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

Cys(2)His(2) zinc finger proteins are composed of modular DNA-binding domai ns and provide an excellent framework for the design and selection of prote ins with novel site specificity. Crystal structures of zinc finger-DNA comp lexes have shown that many Cys(2)His(2) zinc fingers use a conserved dockin g arrangement that juxtaposes residues at key positions in the "recognition helix" with corresponding base positions in the three to four base-pair su bsite. 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 th ey have affinities and specificities for their sites similar to those of na turally occurring DNA-binding proteins. Comparisons are facilitated by the fact that only key DNA-binding residues are varied in each finger while lea ving 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 di splay protocol to look for evidence of context-dependent effects; and (3) b y reselecting finger 1 in the presence of the optimized finger 2/finger 3 d omains to obtain further data on finger modularity. Our data for optimized fingers and binding sites demonstrate a clear correlation with contacts tha t would be predicted from a code. However, there are enough examples of con text-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. (C) 1999 Academic Press.