Proteomimetic Zinc Finger Domains with Modified Metal-binding β-Turns

Abstract

The mimicry of protein tertiary folds by chains artificial in backbone chemical composition leads to proteomimetic analogues with potential utility as bioactive agents and as tools to shed light on biomacromolecule behavior. Notable successes toward such molecules have been achieved; however, as protein structural diversity is vast, design principles must be continually honed as they are applied to new prototype folding patterns. One specific structure where a gap remains in understanding how to effectively generate modified backbone analogues is the metal-binding β-turn found in zinc finger domains. Literature precedent suggests several factors that may act in concert, including the artificial moiety used to modify the turn, the sequence in which it is applied, and modifications present elsewhere in the domain. Here, we report efforts to gain insights into these issues and leverage these insights to construct a zinc finger mimetic with backbone modifications throughout its constituent secondary structures. We first conduct a systematic comparison of four turn mimetics in a common host sequence, quantifying relative efficacy for use in a metal-binding context. We go on to construct a proteomimetic zinc finger domain in which the helix, strands, and turn are simultaneously modified, resulting in a variant with 23% artificial residues, a tertiary fold indistinguishable from the prototype, and a folded stability comparable to the natural backbone on which the variant is based. Collectively, the results reported provide new insights into the effects of backbone modification on structure and stability of metal-binding domains and help inform the design of metalloprotein mimetics.

Figure 1.

(A) Comparison of the NMR structures of zinc finger domains Sp1-2 (PDB 1SP2) and Sp1-3 (PDB 1SP1).^[40] The metal-binding β-turn is boxed in the overlay on the left and shown in zoomed views from the individual structures on the right. (B) Sequences of Sp1-2, Sp1-3, YY1-3, and peptides 1-5 alongside chemical structures of unnatural amino acid residues used in variants 2-5. In the sequences, metal-coordinating Cys and His residues are underlined and positions i+1 and i+2 in the metal-binding β-turn are highlighted in gray; B = norleucine. BTD and δ-Orn are treated as dipeptides in sequence numbering.

Figure 2.

NMR structures of parent peptide 1 and turn variants 2-5. (A) Ensemble of 10 lowest energy structures for each sequence. (B) Zoomed view of the metal-binding β-turn from the lowest energy conformer from each ensemble. BTD and δ-Orn are treated as dipeptides in sequence numbering.

Figure 3.

Per-residue backbone rmsd values from the overlay of the NMR ensembles for turn variants 2-5 with that of parent peptide 1. For each pairwise comparison, the corresponding intra-ensemble rmsd plots for the two individual sequences are shown. The four-residue β-turn is highlighted in gray, and the rmsd value for Cys¹⁰ at position i+3 labeled. Data for the disordered termini (residues 1, 29-31) are not shown.

Figure 4.

(A) Sequences of Sp1-2 peptide 1 and variant 6 alongside chemical structures of unnatural amino acid residues used in the variant. An R group, when present, matches that of the α-amino acid denoted by the corresponding single letter code in the sequence. Metal-coordinating Cys and His residues are underlined in the sequence; B = norleucine. (B) Ensemble of 10 lowest energy coordinate sets from the NMR structure of variant 6. (C) Overlay of the NMR structure of prototype domain 1 and variant 6. A single representative member of each ensemble is shown. Coloring of carbons in the structure of 6 matches the color scheme from panel (A). (D) Per-residue backbone rmsd values from the overlay of the NMR ensembles of variant 6 and prototype 1; the corresponding intra-ensemble rmsd plots for the two individual sequences are shown. Data for the disordered termini (residues 1, 29-31) are not shown.