Zinc finger

Cartoon representation of the Cys2His2 zinc finger motif, consisting of an α helix and an antiparallel β sheet. The zinc ion (green) is coordinated by two histidine residues and two cysteine residues.

Cartoon representation of the protein Zif268 (blue) containing three zinc fingers in complex with DNA (orange). The coordinating amino acid residues and zinc ions (green) are highlighted.

Zinc fingers are small protein domains that can coordinate one or more zinc ions to help stabilize their folds. They can be classified into several different structural families and typically function as interaction modules that bind DNA, RNA, proteins or small molecules. The name "zinc finger" was coined to describe the hypothesized structure of the repeated unit in Xenopus laevis transcription factor IIIA.

Classes

Zinc fingers coordinate zinc ions with a combination of cysteine and histidine residues. They can be classified by the type and order of these zinc coordinating residues (e.g. Cys₂His₂, Cys₄, and Cys₆). A more systematic method classifies them into different "fold groups" based on the overall shape of the protein backbone in the folded domain. The most common "fold groups" of zinc fingers are the Cys₂His₂-like (the "classic zinc finger"), treble clef, and zinc ribbon. ^[1]

Cys₂His₂

The Cys₂His₂-like fold group is by far the best characterized class of zinc fingers and are extremely common in mammalian transcription factors. These domains adopt a simple ββα fold and have the amino acid Sequence motif: X₂-Cys-X_2,4-Cys-X₁₂-His-X_3,4,5-His ^[2] This class of zinc fingers can have a variety of functions such as binding RNA and mediating protein-protein interactions, but is best known for its role in sequence specific DNA-binding proteins such as Zif268. In such proteins, individual zinc finger domains typically occur as tandem repeats with two, three or more fingers comprising the DNA-binding domain of the protein. These tandem arrays can bind in the major groove of DNA and are typically spaced at 3-bp intervals. The α-helix of each domain (often called the "recognition helix") can make sequence specific contacts to DNA bases; residues from a single recognition helix can contact 4 or more bases to yield an overlapping pattern of contacts with adjacent zinc fingers.

Gag knuckle

This fold group is defined by two short β-strands connected by a turn (zinc knuckle) followed by a short helix or loop and resembles the classical Cys₂His₂ motif with a large portion of the helix and β-hairpin truncated. The retroviral nucleocapsid (NC) protein from HIV and other related retroviruses are examples of proteins possessing these motifs. The gag knuckle zinc finger in the HIV NC protein is the target of a class of drugs known as zinc finger inhibitors.

Treble clef

The treble clef motif consists of a β-hairpin at the N-terminus and an α-helix at the C-terminus that each contribute two ligands for zinc binding, although a loop and a second β-hairpin of varying length and conformation can be present between the N-terminal β-hairpin and the C-terminal α-helix. These fingers are present in a diverse group of proteins that frequently do not share sequence or functional similarity with each other. The best characterized proteins containing treble clef zinc fingers are the [nuclear hormone receptors].

Zn₂/Cys₆

The canonical members of this class contain a binuclear zinc cluster in which two zinc ions are bound by six cysteine residues. These zinc fingers can be found in several transcription factors including the yeast Gal4 protein.

Engineered zinc finger arrays

Various strategies have been developed to engineer Cys₂His₂ zinc fingers to bind desired sequences. These include both "modular assembly" and selection strategies that employ either phage display or cellular selection systems. Such engineered zinc finger arrays can then be used in numerous applications such as artificial transcription factors, zinc finger methylases, zinc finger recombinases, and Zinc finger nucleases.^[3] Artificial transcription factors with engineered zinc finger arrays have been used in numerous scientific studies and an artificial transcription factor that activates expression of VEGF is currently being evaluated in humans as a potential treatment for several indications. Zinc finger nucleases have become useful reagents for manipulating genomes of many higher organisms including Drosophila melanogaster, Caenorhabditis elegans, tobacco, various types of mammalian cells^[4] and zebrafish^[5]. An ongoing clinical trial is evaluating Zinc finger nucleases that disrupt the CCR5 gene in CD4+ human T-cells as a potential treatment for HIV/AIDS.

Modular assembly

The most straightforward method to generate new zinc finger arrays is to combine smaller zinc finger "modules" of known specificity. This concept was first described by Pavletich and Pabo in their 1991 publication describing the structure of the zinc finger protein Zif268 bound to DNA. ^[6] In 1994 and 1995 a number of groups developed selection methods ^{[7] [8] [9] [10]} for modifying zinc finger specificity in order to assemble novel zinc finger domains into proteins for use in transcription factors, nucleases and other enzymatic fusion approaches. The most common modular assembly process involves combining separate zinc fingers that can each recognize a 3 basepair DNA sequence to generate a 3-finger, 4-, 5- or 6-finger arrays that recognize target sites ranging from 9 basepairs to 18 basepairs in length. While natural zinc finger proteins typically use three contiguous zinc finger domains to recognize 9 bp target sites, studies published in 1997 demonstrated that unnatural 6-finger arrays could be constructed to bind 18 bp sites. Later crystallographic studies of a 6-finger protein constructed from modular domains supported structural predictions made in the '97 study. The Barbas Laboratory of The Scripps Research Institute has developed and characterized zinc finger domains that recognize most DNA triplet sequences. Assembly of these predefined domains has proven very successful in the construction of artificial transcription factors. Numerous endogenous genes have been regulated using designed zinc finger transcription factors that typically consist of a zinc finger protein consisting of 6 zinc finger domains fused to either an activation or repression domain. Such 6-finger proteins typically bind their 18bp DNA target with nanomolar affinity. Other procedures can utilize 2-finger modules to generate zinc finger arrays with six or more individual zinc fingers. A potential drawback with this procedure is that specificities of individual zinc finger can overlap and can depend on the context of the surrounding zinc fingers and DNA. A recent study demonstrated that a high proportion of 3-finger zinc finger arrays generated by modular assembly fail to bind their intended target in a bacterial two hybrid assay, but the success rate was somewhat higher when sites of the form GNNGNNGNN were targeted. ^[11] Often arrays of 4 to 6 zinc finger domains are required to create a protein that binds its target with nanomolar affinity. When this approach is used to create zinc finger nucleases, the use of 4- to 6-finger proteins is recommended.

Selection methods

Numerous selection methods have been used to generate zinc finger arrays capable of targeting desired sequences. Initial selection efforts utilized phage display to select proteins that bound a given DNA target from a large pool of partially randomized zinc finger arrays. More recent efforts have utilized yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. A promising new method to select novel 3 finger zinc finger arrays utilizes a bacterial two-hybrid system and has been dubbed "OPEN" by its creators. ^[12] This system combines pre-selected pools of individual zinc fingers that were each selected to bind a given triplet and then utilizes a second round of selection to obtain 3-finger arrays capable of binding a desired 9-bp sequence. This system was developed by the Zinc Finger Consortium as an alternative to commercial sources of engineered zinc finger arrays. It is somewhat difficult to directly compare the binding properties of proteins generated with this method to proteins generated by modular assembly as the specificity profiles of proteins generated by the OPEN method have never been reported.

See also

• Zinc finger nuclease
• Zinc finger inhibitor
• Steroid hormone receptor
• DNA-binding protein
• Sequence motif
• Structural motif

References

External links

• McDowall J. "Protein of the Month: Zinc Fingers". European Molecular Biology Laboratory - European Bioinformatics Institute (EMBL-EBI). http://www.ebi.ac.uk/interpro/potm/2007_3/Page1.htm. Retrieved 2008-01-13. 
• Goodsell DS. "Molecule of the Month: Zinc Fingers". Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB). http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb87_1.html. Retrieved 2008-01-13. 
• Zinc Finger Tools design and information site
• Human KZNF Gene Catalog
• Zinc finger C2H2-type domain in PROSITE
• Entry for zinc finger class C2H2 in the SMART database
• The Zinc Finger Consortium
• ZiFiT- Zinc Finger Design Tool
• Zinc Finger Consortium Materials from Addgene