Tunable Helicity, Stability and DNA-Binding Properties of Short Peptides with Hybrid Metal Coordination Motifs

Abstract

Given the prevalent role of α-helical motifs on protein surfaces in mediating protein-protein and protein-DNA interactions, there have been significant efforts to develop strategies to induce α-helicity in short, unstructured peptides to interrogate such interactions. Toward this goal, we have recently introduced hybrid metal coordination motifs (HCMs). HCMs combine a natural metal-binding amino acid side chain with a synthetic chelating group that are appropriately positioned in a peptide sequence to stabilize an α-helical conformation upon metal coordination. Here, we present a series of short peptides modified with HCMs consisting of a His and a phenanthroline group at i and i+7 positions that can induce α-helicity in a metal-tunable fashion as well as direct the formation of discrete dimeric architectures for recognition of biological targets. We show that the induction of α-helicity can be further modulated by secondary sphere interactions between amino acids at the i+4 position and the HCM. A frequently cited drawback of the use of peptides as therapeutics is their propensity to be quickly digested by proteases; here, we observe an enhancement of up to ∼100-fold in the half-lifes of the metal-bound HCM-peptides in the presence of trypsin. Finally, we show that an HCM-bearing peptide sequence, which contains the DNA-recognition domain of a bZIP protein but is devoid of the obligate dimerization domain, can dimerize with the proper geometry and in an α-helical conformation to bind a cognate DNA sequence with high affinities (K_d≥ 65 nM), again in a metal-tunable manner.

Fig. 1. Design of peptides P1–P7. (a) Cartoon showing the proposed mode of helix induction by metal binding to the HCM. The peptides include two pairs of salt bridging side chains (cyan), the HCM motif with His at position i and Cys-Phen at position i + 7 (green), and various amino acids incorporated at the i + 4 position (red). (b) Sequences of P1–P7.

Fig. 2. (a) CD spectra showing the induction of helicity upon addition of various metal ions. (b) Model showing an Ile residue at the i + 4 (pink) position in contact distance with the Phen functionality in the metal-bound HCM. (c) Molar ellipticity of peptide variants (metal-free, bound to Ni, or in the presence of TFE) monitored at 222 nm.

Fig. 3. Kinetics of the tryptic digestion of P3 in the presence or absence of Ni^II. Additional relevant data (effects of other metal ions, LC-MS analysis) are shown in Fig. S16–S18.

Fig. 4. Design of the DNA-binding peptide P8. (a) The V-shaped cyt cb₅₆₂ dimer dictated by Ni^II coordination to the His/Quin HCMs (green) (PDB ID: ; 3L1M). Adapted from ref. 39. (b) Backbone superposition of the Helix3 domains of HCM-modified cyt cb₅₆₂ (black) onto the basic domain of Jun bZip homodimer (magenta) complexed with cAMP responsive element (CRE) (brown) (PDB ID = ; 1JNM). (c) Overall architecture and structural components of P8, and its proposed Ni-induced dimerization geometry based on the structural model in (b).

Fig. 5. (a) Induction of helicity upon metal binding in P8. (b) Ni-binding titration of P8 monitored by changes in the absorption spectrum of Phen at 280 nm. The dotted black line indicates saturation of binding at 0.5 equiv. of Ni^II per P8, while the small absorbance change in the shaded area is attributed to a transient tris-Phen species. (c) Sedimentation velocity data for P8 in the presence of different amounts of Ni^II determined by AUC.

Fig. 6. Electrophoretic mobility shift assay for monitoring P8-CRE binding in the absence (a) and the presence (b) of 0.5 equiv. of Ni^II. Lane Q contains CRE without any added peptide; DNA concentration is kept constant at 1 nM while the P8 concentrations varies: (A) 1 nM, (B) 2 nM, (C) 5 nM, (D) 10 nM, (E) 15 nM, (F) 20 nM, (G) 25 nM, (H) 30 nM, (I) 40 nM, (J) 50 nM, (K) 75 nM, (L) 100 nM, (M) 150 nM, (N) 200 nM, (O) 250 nM, (P) 500 nM. The intensities of the radioactively labelled CRE bands were measured by phosphorimaging. (c) Effects of different metal ions (0.5 equiv.) on CRE binding by P8, determined by electrophoretic mobility shift assays. See Fig. S23 and S24 for additional data. (d) DNA sequence specificity of P8 binding.

Fig. 7. Scheme for the possible modes of P8 binding to DNA. In this case, the DNA can act as a template for dimerization.