Zinc finger

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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.

Contents

[edit] 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]

[edit] Cys2His2 Zinc Fingers

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-X2,4-Cys-X12-His-X3,4,5-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.

[edit] Gag Knuckle Zinc Fingers

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 best characters members of this family are found in the retroviral nucleocapsid (NC) protein from HIV and other related retroviruses. The gag knuckle zinc finger in the HIV NC protein is the target of a class of drugs known as zinc finger inhibitors.

[edit] Treble Clef Zinc Fingers

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].

[edit] Zn2/Cys6 Zinc Fingers

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.

[edit] 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, C 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.

[edit] Modular Assembly

The most straightforward method to generate new zinc finger arrays is to combine smaller zinc finger "modules" of known specificity. The most common modular assembly process involves combining three separate zinc fingers that can each recognize a 3 basepair DNA sequence to generate a 3-finger array that can recognize a 9 basepair target site. Other procedures can utilize either 1-finger or 2-finger modules to generate zinc finger arrays with six or more individual zinc fingers. The main drawback with this procedure is the specificities of individual zinc finger can overlap and can depend on the context of the surrounding zinc fingers and DNA. Without methods to account for this "context dependence", the standard modular assembly procedure often fails unless it is used to recognize sequences of the form (GNN)N.

[edit] 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 zinc finger arrays utilizes a baterial two-hybrid system and has been dubbed "OPEN" by its creators. [6] 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.


[edit] See also

[edit] References

  1. ^ S.E. Krishna; I.Majumdar; N.V. Grishin (January 2003). "SURVEY AND SUMMARY: Structural classification of zinc fingers". Nucleic Acids Res. 31: 532-550. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=12527760. 
  2. ^ C.O. Pabo; E.Peisach; R.A. Grant (2001). "Design and Selection of Novel Cys2His2 Zinc Finger Proteins". Annu. Rev. Biochem. 70: 313-40. http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.biochem.70.1.313?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dncbi.nlm.nih.gov. 
  3. ^ A.C. Jamieson; J.C. Miller; C.O. Pabo (2003). "Drug Discovery with Engineered zinc-finger proteins". Nat. Rev. Drug Discov. 2: 361-8. http://www.nature.com/nrd/journal/v2/n5/abs/nrd1087.html. 
  4. ^ , D. Carroll (2008). "Progress and prospects: Zinc-finger nucleases as gene therapy agents". Gene Therapy 15: 1463-1468. http://www.nature.com/gt/journal/v15/n22/abs/gt2008145a.html. 
  5. ^ S.C. Ekker (2008). "Zinc finger-based knockout punches for zebrafish genes". Zebrafish 5: 1121-3. http://www.liebertonline.com/doi/abs/10.1089/zeb.2008.9988. 
  6. ^ M.L. Maeder et al. (2008). "Rapid "Open-Source" Engineering of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification". Mol. Cell 31: 294-301. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18657511. 

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