Dynamic Stereoselection of Peptide Helicates and Their Selective Labeling of DNA Replication Foci in Cells**

Article abstract of DOI:10.1002/anie.202013039

Although largely overlooked in peptide engineering, coordination chemistry offers a new set of interactions that opens unexplored design opportunities for developing complex molecular structures. In this context, we report new artificial peptide ligands t

Full text of DOI:10.1002/anie.202013039

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Dynamic Stereoselection of Peptide Helicates and Their Selective
Labeling of DNA Replication Foci in Cells
Authors: Jacobo Gómez-González, Yolanda Pérez, Giuseppe
Sciortino, Lorena Roldan-Martín, José Martínez-Costas, Jean
Didier Maréchal, Ignacio Alfonso, Miguel Vázquez, and M.
Eugenio Vazquez
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202013039
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10.1002/anie.202013039
Angewandte Chemie International Edition
RESEARCH ARTICLE
Dynamic Stereoselection of Peptide Helicates and Their Selective
Labeling of DNA Replication Foci in Cells
Jacobo Gómez-González,[a] Yolanda Pérez,[b],§ Giuseppe Sciortino,[c,d],§ Lorena Roldan-Martín,[c] José
Martínez-Costas,[e] Jean-Didier Maréchal,[c] Ignacio Alfonso,[f] Miguel Vázquez López,*,[g] M. Eugenio
Vázquez*,[a]
Dedicated to baby Vega
Abstract: Although largely overlooked in peptide engineering,
Introduction
coordination chemistry offers a new set of interactions that opens
unexplored design opportunities for developing complex molecular
Metal ions offer vast opportunities for the structural control at the
structures. In this context, we report new artificial peptide ligands that
molecular scale; exploiting their different coordinative properties,
fold into chiral helicates in the presence of labile metal ions such as
researchers can create complex assemblies of great beauty and
Fe(II) and Co(II). Heterochiral β-turn promoting sequences encode
unique properties,1 on par with the extraordinary structural and
the stereoselective folding of the peptide ligands and define the
2
functional complexity displayed by peptides. Bridging the
physicochemical properties of their corresponding metal complexes.
architectural potential of coordination chemistry with the structural
CD and NMR spectroscopy in combination with computational
predictability of peptide scaffolds would allow greater complexity
methods allowed us to identify and determine the structure of two
in (supra)molecular designs by combining well-established
isochiral ΛΛ-helicates, folded as topological isomers. Finally, in
peptide engineering concepts and tools with the supramolecular
addition to the in vitro characterization of their selective binding to
organization mediated by metal ions.3
DNA three-way junctions, cell microscopy experiments demonstrated
Helicates are discrete metal complexes in which one or more
that
a rhodamine-labeled Fe(II) helicate was internalized and
organic ligands coil around—and coordinate—two or more metal
ions. Helicates display helical chirality, according to the
selectively stains DNA replication factories in functional cells.
4
orientation in which the ligands twist around the axis defined by
the metal centers. Besides their fundamental interest in
supramolecular chemistry,5 helicates have also shown exciting
6
properties as antifreeze agents, as inhibitors of amyloid
aggregation, 7 as well as G-quadruplex and three-way DNA
binding properties that result in promising antimicrobial and
antitumoral activities.8 Therefore, as a challenging and relevant
model system to demonstrate the potential of peptide platforms
for engineering metallo-supramolecular entities, we decided to
approach the stereoselective synthesis of peptide helicates.
[a]
Dr. J. Gómez-González., A. Prof. M. E. Vázquez*
Centro Singular de Investigación en Química Biolóxica e Materiais
Moleculares (CiQUS), Departamento de Química Orgánica,
Universidade de Santiago de Compostela, Spain.
E-mail: eugenio.vazquez@usc.es
[b]
[c]
[d]
[e]
Dr. Y. Pérez
NMR Facility, Institute of Advanced Chemistry of Catalonia (IQAC-
CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain.
L. Roldán-Martín, Dr. G. Sciortino, Prof. J.-D. Maréchal
Departament de Química, Universitat Autònoma de Barcelona,
08193 Cerdanyola, Spain
We have previously described the synthesis of peptide
ligands containing a O1Pen-2,2'-bipyridine amino acid derivative,
which predictably fold into coordination compounds with defined
chirality,9 including self-assembled helicates,10 in the presence of
metal ions. Unfortunately, the two hydrophobic Pro-Gly β-turns
that encoded the folding of these peptide ligands,11 also made our
designs poorly soluble and prone to aggregation. Moreover, these
helicates, based on the kinetically labile Fe(II)-trisbipyridine
coordination, were inherently dynamic and displayed relatively
weak metal binding constants. Herein we describe the rational
design of alternative oligocationic peptide helicates with improved
solubility and thermodynamic stability, and the synthesis of
kinetically inert Co(III) helicates through oxidation of their
dynamically assembled Co(II) precursors. We also show that
these optimized self-assembled Fe(II) helicates bind to three-way
DNA junctions in vitro and in functional cells, selectively labelling
DNA replication foci in the cell nuclei.
Dr. G. Sciortino
Institute of Chemical Research of Catalonia (ICIQ)
Avgda. Països Catalans, 16, 43007 Tarragona, Spain.
Prof. J. Martínez-Costas
Centro Singular de Investigación en Química Biolóxica e Materiais
Moleculares (CiQUS), Departamento de Bioquímica y Biología
Molecular, Universidade de Santiago de Compostela, Spain.
Dr. I. Alfonso
Department of Biological Chemistry, Institute of Advanced
Chemistry of Catalonia (IQAC-CSIC), Jordi Girona 18-26, 08034
Barcelona, Spain
[f]
[g]
Prof. M. Vázquez López*
Centro Singular de Investigación en Química Biolóxica e Materiais
Moleculares (CiQUS), Departamento de Química Inorgánica,
Universidade de Santiago de Compostela, Spain.
E-mail: miguel.vazquez.lopez@usc.es
§
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.202013039
Angewandte Chemie International Edition
RESEARCH ARTICLE
with the formation of the Fe(II)2LLD and Co(II)2LLD helicates,
respectively (See Figure S5 and S7 in the Supporting information).
As expected, the fluorescence titrations with the DDL ligand
reproduced these results.
Results and Discussion
Rationale
In order to improve the solubility of the peptide ligands (and that
of the final helicates) we decided to replace the original β-turn
modules, (D/L)-Pro-Gly, with more polar sequences that were also
capable of inducing a reverse β-turn to direct the folding of the
peptide chain into the desired three-stranded helicates.12 Based
on the known tendency of heterochiral sequences to promote the
formation of type II or type II' β-turns,13 we focused our attention
on the combination of residues with mixed chirality, D-D-L (or its
mirror image L-L-D) which were reported to increase turn formation
and stability in short peptides.14 This pattern was very attractive
for our purposes, because it allowed us great freedom in the
choice of the residues in the loops, as long as they maintained
their relative chirality. Thus, in order to maximize the solubility,
and considering the prevalence of Arg residues in protein-DNA
complexes,15 as well as the known beneficial effects of arginine
residues in DNA-binding helicates,16 we selected the sequences
L-Arg–L-Pro–D-Arg and D-Arg–D-Pro–L-Arg to define the β-turns
and direct the folding of the peptide ligands into two enantiomeric
peptide helicates, LLD and DDL respectively (Scheme 1).
Additionally, we replaced the original O1PenBpy building block
O
O
H
O
Earlier work
–GPG– or –GpG– hydrophobic β-turns
flexible O1PenBpy coordinating residues
N
N
N
H
N
N
O
N
H
N
O
lead to…
N
M
M(II)
O
NH
N
NH2
NH2
H
Weak metal binding
Low solubility
N
O
N
HN
H2N
O
O
O
O
FmocHN
N
H
CO2H
H2N
NH
N
N
Fmoc-βAlaBpy-OH (1)
HN
FmocHN
HATU
DIEA 0.195 M /DMF
SPPS, 12 cycles
TFA cleavage
L-Pro
L-Arg
NH
O
N
O
O
O
O
H2N
N
O
NH
H
N
H
N
N
N
N
H
NH
N
H
N
O
H2N
O
N
H
H
N
N
L-Arg
N
N
D-Arg
NH
N
H
N
NH
NH2
NH
N
O
HN
O
L-Pro
HN
O
O
O
O
NH2
O
H
N
with
a shorter β-Ala derivative (βAlaBpy) to reduce the
O
N
H
O
conformational freedom of the loops and therefore increase the
preorganization and thermodynamic stability of the assembled
helicates.
N
N
N
HN
O
N
H
N
D-Arg
NH
NH
H2N
LLD
H
(βAlaBpy)2-RPr-(βAlaBpy)2-RPr-(βAlaBpy)2 NH2
Synthesis and characterization of the peptide helicates
H
N
Fe(II) or Co(II)
phosphate buffer 1 mM
pH 6.5
H2N
The precursor peptide ligands LLD and DDL were obtained by
standard Fmoc solid-phase peptide synthesis protocols.17 The
Bpy ligands were introduced in the form of an Fmoc-protected
building block, Fmoc-βAlaBpy-OH (1, Scheme 1), which was
synthesized with minor modifications of reported procedures
reported for the synthesis of Fmoc-O1PenBpy-OH.9 The final
peptide ligands were purified by HPLC, and their identity
confirmed by mass spectrometry (See the Experimental Section
and Supporting Information for details). As expected, the four
charged arginine residues in the sequence made these new
peptide ligands readily soluble in water, thus radically improving
the poor solubility of our previous design.10
H2N
NH2
NH
NH2
H2N
HN
NH2
O
L-Arg
L-Arg
O
O
O
N
HN
N
N
O
N
H
H
O
L-Pro
NH
N
HN
HN
O
N
L-Pro
NH
N
O
N
D-Arg
N
O
O
N
O
M(II)
H
N
M(II)
N
NH
N
O
N
H
N
H
O
H
N
N
N
N
H
N
N
NH2
HN
O
HN
H3N
O
NH
NH2
H2N
O
O
O
D-Arg
M(II)2LLD, M = Fe or Co
This work
–RPr– or –rpR– ionizable loops
shorter βAlaBpy coordinating residues
Tight metal binding
High solubility
enhanced twDNA binding
lead to…
Scheme 1. Top: Key elements in our first-generation design using (D/L)-Pro-Gly
and flexible O1PenBpy building block. Bottom: Solid-phase peptide synthesis of
the helicate ligand LLD using the Fmoc-βAlaBpy-OH building block. β-turn
sequences (RPr) are highlighted in light blue, and one βAlaBpy building block
in green. After cleavage and purification, the helicate is stereoselectively folded
under thermodynamic control in the presence of coordinating metal ions, Fe(II)
or Co(II). The LLD ligand contains two loops featuring the heterochiral L-Arg–L-
Pro–D-Arg sequence, while the DDL contains enantiomeric D-Arg–D-Pro–L-Arg
loops (H-(βAlaBpy)2-rpR-(βAlaBpy)2-rpR-(βAlaBpy)2-NH2). L-amino acids are
indicated in upper-case, and D-amino acids in lower case (i.e., R, P for L-Arg
and L-Pro, and r, p for D-Arg and D-Pro).
The assembly of the Fe(II)2LLD and Co(II)2LLD helicates in
solution was characterized by monitoring the quenching in the
fluorescence emission intensity of the 5'-amido-[2,2'-bipyridine]-
5-carboxamide upon metal coordination (Figure 1a).18 , 19 The
resulting titration profile was successfully fitted to a 1:2 binding
mode with dissociation constants of about 0.5 and 0.4 µM for the
first, and second metal ion coordination for both Fe(II) and
Co(II).20 These values are two orders of magnitude smaller than
those of the original O1Pen-based peptide helicates,10
demonstrating the beneficial effect of the reduced conformational
freedom of the βAlaBpy building block leading to helicates high
thermodynamic stability. Furthermore, the observed positive
cooperativity suggests that the coordination of the first metal ion
further preorganizes the peptide for the binding of the second
ion.21 MALDI spectra of the solutions at saturating concentrations
of metal ions showed peaks at 2555.06 and 2561.02, consistent
Importantly, the LLD and DDL peptide ligands displayed mirror
image circular dichroism spectra. The CD was dominated by a
strong Cotton effect band at c.a. 320 nm, which pointed to a
significant preorganization of the peptide chain and the effective
chiral induction by the heterochiral β-turn modules in combination
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10.1002/anie.202013039
Angewandte Chemie International Edition
RESEARCH ARTICLE
with π-stacking interactions between the bipyridine units.9a, 22
Addition of Fe(II) ions to a LLD solution resulted in a large
enhancement of the positive Cotton effect band at c.a. 320 nm,
as well as the appearance of a weaker negative Cotton effect
band about 525 nm, both bands consistent with the formation of
a ΛΛ- (or M) helicate (Figure 1b). Likewise, the enantiomeric
peptide ligand DDL gave rise to opposite bands at 320 and 525
nm, as expected for the induction of the mirror image ΔΔ- (or P)
helicate (Figure 1b). Taken together, these data are consistent
with a dynamic process in which the mixture of the peptide ligands
and the Fe(II) ions evolves towards the more stable folded
helicates under thermodynamic control.
Structural determination of two helicate topoisomers
The kinetic stability of this new Co(III)2LLD helicate allowed us to
obtain 2D NMR spectra with higher quality than those with the
1
complex with Fe(II), and the 1D H NMR spectra confirmed the
folding of the peptide ligand into stable and ordered structures
(Figure S13, Supporting Information), which encouraged us to
pursue their detailed structural characterization in solution. Based
on our previous work with metallopeptides and NMR studies of
dinuclear ruthenium (II) polypyridyl complex, 27 we decided to
apply the standard approach for peptide sequence assignment
and structural characterization,28 which consists in 2D NMR data
acquisition in protonated solvent
(90% H2O/10% D2O),
proton/carbon resonance assignment and the use of the identified
NOEs from the amino acid/bAla residues to obtain distance
restraints for molecular dynamics (See the Supporting Information
for a more detailed description of this strategy, 1D/2D spectra and
NOEs restraints). Curiously, the isolated fraction (Supporting
Information, Figure S8) showed two main components in solution,
isomers, A and B, in slow exchange in the NMR chemical shift
timescale (Figure 2). The resonances corresponding to 1D
aromatic/amide region and 2D 1H-1H NOESY correlations for
NH-a/bCH2 (in particular, for b-Ala3/5 at helicate turns) revealed
the conformational flexibility of the peptidic turns in the metal
complex. In contrast, the region of the Bpy ligands coordinated to
the metal ions is more rigid. For this reason, due to 15N T1 long
relaxation times, the 15N bipyridine resonances could not be
observed in the 15N-1H HMBC spectrum (data not shown). Based
on the NMR experimental data,29 and considering the similar
coordination geometries of the Co(III) and Fe(II) trisbipyridyl
complexes, we generated two reasonable starting point models of
the Fe(II) isomers A and B, and assessed their stability by
Molecular Dynamics (MD) simulations in explicit solvent and
periodic boundary conditions (See the Supporting Information for
details). During the simulations, the helical twist of the helicates,
as well as the octahedral coordination geometry of the Fe(II) metal
centers with the Bpy units were conserved. The root-mean square
deviation (RMSD) on the heavy atoms (excluding the side chains)
was computed during the trajectories using the minimized initial
structures as a reference. In both cases the trajectories stabilize
after the first 25 ns, reaching relatively stable RMSDs of 1.5 and
2.5 Å in average for isomers A and B, respectively. A cluster
analysis performed on the full-length MDs showed a predominant
geometry occupied about 64% and 89% of the total conformation
repartition for A and B. Starting from the representative structures
of these clusters, further molecular dynamics refinements where
carried out along 60 additional ns under NOE’s distance restraints
(Tables S3 and S4) in explicit solvent. The NMR refinement was
performed using SANDER including flat-bottom NOE-derived
distance potential function.30 Overall, the results show that the
two model structures, A and B, are valid candidates consistent
with the experimental observations (see Experimental Section for
a detailed analysis). Helicates A and B are topoisomers, that is,
they have the same chirality but different connectivity between the
Bpy units around the metal centers. In isomer A, the N-terminal
(blue, Figure 2) and the C-terminal (red, Figure 2) bipyridine units
are placed in the same side of the metallocylinder, whereas in
isomer B, both terminal Bpy units are disposed in opposite sides
of the helicate.31
a)
b)
10
5
1.0
0.5
0.0
0
10 20
0
[Fe(II)] (µM)
-5
-10
350 400 450 500 550 600
300 400 500 600
wavelength (nm)
wavelength (nm)
Figure 1. Titration of the LLD peptide ligand with Fe(II) ions. a) Normalized
emission spectrum of a 2 µM solution of LLD in 1 mM phosphate buffer NaCl
10 mM, pH 6.5 (thick line), and spectra of the same solution in the presence of
increasing concentrations of Fe(II) ions (thin lines); inset showing the titration
profile of three independent experiments and best fit to a one to two binding
model (same range as main plot); b) circular dichroism spectra of 5 µM solutions
of LLD (black dashed line) and DDL (red dashed line) in 1 mM phosphate buffer
NaCl 10 mM, pH 6.5, and in the presence of 25 µM Fe(II) ions (same colors,
continuous lines).
Locking the dynamic equilibrium by oxidizing Co(II) to Co(III)
The dynamic self-assembly of the helicates limits their potential
applications and prevents their detailed structural characterization,
because these self-assembled species are intrinsically dynamic
and could disassemble, exchange ligands, or modify their
structure or association state in response to changes in their
environment.23 To overcome these limitations we tested the in situ
formation of robust Co(III) helicates by oxidation of the
dynamically assembled Co(II) analogs.24 This strategy has been
extensively used for the assembly of kinetically-inert metal
complexes and described for the immobilization of His-tagged
proteins ,25 but, to our knowledge, has never been used in the
case of artificial metallopeptides. Thus, preformed solution of the
Co(II)2LLD helicate was treated with ceric ammonium nitrate,
(NH4)2Ce(NO3)6, and analyzed by HPLC. Gratifyingly, while the
labile helicates Fe(II)2LLD and Co(II)2LLD could never be
observed in the acidic RP-HPLC conditions (0.1% TFA
H2O/CH3CN), after oxidation we could isolate a new peak with
mass consistent with the formation of the Co(III)2LLD helicate,
and a circular dichroism spectrum indicating the presence of
species with the expected ΛΛ-chirality (Figure S10).26
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10.1002/anie.202013039
Angewandte Chemie International Edition
RESEARCH ARTICLE
b)
a)
1.0
1.0
0.5
0.0
0.5
0.0
0
5 10 15
[Fe(II)2LLD]
(µM)
500 550 600 650
0
10
20
[Co(III)2LLD] (µM)
wavelength (nm)
Figure 3. a) Normalized emission spectra of a titration of a 2 µM solution of the
fluorescein-labeled three-way junction DNA, Flu-twDNA, in 1 mM phosphate
buffer, 10 mM NaCl, pH 6.5 (thick line) with increasing concentrations of the
Fe(II)2LLD helicate (thin lines); inset: corresponding normalized titration profile
of the fluorescein quenching at 515 nm and best fit to a one to one binding
model; b) Normalized emission of a 2 µM solution of the fluorescein-labeled
three-way junction DNA, Flu-twDNA, in 10 mM phosphate buffer, 100 mM NaCl,
10 mM MgCl2, pH 6.5 with increasing concentrations of the Co(III)2LLD; the best
fit to a simple one to one binding model is shown as a dashed line, and the fit to
a model including non-specific binding is shown as a continuous line. Titration
data are the average of three independent experiments. Flu-twDNA, Y1 Flu-5'–
TTTT CAC CGC TCT GGT CCT C–3'; Y2 5'–CAG GCT GTG AGC GGT G–3';
Y3 5'–GAG GAC CAA CAG CCT G–3'.
Figure 2. Top: assembly-followed-by-fixing approach for the construction of
kinetically-inert Co(III)2LLD peptide helicates. The initial dynamic selection
under thermodynamic control is followed by the oxidation of Co(II) to Co(III) to
yield kinetically-inert complexes A and B, which have the same chirality, but
different topology. Bottom: representative structures for the Fe(II) helicates of
the two most populated clusters of the MD trajectories (stick) together with 100
frames of each trajectory. N-terminal bipyridine in blue, and C-terminal
bipyridine in red.
To better understand the interaction of the Fe(II) and Co(III)
helicates with the DNA, we carried out electrophoretic mobility
shift assays (EMSA) in polyacrylamide gel under non-denaturing
conditions and using SYBRGold as the DNA stain.34 In agreement
with the fluorescence studies, we found that incubation of twDNA
with increasing concentrations of the preformed Fe(II)2LLD
helicate resulted in the appearance of new slow-migrating band,
consistent with the formation of the twDNA/Fe(II)2LLD complex
(Figure 4a). As expected for a high-affinity interaction, complete
band shift is observed already at the lowest concentrations tested,
and gives rise to a single new band with no smearing, even at
high concentration of the Fe(II)2LLD helicate. On the other hand,
no new slow-migrating bands were observed upon incubation of
Fe(II)2LLD with a double-stranded DNA (dsDNA), which confirms
the low affinity of this metallopeptide for regular B-DNA (Figure
4b). In agreement with the fluorescence titrations, the EMSA
assays showed reduced affinity of the Co(III)2LLD helicate for the
twDNA, so that the saturation of the twDNA is only observed at
the highest concentrations of Co(III)2LLD (500 nM, Figure 4c).
Remarkably, these assays also confirmed the tendency of the
highly charged Co(III)2LLD helicate to form non-specific
complexes, as shown by the disappearance of the dsDNA band
upon incubation with this helicate (Figure 4d).35
The peptide helicates selectively recognize three-way DNA
junctions in vitro
Helicates are known to selectively interact with DNA three-way
junctions by insertion into the hydrophobic cavity created by the
surface of the base pairs at the center of the DNA junction.8d,32 To
study the DNA binding properties of these new peptide helicates
we prepared a 2 µM solution of a fluorescein-labeled three-way
junction DNA (Flu-twDNA), and measured its luminescence after
the addition of successive aliquots of a solution of the preformed
helicate Fe(II)2LLD. The progressive quenching of the fluorescein
emission at 515 nm could be fitted to a simple 1:1 binding mode
(Flu-twDNA/Fe(II)2LLD) with an apparent dissociation constant
KD ≈ 0.45 µM,33 which is in line with the data obtained for the
original Pro-Gly Fe(II) peptide helicate (Figure 3). Curiously, in the
case of the helicate Co(III)2LLD, the titration profile of the
emission intensity at 515 nm had to be fitted to a more complex
model with a lower-affinity binding site, likely corresponding to the
insertion of the Co(III)2LLD metallopeptide in the hydrophobic
cavity, with a KD ≈ 7.9 µM, and three equivalent higher-affinity
sites (KD ≈ 4.9 µM), which based on reported X-ray structures of
related helicate/DNA complexes could correspond to nonspecific
stacking interactions of the helicate with the blunt ends of the
twDNA arms.8d
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10.1002/anie.202013039
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RESEARCH ARTICLE
A rhodamine-labelled Fe(II)-helicate selectively labels DNA
replication sites in functional cells
Having demonstrated the binding of these helicates to twDNA in
vitro, we envisioned the application of these helicates to study
these DNA structures in cells. Preliminary experiments in which
the tetramethylrhodamine- and fluorescein-labeled Fe(II)
37
helicates, Fe(II)2TAMRA-LLD or Fe(II)2Flu-LLD,
were
incubated overnight with HeLa cells showed that these complexes
Figure 4. a) EMSA analysis of 200 nM twDNA in the presence of 0, 50, 100,
250, 500 nM of Fe(II)2LLD. The new, slow-migrating band (indicated with an
arrow), corresponds to the three-way DNA structure assembled in the presence
of the Fe(II) helicate; b) 75 nM dsDNA with 0, 1000, and 1500 nM of Fe(II)2LLD;
gels in c) and d) correspond to the same concentrations of twDNA and dsDNA
as a) and b) incubated with the same concentrations of the Co(III)2LLD helicate
(dsDNA 5'–AAC ACA TGC AGG ACG GCG CTT–3'; twDNA, Y1 5'–CAC CGC
TCT GGT CCT C–3'; Y2 5'–CAG GCT GTG AGC GGT G–3'; Y3 5'–GAG GAC
CAA CAG CCT G–3').
remain trapped in endosomes (see Figure S28 in the Supporting
38
information).
However, if the cells were pretreated with
Digitonin,39 then Fe(II)2TAMRA-LLD was effectively internalized
and showed in the red emission channel as a punctuated pattern
in the cytoplasm and the nucleus (Figure 6a). Remarkably, the
nuclear distribution of the helicate partially matched the
localization of the DNA replication sites labeled with the
proliferating cell nuclear antigen (PCNA) fused to GFP (Figure
6b), 40 which demonstrates that Fe(II) helicate is capable of
targeting the three-way junctions transiently formed during DNA
replication in functional cells (Figure 6c).41 The staining of what
appears to be the cell nucleolus is consistent with
Fe(II)2TAMRA-LLD binding to three-way RNA structures, which
are also potential biological targets of helicates. 42 To our
knowledge, this is the first demonstration of in cellula twDNA
staining with designed fluorescent probes. As expected from the
in vitro experiments, the Co(III)2TAMRA-LLD helicate displayed
poor specificity, leading to indiscriminate labeling in Digitonin
treated cells (data not shown).
Based on the previous structural characterization, we used the
new GaudiMM platform to obtain plausible models for two
Fe(II)2LLD topoisomers threaded into the DNA three-way
junction.36 The structures of the Fe(II) helicates extracted from the
MD simulation under NOE constraints were docked to the DNA
extracted from the X-ray structure of the helicate/three-way DNA
junction complex reported by Hannon et al. (PDB ID: 2ET0).8d
Energetically preferred orientations of the helicates inside the
central cavity of the DNA three-way junction were found when
considering displacements of up to 2.5 Angstroms along the ten
lowest-energy normal mode vectors of the breathing modes of the
twDNA (see Docking studies in the Experimental section), thus
demonstrating that the accommodation of the helicates requires
adaptation of the DNA target to the guests and that leads to a
slightly different conformation than in the available X-ray structure
taken as reference. Calculations with the two different isomers
also showed a better accommodation of isomer B into the three-
way DNA cavity showing higher scoring and lower clashes
respect to the isomer A (see Supporting Information for further
details). No clear penalizing interactions between the three-way
DNA junction and the helicate units are observed although the
center of mass of B aligned better with the twDNA and the entire
complex fits more deeply into the DNA receptor. This is likely
related to the smaller volume of B and its slightly more stretched
geometry (Figure 5 and S17).
Figure 5. Fe(II)2TAMRA-LLD selectively stains DNA replication sites. HeLa
cells expressing protein GFP-PCNAL2 were incubated with 25 µg/ml Digitonin
for
3 min, then 5 µM of TAMRA-LLD peptide ligand and 25 µM of
(NH4)2Fe(SO4)2 for 30 min. a) Red channel emission showing the distribution of
the rhodamine-labeled helicate; b) green channel, corresponding to the
emission of the GFP-PCNAL2 probe labeling the DNA replication foci; c) overlay
of the green and red channels of the square region shown in a) Arrows highlight
some foci where the staining of GFP-PCNAL2 and Fe(II)2TAMRA-LLD overlap.
Conclusion
We have demonstrated that peptide design rules can be applied
to encode both structural and chiral information in de novo
metallopeptides. In particular, we report new oligocationic
peptides containing bipyridine ligands that fold into three-stranded
helicates in the presence of Fe(II) ions and bind with high affinity
and specificity to DNA three-way junctions. For the first time, we
have determined the atomic structure of these peptide helicates
by a combination of CD, NMR and sophisticated computational
methods, and applied a new theoretical approach to explore the
nature of their DNA complexes. Finally, we also demonstrate that
the Fe(II)2TAMRA-LLDhelicate binds to DNA three-way junctions
Figure 5. Lowest energy pose of isomer B of the Fe(II)2LLD helicate in the
central cavity of the twDNA. Left: top view showing the tight fit between the
helicate and the twDNA hydrophobic site, which appears to be too narrow to
comfortably accommodate isomer A. Right view of the same complex.
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10.1002/anie.202013039
Angewandte Chemie International Edition
RESEARCH ARTICLE
in functional cells, selectively labeling DNA replication foci in the
nuclei, thus demonstrating for the first time the selective staining
of three-way DNA in cellula with designed fluorescent probes.
Innovation/Spanish Research Agency (MCI/AEI/FEDER, RTI218-
096182-B-I00, CSIC13-4E-2076) and AGAUR (2017 SGR 208).
G.S. L.R.-M. and J.-D.M. thank Spanish MINECO (grant
CTQ2017-87889-P) and the Generalitat de Catalunya
(2017SGR1323). G.S., thanks Regione Autonoma della
Sardegna (grant RASSR79857) for his postdoctoral fellowship. L.
R.-M. thanks the Generalitat de Catalunya for her FI fellowship.
plasmid GFP-PCNAL2 was generously provided by Dr. Cristina
Cardoso. Molecular graphics with UCSF Chimera, developed by
the Resource for Biocomputing, Visualization, and Informatics at
the University of California, San Francisco, with support from NIH
P41-GM103311. 43 We are also grateful to Prof. Peter Scott of the
IAS at the University of Warwick for his insights and advice during
the preparation of this manuscript.
Acknowledgements
Financial support from the Spanish grant RTI2018-099877-B-I00,
the Xunta de Galicia (grupos con potencial de crecemento
ED431B 2018/04, Centro singular de investigación de Galicia
accreditation 2016–2019, ED431G/09, and ED431B 2018/04)
and the European Union (European Regional Development Fund
- ERDF) are gratefully acknowledged. J. G.-G. thanks the Spanish
Ministry of Science and Innovation/Spanish Research Agency for
his FPI fellowship. We also wish to express our gratitude to
Soraya Learte-Aymamí for her assistance in the optimization of
the EMSA assays. I. A. thanks Spanish Ministry of Science and
Keywords: DNA recognition • peptide design • metallopeptides •
supramolecular chemistry • coordination chemistry
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Entry for the Table of Contents
COMMUNICATION
J. Gómez-González, Y. Pérez, G.
Sciortino, L. Roldán-Martín, J. Martínez-
Costas, J.-D. Maréchal, I. Alfonso, M.
Vázquez López,* M. E. Vázquez*
Page No. – Page No.
Dynamic Stereoselection of Peptide
Helicates and Their Selective
Labeling of DNA Replication Foci in
Cells
Artificial bipyridine-containing peptide ligands encode in their sequence the
stereoselective folding into chiral three-stranded Fe(II) or Co(II) peptide helicates,
and as kinetically-inert dinuclear Co(III) complexes by in situ oxidation. These
peptide helicates selectively label DNA replication foci in functional cells.
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