Peptides are excellent supramolecular building blocks that encode precise structural and functional information within their amino acid sequence. Accordingly, researchers have explored diverse peptide motifs, such as coiled-coils, β-hairpins, or peptide amphiphiles, as the basis of biofunctional devices and materials (Matsuura et al., 2005, 2010; Gazit, 2007; Ulijn and Smith, 2008; Apostolovic et al., 2010; Robson Marsden and Kros, 2010; Boyle and Woolfson, 2011; Lai et al., 2012; Pazos et al., 2016). Curiously, despite the enormous potential for controlling stereochemistry, nuclearity and stoichiometry, the controlled supramolecular assembly of inorganic complexes with peptide motifs has been somewhat overlooked, and only a handful of systems based on modified coiled-coil motifs have been reported (Lieberman and Sasaki, 1991; Ghadiri et al., 1992; Li et al., 2000; Peacock et al., 2012; Ball, 2013; Berwick et al., 2014; Luo et al., 2016). On the other hand, helicates are discrete metal complexes in which one or more organic ligands are coiled around—and coordinating—two or more metal ions (Piguet et al., 1997; Albrecht, 2001, 2005) as a result of ligand coiling, helicates are inherently chiral species that can appear as two enantiomers according to the orientation in which the ligands twist around the helical axis defined by the metal centers. Besides their intrinsic interest in basic supramolecular chemistry, helicates have shown promising DNA-binding properties that have been associated with antimicrobial and antitumoral effects (Howson et al., 2012; Kaner et al., 2015). However, more than 20 years after the pioneering studies by Prof. Jean-Marie Lehn (Lehn et al., 1987; Ulijn and Smith, 2008), helicates are still not viable alternatives to traditional DNA-binding agents. The slow development in the applied chemistry of metal helicates ultimately derives from the shortcomings associated with the classic synthetic approaches with organic ligands that complicate the structural control of the final helicates (i.e., oligomerization state, relative orientation of asymmetric ligands, supramolecular helicity) and hampers their efficient structural and functional optimization. Indeed, despite some noteworthy examples (Haino et al., 2009; Cardo et al., 2011; Howson et al., 2012; Chen et al., 2017; Mitchell et al., 2017; Guan et al., 2018), no general approach for the efficient and versatile stereoselective synthesis of helicates is yet available, making of these systems a challenging test case to demonstrate the potential of peptides for the controlled assembly of metallostructures.

Our strategy relied in the selection of a synthetically-accessible and structurally well-defined trimeric peptide domain as scaffold for the programmed assembly of the helicate. As an alternative (and orthogonal) platform to the ubiquitous leucine zippers, we focused our attention on the C-terminal domain of the bacteriophage T4 Fibritin foldon (T4Ff), a trimeric β-propeller-like structure formed by the self-assembly of a short 27-amino acid peptide (Tao et al., 1997; Papanikolopoulou et al., 2004; Habazettl et al., 2009). The intrinsic stability and structural resilience of the T4Ff scaffold has been exploited for the stabilization of trimeric structures of a number of peptides and engineered proteins (Stetefeld et al., 2003; Du et al., 2011; Berthelmann et al., 2014; Kobayashi et al., 2015), and given those precedents we envisioned that the T4Ff could also be used as a robust platform for the programmed assembly of chiral dinuclear helicates, thus offering an alternative for the integration of coordination and peptide chemistry beyond other widely explored peptide scaffolds.

As metal-chelating unit we chose 2,2′-bipyridine, a ligand that has been extensively used in coordination chemistry and yields stable complexes with a variety of metal ions (Kaes et al., 2000). Furthermore, we have previously described an Fmoc-protected 2,2′-bipyridine dipeptide derivative that can be readily implemented into standard Fmoc solid-phase peptide synthesis (SPPS) protocols, and have showed that the structure of this chelating unit, in which the 2,2′-bipyridine ligand is integrated in the peptide backbone, effectively couples the conformational preferences of the peptide chain with the geometry of the resulting metal complexes (Rama et al., 2012; Gamba et al., 2013, 2014, 2016; Salvadó et al., 2016).

The chelating 2,2′-bipyridine residue was obtained following an optimized synthetic route (Rama et al., 2012), based on the work carried out by the Newkome and Imperiali groups (Newkome et al., 1997; Torrado et al., 1998). The key step in the synthesis being the desymmetrization of a diethyl [1,1′-biphenyl]-4,4′-dicarboxylate intermediate with hydrazine monohydrate under conditions that allow the selective precipitation of the monocarbohydrazide, which is oxidized into the corresponding azyl azide, and then transformed into a carbamate through a Curtius rearrangement (Rama et al., 2012). Simultaneous hydrolysis of the carbamate and the ester group gives the desired bipyridine amino acid, which is derivatized in the form of a dipeptide to obtain the Fmoc-βAlaBpy-OH building block for increased solubility, stability, and solubility that allow its use following standard solid-phase peptide synthesis protocols (Ishida et al., 2006).

Inspection of the structure of T4Ff (PDB IDs 4NCU or 1RFO; Güthe et al., 2004; Berthelmann et al., 2014) showed that the N-terminal Gly residues are relatively close to each other and could accommodate the chelating 2,2′-bipyridine units without noticeable distortion of the T4Ff scaffold upon metal coordination. Moreover, we envisioned that the natural twist of the N-terminal polyproline helices in the folded T4Ff trimer should induce a ΛΛ-configuration (M helicity) on its derived helicate (Tao et al., 1997), which would be the preferred chirality for the efficient recognition of three-way DNA junctions (Oleksy et al., 2006; Gamba et al., 2016). Therefore, we synthesized the desired (βAlaBpy)₂-T4Ff helicate precursor ligand following standard Fmoc SPPS methods as outlined in Figure 1 (Coin et al., 2007). The final peptide ligand was purified by HPLC and its identity confirmed by ESI-MS.

Having at hand the desired peptides we proceeded with the study of their metal binding properties. Surprisingly, while 2,2′-bipyridine is weakly emissive, and is even considered non-fluorescent (Dhanya and Bhattacharyya, 1992; Yagi et al., 1994), we found that the asymmetric 5′-amido-[2,2′-bipyridine]-5-carboxamide unit within the βAlaBpy residue was highly emissive, displaying intense band at c.a. 420 nm with a quantum yield of 0.37 (Dong et al., 2017). Additionally, the emission was quenched by coordination to Fe(II) ions, which could be exploited to monitor the formation of the β-annulus helicate. Thus, we recorded the emission spectra of a 3 μM solution (9 μM monomer) of [(βAlaBpy)₂-T4Ff]₃ in phosphate buffer (1 mM, pH 6.5) in the presence of increasing concentrations of (NH₄)₂Fe(SO₄)₂ • 6 H₂O (Mohr salt) as source of Fe(II) ions (λexc = 305 nm), and observed a concentration-dependent quenching of the emission intensity of the bipyridine ligands. The emission intensity profile of the titration nm could be fitted to a 1:2 binding mode with dissociation constants for the first, and second iron coordination of K_D1 = 5.5 ± 3.3 μM and a K_D2 = 6.6 ± 0.7 μM, respectively (Figure 2, left; Kuzmic, 1996, 2009). UV/Vis titrations were also qualitatively consistent with the fluorescence data, showing a weak MLCT at about 535 nm in the presence of Fe(II) ions (See Supplementary Material). The formation of the expected [[(βAlaBpy)₂-T4Ff]₃Fe₂]⁴⁺ complex was also confirmed by mass spectrometry of the final solution of the titrations, which showed a peak at the expected mass of the molecular ion (m/z = 11084.6).

In order to study the chirality induction around the metal centers we measured the circular dichroism spectra of the trimeric [(βAlaBpy)₂-T4Ff]₃ ligand, and its Fe(II) complex [(βAlaBpy)₂-T4Ff]₃Fe2+4. As expected from the original structural analysis, the observed positive Cotton effect at c.a. 330 nm is consistent with the formation of a ΛΛ-helicate. Furthermore, the small change in the CD spectra upon addition of Fe(II) also suggests that the bipyridine ligands are strongly preorganized, even in absence of the metal, and that only a small rearrangement of the chromophores takes place upon coordination (Figure 2, right). This is consistent with earlier computational studies with related bis-bipyridyl peptide ligands, which showed that the bipyridine residues have a large tendency to stack on top of each other (Rama et al., 2012). This stacking interaction will presumably rigidify the bis-bipyridyl trimer and facilitate the helical induction by the foldon domain.

In order to gain some insight into the structure and stability of the peptide helicate we performed Molecular Dynamics (MD) simulations in explicit solvent and periodic boundary conditions (see Methods section for details). The structure of the ΛΛ-[(βAlaBpy)₂-T4Ff]₃Fe2+4 unit appears highly stable along all the MD trajectory retaining its helicity conformation and the Fe(II) octahedral coordination geometry. Moreover, the T4Ff scaffold appears stable during the simulation showing no appreciable deformations as a result of the introduction of the artifical (βAlaBpy)₂ unit. The root-mean square deviation (RMSD) of the whole system was computed along the MD using the minimized initial structures as a reference, the trajectories attain relative stable RMSD after the first ~20 ns, that reach up to 1.99 ± 0.62 Å in average (See Supplementary Material). A cluster analysis was performed on the full length MD experiments showing a predominant conformations occupying about ~40% of the total conformation repartition. Overall, the results highlight that the computed model is very stable along the 100 ns of the MD and results consistent with the experimental data. Interestingly, the MD analysis revealed a hinge region with increased flexibility connecting the more rigid helicate and foldon domains, which suggests the replacement of the N-terminal Gly reside for a more conformationally restricted residue in future designs.

Having made a preliminary characterization of the T4Ff helicate, we studied its DNA binding properties by titrating a 2 μM solution of [(βAlaBpy)₂-T4Ff]₃ (6 μM mononer) in the presence of saturating concentrations of Fe(II) according to the previous fluorescence titrations (20 μM) with increasing concentrations of a three-way DNA junction (tw-DNA), and measuring the fluorescence anisotropy of the bipyridine fluorophores at 420 nm after each addition of DNA. The titration profile could be fitted to a 1:1 binding mode, with a dissociation constant of 2.17 ± 0.45 μM of the [(βAlaBpy)₂-T4Ff]₃Fe₂ complex to tw-DNA. Titrations under the same conditions with a model double stranded DNA (ds-DNA) led to a small, monotonic increase in the anisotropy, which is in tune with the the formation of weak complexes or non-specific binding (Figure 3). The low affinity to dsDNA is consistent with previous studies with other helicates (Figure 4; Tuma et al., 1999; Oleksy et al., 2006; Gamba et al., 2016). Control titrations adding with [(βAlaBpy)₂-T4Ff]₃ foldon in absence of metal did not show any response to added DNA (See Supplementary Material), thus confirming that the formation of the helicate structure is required for DNA recognition, and the foldon only have a structural role in the formation of the helicate.

In addition to the spectroscopic studies, we also studied the DNA binding properties of the [(βAlaBpy)₂-T4Ff]₃Fe₂ helicate by electrophoretic mobility assays (EMSA) in polyacrylamide gel under non-denaturing conditions (Liebler and Diederichsen, 2004), visualizing the DNA in the gel using SybrGold staining (Vázquez et al., 2007). In agreement with the fluorescence titration studies discussed previously, incubation of the target tw-DNA with the [(βAlaBpy)₂-T4Ff]₃Fe₂ helicate resulted in the concentration-dependent appearance of a new retarded band, which is consistent with the formation of the expected tw-DNA/[(βAlaBpy)₂-T4Ff]₃Fe₂ complex (Figure 5, lanes 1–6). Additionally, the overall intensity of the lanes of the gel is progressively reduced in the presence of increasing concentrations of the [(βAlaBpy)₂-T4Ff]₃Fe₂ complex, which suggests the formation of higher-order aggregates with the three-way junction DNA in the gel conditions (Chanvorachote et al., 2009; Thordarson, 2010). On the other hand, incubation of a model double-stranded DNA with the peptide helicate did not show any new slow-migrating bands (Figure 5, lanes 7-10), which is in agreement with the expected low affinity for this form of DNA, and demonstrates that the small increase observed in the fluorescence anisotropy titration of dsDNA (Figure 4) arises from weak interactions that are not seen at the lower concentrations used in the EMSA experiment.

In summary, we have shown the potential of small protein domains for the precise structural organization of coordination complexes. Modification of the T4 Fibritin foldon with metal-chelating bipyridines results allows the assembly of unique three-strand helicates in which the parallel orientation of the three helicate ligands is directed by the self-assembled T4Ff domain, and the chirality of the dinuclear helicate (M helicity or ΛΛ-configuration in the metal complexes) is selected by the relative orientation of the natural polyproline helices at the N-terminus of the T4Ff trimer. The final supramolecular peptide helicate [(βAlaBpy)₂-T4Ff]₃Fe₂ displays good in vitro DNA binding and selectivity toward three-way DNA junctions. We are currently exploring alternative peptide sequences to improve the solubility of the peptide/DNA complexes, and modifications with positively charged residues that might increase the overall affinity.

JG-G and DGP performed the experimental work (synthesis of the bipyridine building block, peptide synthesis, metal and DNA binding studies), GB did preliminary studies with the (βAlaBpy)2-T4Ff peptide. GS and J-DM did the computational work and contributed to the preparation of the final manuscript. MVL and MEV conceived the project, supervised the experimental work. MEV wrote the manuscript with the collaboration of MVL, and prepared the graphic material.