Of folding and function: understanding active-site context through metalloenzyme design.

In the emerging field of biomolecular design, the introduction of metal-binding sites into loop or turn regions of known protein scaffolds has been utilized to create unique metalloprotein and metallopeptide systems for study. This Forum Article highlights examples of the modular-turn-substitution approach to design and the range of structural and mechanistic questions to which this tool can be applied. Examples from the authors' laboratory are given to show that lanthanide-binding metallopeptides, and now a full metallohomeodomain, can be generated by modular substitution of a Ca-binding EF-hand loop into the unrelated scaffold, the engrailed helix-turn-helix motif. We have previously shown that these peptides bind trivalent Ln(III) ions and promote DNA and phosphate hydrolysis, the targeted function. Here, a series of chimeric peptides are presented that differ only in the ninth loop position [given in parentheses; Peptides P3N (Asn), P3E (Glu), P3A (Ala), and P3W(D) (Asp]. This residue, a putative second-shell ligand stabilizing a coordinated water, was found to influence not only metal affinity but also peptide folding. The affinity for Tb(III) was determined by Trp-Tb fluorescence resonance energy transfer and followed the order Ka = P3W(D) > P3A approximately P3E > P3N. However, circular dichroism (CD) titrations with EuCl3 showed that only P3W(D) and P3N folded to any extent upon metal binding, indicating that the Asp/Asn side chains stabilize the central loop structure and thus propagate folding of the peripheral helices, whereas neither Ala nor Glu appears to be interacting with the metal to organize the loop. Finally, we investigated the longer range context of a given loop substitution by cloning and expressing a lanthanide-binding homeodomain (C2), whose loop insertion sequence is analogous to that of peptide P3W(D). We find by CD that apo-C2 has a significant helical structure (approximately 25% alphahelicity), which increases further upon the addition of Tb(III) (approximately 32% alpha helicity). The protein's Tb(III) affinity is similar to that of the chimeric peptides. However, unlike previously reported metallopeptides, we find that EuC2 does not appreciably promote phosphate or DNA cleavage, which suggests a difference in metal accessibility in the context of the full domain. We have demonstrated that substituting beta turns with metal-binding turns does not necessarily require homologous parental scaffolds or small flexible peptides but rather relies on the structural similarity of the motifs flanking the turn.