Combining structure-based design with phage display to create new Cys(2)His(2) zinc finger dimers.
BACKGROUND
Several strategies have been reported for the design and selection of novel DNA-binding proteins. Most of these studies have used Cys(2)His(2) zinc finger proteins as a framework, and have focused on constructs that bind DNA in a manner similar to Zif268, with neighboring fingers connected by a canonical (Krüppel-type) linker. This linker does not seem ideal for larger constructs because only modest improvements in affinity are observed when more than three fingers are connected in this manner. Two strategies have been described that allow the productive assembly of more than three canonically linked fingers on a DNA site: connecting sets of fingers using linkers (covalent), or assembling sets of fingers using dimerization domains (non-covalent).


RESULTS
Using a combination of structure-based design and phage display, we have developed a new dimerization system for Cys(2)His(2) zinc fingers that allows the assembly of more than three fingers on a desired target site. Zinc finger constructs employing this new dimerization system have high affinity and good specificity for their target sites both in vitro and in vivo. Constructs that recognize an asymmetric binding site as heterodimers can be obtained through substitutions in the zinc finger and dimerization regions.


CONCLUSIONS
Our modular zinc finger dimerization system allows more than three Cys(2)His(2) zinc fingers to be productively assembled on a DNA-binding site. Dimerization may offer certain advantages over covalent linkage for the recognition of large DNA sequences. Our results also illustrate the power of combining structure-based design with phage display in a strategy that assimilates the best features of each method.
Wolfe
S A
SA
Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Ramm
E I
EI
Pabo
C O
CO
D000595:Amino Acid Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D015195:Drug Design; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D019151:Peptide Library; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics; D014162:Transfection; D016335:Zinc Fingers / Q000235:genetics
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases