title: DNA targeting and cleavage by an engineered metalloprotein dimer.
abstract: Nature has illustrated through numerous examples that protein dimerization has structural and functional advantages. We previously reported the design and characterization of an engineered "metallohomeodomain" protein (C2) based on a chimera of the EF-hand Ca-binding motif and the helix-turn-helix motif of homeodomains (Lim and Franklin in Protein Sci. 15:2159-2165, 2004). This small metalloprotein binds the hard metal ions Ca(II) and Ln(III) and interacts with DNA with modest sequence preference and affinity, yet exhibits only residual DNA cleavage activity. Here we have achieved substantial improvement in function by constructing a covalent dimer of this C2 module (F2) to create a larger multidomain protein. As assayed via fluorescence spectroscopy, this F2 protein binds Ca(II) more avidly (25-fold) than C2 on a per-domain basis; in gel shift selection experiments, metallated F2 exhibits a specificity toward 5'-TAATTA-3' sequences. Finally, Ca(2)F2 cleaves plasmid DNA and generates a linear product in a Ca(II)-dependent way, unlike the CaC2 monomer. To the best of our knowledge this activation of Ca(II) in the context of an EF-hand binding motif is unique and represents a significant step forward in the design of artificial metallonucleases by utilizing biologically significant metal ions.
authors: Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA.
authors: Siu Wah
authors: SW
authors: Wong-Deyrup
authors: Charulata
authors: C
authors: Prasannan
authors: Cynthia M
authors: CM
authors: Dupureur
authors: Sonya J
authors: SJ
authors: Franklin
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D020832:EF Hand Motifs; D004591:Electrophoresis, Polyacrylamide Gel; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; D008667:Metalloproteins; D004247:DNA