Engineered lanthanide‐binding metallohomeodomains: Designing folded chimeras by modular turn substitution

Abstract

A series of chimeric metallohomeodomains are described, engineered by rational design of a flexible Ca/Ln binding site into a DNA‐binding scaffold. A modular turn‐substitution approach was used to create proteins that both bind DNA and lanthanide ions, while retaining the secondary structure of the full homeodomain (determined by circular dichroism [CD]). Four similar metallohomeodomains were designed (C1–C4), their structural stability predicted by molecular dynamics (MD) simulation of loop‐mutations into the known homeodomain structure, and each designed protein cloned, expressed, and purified using standard molecular biology techniques. Two of the four loop insertions resulted in folded, metal‐ and DNA‐binding proteins (EuC2 K d = 2.1 ± 0.4 μM; EuC4 K d = 3.2 ± 1.0 μM). These results show the successful incorporation of a metal site into a full protein domain, without compromising long‐range structure. This is an important achievement in biomolecular design, as it provides a critical starting point for exploring metallonuclease function and substrate accessibility in a well‐organized chimeric protein domain (rather than only in small HTH peptide systems).