pubs.acs.org/bc Communication Stereoselective Self-Assembly of DNA Binding Helicates Directed by the Viral β‑Annulus Trimeric Peptide Motif Jacobo Gómez-González,∥ David Bouzada,∥ Lidia A. Pérez-Márquez, Giuseppe Sciortino, Jean-Didier Maréchal, Miguel Vázquez López,* and M. Eugenio Vázquez* Cite This: Bioconjugate Chem. 2021, 32, 1564−1569 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Combining coordination chemistry and peptide engineering offers extraordinary opportunities for developing novel molecular (supra)structures. Here, we demonstrate that the β- annulus motif is capable of directing the stereoselective assembly of designed peptides containing 2,2′-bipyridine ligands into parallel three-stranded chiral peptide helicates, and that these helicates selectively bind with high affinity to three-way DNA junctions. P eptides are ideal platforms for the programmed assembly of supramolecular structures, as they encode in their sequences precise structural and functional information in their a short dodecapeptide, G48ISMAPSAQGAM59, from the N- terminus of the C subunits of the coat protein of the Sesbania Mosaic Virus (SeMV) capsid.37 Structurally, the β-annulus is a sequence. Several peptide motifs, such as coiled-coils, β- three-way junction of two-stranded β-sheets formed between hairpins, or amphiphilic peptides, have been studied as the residues 48−52 of each strand with residues 55−58 of the basis for biofunctional devices and materials.1−9 However, symmetric peptide chain.38 The backbone of the polypeptide despite the enormous potential for the control of stereo- displays a 120° turn that allows this arrangement thanks to a chemistry, nuclearity, and stoichiometry, the assembly of metal central residue Pro53 (Figure 1a,b). The β-annulus has been complexes driven by peptide motifs has only started to take previously used for the formation of nanospheres and large off,10−12 and most examples in the literature are restricted to systems based on coiled-coils.13−19 As a test case to show the aggregates,39 but its application to encode the assembly of potential of peptide motifs to direct the assembly of metal discrete supramolecules has not yet been explored. Upon complexes, we focused our attention on helicates,20 which are inspection of the β-annulus structure (PDB code 1X33),40 we discrete metal complexes in which one or more organic ligands realized that the three symmetrically equivalent Ser54 residues coordinate two or more metal ions,21−23 because of their located at the center of the β-annulus were ideally positioned interest in supramolecular chemistry and exciting biological to serve as anchor points for the introduction of the helicate applications.24−30 Helicates are inherently chiral species that strands composed by two 2,2′-bipyridine building blocks can show right-handed (designated as P) or left-handed (M) (βAlaBpy) in tandem (Figure 1c and Scheme 1).41 Exploratory helicity, according to the orientation in which the ligands coil molecular modeling studies confirmed that a mutated peptide around the axis defined by the metal centers. Indeed, one of featuring a set of two chelating βAlaBpy units attached to the the biggest challenges for the synthesis of helicates is their side-chains of mutated Lys residues at that position could stereoselective assembly with controlled supramolecular coordinate a pair of metal ions forming a dinuclear helicate chirality.31−34 In this context, we wanted to test whether a without significantly distorting the β-annulus structure (Figure small trimeric peptide motif could effectively control the self- 1d,e). assembly of three-stranded peptide helicates, selecting a particular orientation of the ligand chains and helical chirality. In contrast to our previous approach to helicate synthesis Received: June 16, 2021 relying on the folding of a single peptide chain,35,36 here we Revised: July 25, 2021 would rely on the assembly of three independent peptide Published: July 28, 2021 monomers to template the formation of the desired helicate. As an alternative platform to the omnipresent coiled-coils, we focused our attention on the C3-symmetric β-annulus motif, © 2021 American Chemical Society https://doi.org/10.1021/acs.bioconjchem.1c00312 1564 Bioconjugate Chem. 2021, 32, 1564−1569 Bioconjugate Chemistry pubs.acs.org/bc Communication The target peptide was built following standard solid-phase peptide synthesis protocols,43 and once the β-annulus strand was fully assembled, the Alloc group was selectively removed from the Lys side chain under catalytic conditions (Pd(PPh3)4, PhSiH3, Scheme 1), and the metal-chelating 2,2′-bipyridine bulding blocks (Fmoc-βAlaBpy-OH) were sequentially at- tached to the orthogonally deprotected Lys ζNH2 (Scheme 1). Finally, the deprotection and release of the peptide from the resin was carried out using standard conditions by treatment with an acidic TFA cocktail. The peptide was purified by reverse-phase HPLC, and the identity confirmed by MS (MALDI-TOF). Having at hand the desired peptide, we next studied the binding of the peptide β-annK(Bpy)2 to Fe(II) and its capacity for templating the formation of the corresponding helicate. For this, we added increasing amounts of (NH4)2Fe- (SO4)2·6 H2O (Mohr’s salt) to a buffered solution of β-annK(Bpy)2 and recorded the progressive decrease in the emission of the Bpy ligand at 402 nm after each addition (Figure 2a). The resulting titration profile could be fitted to a 2:3 interaction model with the DynaFit program,44,45 with tight dissociation constants, KD1 = 5.0 ± 3.3 μM and KD2 = 3.5 ± Figure 1. Design of the β-annulus helicate. (a) Structure of the 0.7 μM for the first and second coordination, respectively. In Sesbania Mosaic Virus (SeMV) capsid, highlighting the β-annulus addition to the spectroscopic data, ESI-MS analysis of the motif (in red), at the center of the C subunit trimer in green (PDB ID saturated solution confirmed the formation of the expected 1X33). (b) Isolated β-annulus showing the relative orientation of the complex Fe(II)2[β-annK(Bpy)2]3 with clear peaks correspond- three peptide chains and the natural residues in one of the symmetric ing to [M+3H]3+ = 1754,5; [M+4H]4+ = 1316.2; [M+5]5+ = peptide strands. Note the position of Ser54 near the center of the 1053.2 species (see Figure S6, Supporting Information). annulus. Bottom: modification of the β-annulus to introduce the Having demonstrated that the β-annK(Bpy)2 peptide self- coordinating βAlaBpy residues. (c) Detail of the β-annulus high- assembles and coordinates Fe(II) ions, we were interested in lighting the distance between Ser54 residues. (d) Ser54 are mutated into Lys residues, that serve as anchor points for the introduction of evaluating if the chirality of the annulus could be translated the chelating βAlaBpy residues to yield the β-annK(Bpy)2 ligand into helical chirality in the complex. In other words, if the precursor of the helicate shown in (e). formation of the helicate Fe(II)2[β-annK(Bpy)2]3 was stereo- selective. Thus, we recorded the circular dichroism (CD) spectra of a 100 μM solution of the β-annK(Bpy)2 peptide and To obtain the helicate precursor, we first synthesized the of the same solution in the presence of saturating peptide Ac-G48IS-£51-AP-Lys(Alloc)54-AQGAK59-NH2 with concentration of Fe(II) (15 equiv). The spectrum of the the orthogonally protected Lys handle in place of Ser54. In peptide by itself displayed the typical signature of a β-sheet addition, the residue Met51 was replaced with an isosteric nor- structure in the far UV region, as well as a clear CD band in the leucine residue (£51) to avoid potential oxidation problems.42 wavelength range of the 2,2′-bipyridine units at ca. 320 nm, Finally, for synthetic reasons, and in order to promote the which indicated a clear preorganization of these ligands. solubility of the helicate precursor peptide, the C-terminal Furthermore, the CD spectrum in the presence of the Fe(II) Met59 was replaced with an ionizable Lys residue (K59). showed a more clear Cotton effect in the bipyridine band, Scheme 1. Solid-Phase Peptide Synthesis (SPPS) of the β-Annulus Helicate Precursor Peptide Ligand β-annK(Bpy)2 1565 https://doi.org/10.1021/acs.bioconjchem.1c00312 Bioconjugate Chem. 2021, 32, 1564−1569 Bioconjugate Chemistry pubs.acs.org/bc Communication Figure 2. Left: emission spectra of a 20 μM β-annK(Bpy)2 peptide Figure 3. Left: Fluorescence spectra of a 2 μM solution of fluorescein- solution (1 mM PBS buffer, 10 mM NaCl, pH 6.5) with increasing labeled twDNA in 1 mM PBS buffer, 10 mM NaCl, pH 6.5 (dashed concentrations of (NH4)2Fe(SO4)2·6H2O. Inset: Titration profile of line), and the same solution in the presence of increasing the maximum emission wavelength at 401 nm with increasing concentrations of the Fe(II)2[β-annK(Bpy)2]3 helicate (full lines). concentrations of Fe(II). The best fit according to the 2:3 model in Right: titration profile (emission intensity at 515 nm) and the best fit DynaFit is also shown. Experimental data points correspond to the to a 1:1 Fe(II)2[β-annK(Bpy)2]3 helicate/twDNA binding. The average of three independent titrations. Right: CD spectra of a 100 experimental points correspond to the mean of three independent μM β-annK(Bpy)2 peptide solution in HEPES 10 mM buffer, NaCl titrations. 100 mM, and pH 6.5 (dashed line), and of the same solution in the presence of 1.5 mM Fe(II) (solid line); the CD intensity of the Fe(II) complex was weaker than that of the isolated peptide so, for clarity purposes, its CD spectrum has been represented multiplied by ten. Fe(II)2[β-annK(Bpy)2]3 complex) with an apparent KD of 308 ± 60 nM (Figure 3, Right). In contrast, the titration profile of the helicate with a model duplex DNA could not be which also showed a bathochromic shift; taken together, these fitted to a simple 1:1 binding model and required the changes in the CD spectrum are consistent with the formation introduction of nonspecific interactions in our analysis, in of a helicate complex with P helicity (ΔΔchirality in both metal agreement with earlier reports with organic ligand helicates.48 centers). Furthermore, the affinity of the helicate for the regular dsDNA The stability of the two possible helicate chiralities (P and was significantly lower than for the three-way junction, with an M) in Fe(II)2[β-annK(Bpy)2]3 was assessed by molecular apparent KD of ∼5 μM). dynamics (MD) simulations in explicit solvent and periodic In addition to the spectroscopic studies, we also analyzed the boundary conditions (see the Supporting Information for DNA binding of the helicate Fe(II)2[β-annK(Bpy)2]3 by computational details). The helical conformation of the electrophoretic mobility assays (EMSA) in polyacrylamide gel helicate unit Fe(II)2[(βAlaBpy)2]3, as well as the octahedral under nondenaturing conditions.49 In agreement with the coordination geometry of the Fe(II) ions, was conserved along fluorescence titration studies, incubation of the target three- the simulations, and the trajectories attain relatively stable way DNA, twDNA, with increasing concentrations of the RMSDs with respect to the initial structures after the first ∼10 peptide helicate resulted in the appearance of a new slow- ns (2.00 ± 0.52 Å and 1.92 ± 0.46 Å as average for P and M migrating band, consistent with the formation of the expected conformations, respectively); the β-annulus is stabilized after twDNA/Fe(II)2[β-annK(Bpy)2]3 complex (Figure 4, lanes 1− 40 ns with an average RMSD of about 11 Å. Cluster analysis 6). Remarkably, no smearing is observed, even at high was performed on the full-length MD experiments showing concentrations of the helicate Fe(II)2[β-annK(Bpy)2]3, and two predominant conformations due to the high mobility of only one band is formed, thus demonstrating the formation of the C-terminal chains that can be directed to the bulk of the a unique complex.50,51 On the other hand, incubation of a solvent or close to the βAlaBpy units, interacting by π-stacking model dsDNA with helicate Fe(II)2[β-annK(Bpy)2]3 did not with the Bpy rings and lipophilic contacts with the βAla induce the formation of new retarded bands, even at high residue. The conformation in which the C-terminal Lys concentrations of the helicate, which clearly confirms the low residues interact with βAlaBpy appear to be more stable for affinity of this complex for regular B-DNA (Figure 4, lanes 7− the P isomer. 10), and supports the model in which the observed Helicates are known to selectively interact with three-way fluorescence quenching is due to of low-affinity nonspecific DNA junctions.46 In order to study the binding of the β- interactions, which are not observed in the more stringent gel annulus helicate to the DNA, we relied on the observation that electrophoresis conditions. the Fe(II) complex has a strong quenching effect in nearby We have demonstrated that the short β-annulus motif from fluorophores.47 Therefore, we prepared a 2 μM solution of a the Sesbania Mosaic Virus can be modified to direct the fluorescein-labeled three-way junction DNA (twDNA, FAM- stereoselective self-assembly of peptide helicates. Modeling 5′-TTTT CAC CGC TCT GGT CCT C-3′; 5′-CAG GCT studies support the proposed assembly in which the β-annulus GTG AGC GGT G-3′; 5′-GAG GAC CAA CAG CCT G-3′) directs the trimerization, the relative orientation of the and recorded its emission upon excitation at 490 nm after the bipyridine ligands, and induces a P helicity in the resulting addition of successive aliquots of a solution containing the helicate. Furthermore, the resulting helicate displays high preformed helicate Fe(II)2[β-annK(Bpy)2]3 (Figure 3, Left). selectivity toward three-way DNA junctions over regular B- The titration profile of the emission intensity at 515 nm could dsDNA, as shown by both spectroscopic and electrophoretic be fitted to an 1:1 binding mode (twDNA/ assays. 1566 https://doi.org/10.1021/acs.bioconjchem.1c00312 Bioconjugate Chem. 2021, 32, 1564−1569 Bioconjugate Chemistry pubs.acs.org/bc Communication ́ Investigaciones Cientificas (CSIC), Avda. Astrofísico Fco. Sánchez 3, 38206 La Laguna, Tenerife, Spain Giuseppe Sciortino − Insilichem, Departament de Química, Universitat Autònoma de Barcelona, 08193 Cerdanyola, Spain; Present Address: Institute of Chemical Research of Catalonia (ICIQ). Avgda. Paisos̈ Catalans, 16, 43007 Tarragona, Spain.; orcid.org/0000-0001-9657-1788 Jean-Didier Maréchal − Insilichem, Departament de Química, Universitat Autònoma de Barcelona, 08193 Cerdanyola, Spain; orcid.org/0000-0002-8344-9043 Complete contact information is available at: Figure 4. EMSA binding studies results for Fe(II)2[β-annK(Bpy)2]3 https://pubs.acs.org/10.1021/acs.bioconjchem.1c00312 helicate. Lanes 1−6, 200 nM twDNA with 0, 150, 250, 500, 1000, and 2000 nM of [β-annK(Bpy)2]3 and 20 equiv of (NH4)2Fe(SO4)2· Author Contributions ∥ 6H2O in each lane; lanes 7−10, 200 nM dsDNA with 0, 500, 1000, J.G.-G. and D.B. contributed equally. and 2000 nM of [β-annK(Bpy)2]3 and 20 equiv of (NH4)2Fe(SO4)2· Notes 6H2O in each lane. Samples were resolved on a 10% nondenaturing The authors declare no competing financial interest. ■ polyacrylamide gel and 0.5 × TBE buffer over 40 min at 25 °C and stained with SyBrGold (5 μL in 50 mL of 1 × TBE) for 10 min,52 followed by fluorescence visualization. Oligonucleotide sequences: ACKNOWLEDGMENTS dsDNA (only one strand), 5′-AAC ACA TGC AGG ACG GCG Financial support from the Spanish grant RTI2018-099877-B- CTT-3′; twDNA, 5′-CAC CGC TCT GGT CCT C-3′; 5′-CAG I00, the Xunta de Galicia (Centro singular de Investigación de GCT GTG AGC GGT G-3′; 5′-GAG GAC CAA CAG CCT G-3′. Galicia accreditation 2016−2019, ED431G/09, and ED431B ■ * ASSOCIATED CONTENT sı Supporting Information 2018/04), and the European Union (European Regional Development Fund - ERDF) is gratefully acknowledged. J.G.- G. thanks the Spanish MINECO for his FPI fellowship. D.B. The Supporting Information is available free of charge at thanks the CIQUS for his 2018 PhD fellowship. G.S. and J.- https://pubs.acs.org/doi/10.1021/acs.bioconjchem.1c00312. D.M. thank Spanish MINECO (grant CTQ2017-87889-P) and Detailed experimental procedures for the synthesis of Generalitat de Catalunya (2017SGR1323) for the financial the 2,2′-bipyridine building block, peptide synthesis and support. Finally, we would like to thank Prof. Kazunori characterization, EMSA experiments, and molecular Matsuura at Tottori University for his valuable input during the preparation of this manuscript. ■ modeling (PDF) ■ AUTHOR INFORMATION Corresponding Authors REFERENCES (1) Ulijn, R. V., and Smith, A. M. (2008) Designing Peptide Based Nanomaterials. Chem. Soc. Rev. 37 (4), 664−675. Miguel Vázquez López − Centro Singular de Investigación en (2) Gazit, E. (2007) Self-Assembled Peptide Nanostructures: The Química Biolóxica e Materiais Moleculares (CiQUS), Design of Molecular Building Blocks and Their Technological Departamento de Química Inorgánica, Universidade de Utilization. Chem. Soc. 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