations used in
the case of our MYC analogue (see the ESI†).
20 N. Chuard, G. Gasparini, D. Moreau, S. Lörcher, C. Palivan, W.
Meier, N. Sakai and S. Matile, Angew. Chem. Int. Ed Engl.
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