Engineered lanthanide-binding metallohomeodomains: designing folded chimeras by modular turn substitution.
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 Kd = 2.1 +/- 0.4 microM; EuC4 Kd = 3.2 +/- 1.0 microM). 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).
Lim
Sunghyuk
S
Department of Chemistry, University of Iowa, Iowa 52242, USA.
Franklin
Sonya J
SJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA; D005063:Europium / Q000737:chemistry; D019077:Helix-Turn-Helix Motifs; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D028581:Lanthanoid Series Elements; D008667:Metalloproteins; D005063:Europium; D004247:DNA