Analysis of zinc fingers optimized via phage display: evaluating the utility of a recognition code.

Cys2His2 zinc finger proteins are composed of modular DNA-binding domains and provide an excellent framework for the design and selection of proteins with novel site specificity. Crystal structures of zinc finger-DNA complexes have shown that many Cys2His2 zinc fingers use a conserved docking arrangement that juxtaposes residues at key positions in the "recognition helix" with corresponding base positions in the three to four base-pair subsite. Several groups have proposed that specificity can be explained with a zinc finger-DNA recognition code that correlates specific amino acids at these key positions in the alpha-helix with specific bases in each position of the corresponding subsite. Here, we explore the utility of such a code through detailed studies of zinc finger variants selected via phage display. These proteins provide interesting systems for detailed analysis since they have affinities and specificities for their sites similar to those of naturally occurring DNA-binding proteins. Comparisons are facilitated by the fact that only key DNA-binding residues are varied in each finger while leaving all other regions of the structure unchanged. We study these proteins in detail by (1) selecting their optimal binding sites and comparing these binding sites with sites that might have been predicted from a code; (2) by examining the "evolutionary history" of these proteins during the phage display protocol to look for evidence of context-dependent effects; and (3) by reselecting finger 1 in the presence of the optimized finger 2/finger 3 domains to obtain further data on finger modularity. Our data for optimized fingers and binding sites demonstrate a clear correlation with contacts that would be predicted from a code. However, there are enough examples of context-dependent effects (not explained by any existing code) that selection is the most reliable method for maximizing the affinity and specificity of new zinc finger proteins.

[1]  A Klug,et al.  Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[2]  C. Pabo,et al.  High-resolution structures of variant Zif268-DNA complexes: implications for understanding zinc finger-DNA recognition. , 1998, Structure.

[3]  R. Sauer,et al.  Transcription factors: structural families and principles of DNA recognition. , 1992, Annual review of biochemistry.

[4]  S H Kim,et al.  A zinc finger directory for high-affinity DNA recognition. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. Berg,et al.  Redesigning the DNA‐binding specificity of a zinc finger protein: A data base‐guided approach , 1992, Proteins.

[6]  John W. R. Schwabe,et al.  The crystal structure of a two zinc-finger peptide reveals an extension to the rules for zinc-finger/DNA recognition , 1993, Nature.

[7]  A. Travers,et al.  Sequence-specific DNA binding by a two zinc-finger peptide from the Drosophila melanogaster Tramtrack protein. , 1992, Journal of molecular biology.

[8]  N. Pavletich,et al.  Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A , 1991, Science.

[9]  J R Desjarlais,et al.  Length-encoded multiplex binding site determination: application to zinc finger proteins. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[10]  A Klug,et al.  Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[11]  E. Jennings,et al.  DNA binding sites for the transcriptional activator/repressor YY1. , 1995, Nucleic acids research.

[12]  C. Pabo,et al.  Zif268 protein-DNA complex refined at 1.6 A: a model system for understanding zinc finger-DNA interactions. , 1996, Structure.

[13]  N. Corbi,et al.  Synthesis of a new zinc finger peptide; comparison of its `code' deduced and `CASTing' derived binding sites , 1997, FEBS letters.

[14]  Y. Choo.  End effects in DNA recognition by zinc finger arrays. , 1998, Nucleic acids research.

[15]  J R Desjarlais,et al.  Toward rules relating zinc finger protein sequences and DNA binding site preferences. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[16]  J. Gogos,et al.  Sequence discrimination by alternatively spliced isoforms of a DNA binding zinc finger domain. , 1992, Science.

[17]  J. Berg,et al.  Serine at position 2 in the DNA recognition helix of a Cys2-His2 zinc finger peptide is not, in general, responsible for base recognition. , 1995, Journal of molecular biology.

[18]  J. Berg,et al.  The Galvanization of Biology: A Growing Appreciation for the Roles of Zinc , 1996, Science.

[19]  J. Berg,et al.  A 2.2 Å resolution crystal structure of a designed zinc finger protein bound to DNA , 1996, Nature Structural Biology.

[20]  S. Harrison,et al.  Differing roles for zinc fingers in DNA recognition: structure of a six-finger transcription factor IIIA complex. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[21]  L. Lau,et al.  A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with "zinc finger" sequences. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Aaron Klug,et al.  In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence , 1994, Nature.

[23]  C. Pabo,et al.  Crystal structure of a five-finger GLI-DNA complex: new perspectives on zinc fingers. , 1993, Science.

[24]  P. Tsichlis,et al.  repressor . binds DNA and functions as a transcriptional Gfi-1 encodes a nuclear zinc finger protein , 1996 .

[25]  G. Jacobs,et al.  Determination of the base recognition positions of zinc fingers from sequence analysis. , 1992, The EMBO journal.

[26]  J R Desjarlais,et al.  Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding proteins. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[27]  P E Wright,et al.  Solution structure of the first three zinc fingers of TFIIIA bound to the cognate DNA sequence: determinants of affinity and sequence specificity. , 1997, Journal of molecular biology.

[28]  J. Milbrandt,et al.  DNA-binding specificity of NGFI-A and related zinc finger transcription factors , 1995, Molecular and cellular biology.

[29]  S K Burley,et al.  Cocrystal structure of YY1 bound to the adeno-associated virus P5 initiator. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[30]  C. Barbas,et al.  Building zinc fingers by selection: toward a therapeutic application. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[31]  A Klug,et al.  Physical basis of a protein-DNA recognition code. , 1997, Current opinion in structural biology.

[32]  C. Pabo,et al.  Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. , 1994, Science.

[33]  S H Kim,et al.  In vitro selection of zinc fingers with altered DNA-binding specificity. , 1994, Biochemistry.