CiteULike: msuarezdiez Barbas

Insights into the molecular recognition of the 5'-GNN-3' family of DNA sequences by zinc finger domains

Birgit Dreier — 2008-05-15T09:45:30-00:00

Journal of Molecular Biology, Vol. 303, No. 4. (3 November 2000), pp. 489-502.

In order to construct zinc finger domains that recognize all of the possible 64 DNA triplets, it is necessary to understand the mechanisms of protein/DNA interactions on the molecular level. Previously we reported 16 zinc finger domains which had been characterized in detail to bind specifically to the 5'-GNN-3' family of DNA sequences. Artificial transcription factors constructed from these domains can regulate the expression of endogenous genes. These domains were created by phage-display selection followed by site-directed mutagenesis. A total of 84 mutants of a three-domain zinc finger protein have been analyzed for their DNA-binding specificity. Here, we report the results of this systematic and extensive mutagenesis study. New insights into zinc finger/DNA interactions were obtained by combining specificity data with computer modeling and comparison with known structural data from NMR and crystallographic studies. This analysis suggests that unusual cross-strand and inter-helical contacts are made by some of these proteins, and the general orientation of the recognition helix to the DNA is flexible, even when constrained by flanking zinc finger domains. These findings disfavor the utility of existing simple recognition codes and suggest that highly specific domains cannot be obtained from phage display alone in most cases, but only in combination with rational design. The molecular basis of zinc finger/DNA interaction is complex and its understanding is dependent on the analysis of a large number of proteins. This understanding should enable us to refine rapidly the specificity of other zinc finger domains, as well as polydactyl proteins constructed with these domains to recognize extended DNA sequences.

Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences.

DJ Segal — 2008-05-15T08:52:40-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 96, No. 6. (16 March 1999), pp. 2758-2763.

We have taken a comprehensive approach to the generation of novel DNA binding zinc finger domains of defined specificity. Herein we describe the generation and characterization of a family of zinc finger domains developed for the recognition of each of the 16 possible 3-bp DNA binding sites having the sequence 5'-GNN-3'. Phage display libraries of zinc finger proteins were created and selected under conditions that favor enrichment of sequence-specific proteins. Zinc finger domains recognizing a number of sequences required refinement by site-directed mutagenesis that was guided by both phage selection data and structural information. In many cases, residues not expected to make base-specific contacts had effects on specificity. A number of these domains demonstrate exquisite specificity and discriminate between sequences that differ by a single base with >100-fold loss in affinity. We conclude that the three helical positions -1, 3, and 6 of a zinc finger domain are insufficient to allow for the fine specificity of the DNA binding domain to be predicted. These domains are functionally modular and may be recombined with one another to create polydactyl proteins capable of binding 18-bp sequences with subnanomolar affinity. The family of zinc finger domains described here is sufficient for the construction of 17 million novel proteins that bind the 5'-(GNN)6-3' family of DNA sequences. These materials and methods should allow for the rapid construction of novel gene switches and provide the basis for a universal system for gene control.

De Novo Computational Design of Retro-Aldol Enzymes

Lin Jiang — 2008-03-07T02:58:37-00:00

Science, Vol. 319, No. 5868. (7 March 2008), pp. 1387-1391.

The creation of enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Using new algorithms that rely on hashing techniques to construct active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retro-aldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model. 10.1126/science.1152692