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 structural motifs that can coordinate one or more zinc ions to help stabilize their folds. They can be classified into several different structural families (zinc finger proteins) and typically function as interaction modules that bind DNA, RNA, proteins, or small molecules. The name "zinc finger" was originally coined to describe the finger-like appearance of a diagram showing the hypothesized structure of the repeated unit in Xenopus laevis transcription factor IIIA.[1]

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.[2]

The following table[2] shows the different structures and their key features:

Fold Group Representative structure Ligand placement
Cys2His2 PDB 1zaa EBI.jpg Two ligands form a knuckle and two more form the c terminus of a helix.
Gag knuckle PDB 1ncp EBI.jpg Two ligands form a knuckle and two more form a short helix or loop.
Treble clef Two ligands form a knuckle and two more form the N terminus of a helix.
Zinc ribbon PDB 1pft EBI.jpg Two ligands each form two knuckles.
Zn2/Cys6 PDB 1d66 EBI.jpg Two ligands form the N terminus of a helix and two more form a loop.
TAZ2 domain like Two ligands form the termini of two helices.


[edit] Cys2His2

Zinc finger, C2H2 type
Identifiers
Symbol zf-C2H2
Pfam PF00096
Pfam clan CL0361
InterPro IPR007087
PROSITE PS00028

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:[3]

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

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 knuckle
Identifiers
Symbol zf-CCHC
Pfam PF00098
InterPro IPR001878
SMART SM00343
PROSITE PS50158

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.

[edit] 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.

[edit] Zinc Ribbon

TFIIB zinc-binding
Identifiers
Symbol TF_Zn_Ribbon
Pfam PF08271
Pfam clan Zn_Beta_Ribbon
InterPro IPR013137
PROSITE PS51134

The zinc ribbon fold is characterised by two beta-hairpins forming two structurally similar zinc-binding sub-sites.


[edit] Zn2/Cys6

Fungal Zn(2)-Cys(6) binuclear cluster domain
Identifiers
Symbol Zn_clus
Pfam PF00172
InterPro IPR001138
SMART GAL4
PROSITE PS00463
CDD cd00067

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

Generating arrays of engineered Cys2His2 zinc fingers is the most developed method for creating proteins capable of targeting desired genomic DNA sequences. The majority of engineered zinc finger arrays are based on the zinc finger domain of the murine transcription factor Zif268, although some groups have used zinc finger arrays based on the human transcription factor SP1. Zif268 has three individual zinc finger motifs that collectively bind a 9 bp sequence with high affinity.[4] The structure of this protein bound to DNA was solved in 1991[5] and stimulated a great deal of research into engineered zinc finger arrays. In 1994 and 1995, a number of groups used phage display to alter the specificity of a single zinc finger of Zif268.[6][7][8][9] Carlos F. Barbas et al. also reported the development of zinc finger technology in the patent literature and have been granted a number of patents that have been important for the commercial development of zinc finger technology.[10][11] Typical engineered zinc finger arrays have between 3 and 6 individual zinc finger motifs and bind target sites ranging from 9 basepairs to 18 basepairs in length. Arrays with 6 zinc finger motifs are particularly attractive because they bind a target site that is long enough to have a good chance of being unique in a mammalian genome.[12] There are two main methods currently used to generate engineered zinc finger arrays, modular assembly and a bacterial selection system, and there is some debate about which method is best suited for most applications.[13][14]

[edit] Modular assembly

The most straightforward method to generate new zinc finger arrays is to combine smaller zinc finger "modules" of known specificity. The structure of the zinc finger protein Zif268 bound to DNA described by Pavletich and Pabo in their 1991 publication has been key to much of this work and describes the concept of obtaining fingers for each of the 64 possible base pair triplets and then mixing and matching these fingers to design proteins with any desired sequence specificity.[5] The most common modular assembly process involves combining separate zinc fingers that can each recognize a 3 basepair DNA sequence to generate 3-finger, 4-, 5-, or 6-finger arrays that recognize target sites ranging from 9 basepairs to 18 basepairs in length. Another method uses 2-finger modules to generate zinc finger arrays with up to six individual zinc fingers.[15] The Barbas Laboratory of The Scripps Research Institute used phage display to develop and characterize zinc finger domains that recognize most DNA triplet sequences[16][17][18] while another group isolated and characterized individual fingers from the human genome.[19] A potential drawback with modular assembly in general 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 with sufficient affinity in a bacterial two-hybrid assay and fail to function as zinc finger nucleases, but the success rate was somewhat higher when sites of the form GNNGNNGNN were targeted.[20] A subsequent study used modular assembly to generate zinc finger nucleases with both 3-finger arrays and 4-finger arrays and observed a much higher success rate with 4-finger arrays.[21] A variant of modular assembly that takes the context of neighboring fingers into account has also been reported and this method tends to yield proteins with improved performance relative to standard modular assembly.[22]

[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. This technique is difficult to use on more than a single zinc finger at a time, so a multi-step processes that generated a completely optimized 3-finger array by adding and optimizing a single zinc finger at a time was developed.[23] 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.[24] 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.

[edit] Applications

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.[25] While initial studies with another DNA-binding domain from bacterial TAL effectors show promise,[26][27][28][29] it remains to be seen whether these domains are suitable for some or all of the applications where engineered zinc fingers are currently used. 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 clinical indications. Zinc finger nucleases have become useful reagents for manipulating genomes of many higher organisms including Drosophila melanogaster, Caenorhabditis elegans, tobacco, corn,[15] zebrafish,[30] various types of mammalian cells,[31] and rats.[32] 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.[33]

[edit] See also

[edit] References

  1. ^ Miller J, McLachlan AD, Klug A (June 1985). "Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes". EMBO J. 4 (6): 1609–14. PMC 554390. PMID 4040853. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=554390. 
  2. ^ a b Krishna SS, Majumdar I, Grishin NV (January 2003). "Structural classification of zinc fingers: survey and summary". Nucleic Acids Res. 31 (2): 532–50. doi:10.1093/nar/gkg161. PMC 140525. PMID 12527760. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=140525. 
  3. ^ C.O. Pabo; E.Peisach; R.A. Grant (2001). "Design and Selection of Novel Cys2His2 Zinc Finger Proteins". Annu. Rev. Biochem. 70: 313–40. doi:10.1146/annurev.biochem.70.1.313. PMID 11395410. http://arjournals.annualreviews.org/doi/abs/10.1146%2Fannurev.biochem.70.1.313. 
  4. ^ Christy B, Nathans D (November 1989). "DNA binding site of the growth factor-inducible protein Zif268". Proc. Natl. Acad. Sci. U.S.A. 86 (22): 8737–41. doi:10.1073/pnas.86.22.8737. PMC 298363. PMID 2510170. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=298363. 
  5. ^ a b Pavletich NP, Pabo CO (May 1991). "Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A". Science 252 (5007): 809–17. doi:10.1126/science.2028256. PMID 2028256. http://www.sciencemag.org/cgi/pmidlookup?view=long&pmid=2028256. 
  6. ^ Rebar EJ, Pabo CO (February 1994). "Zinc finger phage: affinity selection of fingers with new DNA-binding specificities". Science 263 (5147): 671–3. doi:10.1126/science.8303274. PMID 8303274. http://www.sciencemag.org/cgi/pmidlookup?view=long&pmid=8303274. 
  7. ^ Jamieson AC, Kim SH, Wells JA (May 1994). "In vitro selection of zinc fingers with altered DNA-binding specificity". Biochemistry 33 (19): 5689–95. doi:10.1021/bi00185a004. PMID 8180194. 
  8. ^ Choo Y, Klug A (November 1994). "Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage". Proc. Natl. Acad. Sci. U.S.A. 91 (23): 11163–7. doi:10.1073/pnas.91.23.11163. PMC 45187. PMID 7972027. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=45187. 
  9. ^ Wu H, Yang WP, Barbas CF (January 1995). "Building zinc fingers by selection: toward a therapeutic application". Proc. Natl. Acad. Sci. U.S.A. 92 (2): 344–8. doi:10.1073/pnas.92.2.344. PMC 42736. PMID 7831288. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=42736. 
  10. ^ United States Patents 6,140,466, 6,140,081; 6,242,568; 6,610,512; 6,790,941; 7,011,972; 7,067,617; 7,101,972; 7,329,541; 7,151,201; 7,329,728; 7,378,510; 7,442,784; 7,741,110; 7,781,645; 7,833,784; Barbas et al. inventors
  11. ^ Scott, Christopher Thomas (2005). "The zinc finger nuclease monopoly". Nature Biotechnology 23 (8): 915–918. doi:10.1038/nbt0805-915. PMID 16082353.  edit
  12. ^ Liu Q, Segal DJ, Ghiara JB, Barbas CF (May 1997). "Design of polydactyl zinc-finger proteins for unique addressing within complex genomes". Proc. Natl. Acad. Sci. U.S.A. 94 (11): 5525–30. doi:10.1073/pnas.94.11.5525. PMC 20811. PMID 9159105. http://www.pnas.org/cgi/pmidlookup?view=long&pmid=9159105. 
  13. ^ Kim JS, Lee HJ, Carroll D (February 2010). "Genome editing with modularly assembled zinc-finger nucleases". Nat. Methods 7 (2): 91; author reply 91–2. doi:10.1038/nmeth0210-91a. PMC 2987589. PMID 20111032. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2987589. 
  14. ^ Joung JK, Voytas DF, Cathomen T (February 2010). "Reply to "Genome editing with modularly assembled zinc-finger nucleases"". Nat. Methods 7 (2): 91–2. doi:10.1038/nmeth0210-91b. PMID 20111031. 
  15. ^ a b Shukla VK, Doyon Y, Miller JC, et al. (May 2009). "Precise genome modification in the crop species Zea mays using zinc-finger nucleases". Nature 459 (7245): 437–41. Bibcode 2009Natur.459..437S. doi:10.1038/nature07992. PMID 19404259. 
  16. ^ Segal DJ, Dreier B, Beerli RR, Barbas CF (March 1999). "Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences". Proc. Natl. Acad. Sci. U.S.A. 96 (6): 2758–63. doi:10.1073/pnas.96.6.2758. PMC 15842. PMID 10077584. http://www.pnas.org/cgi/pmidlookup?view=long&pmid=10077584. 
  17. ^ Dreier B, Fuller RP, Segal DJ, et al. (October 2005). "Development of zinc finger domains for recognition of the 5'-CNN-3' family DNA sequences and their use in the construction of artificial transcription factors". J. Biol. Chem. 280 (42): 35588–97. doi:10.1074/jbc.M506654200. PMID 16107335. http://www.jbc.org/cgi/pmidlookup?view=long&pmid=16107335. 
  18. ^ Dreier B, Beerli RR, Segal DJ, Flippin JD, Barbas CF (August 2001). "Development of zinc finger domains for recognition of the 5'-ANN-3' family of DNA sequences and their use in the construction of artificial transcription factors". J. Biol. Chem. 276 (31): 29466–78. doi:10.1074/jbc.M102604200. PMID 11340073. http://www.jbc.org/cgi/pmidlookup?view=long&pmid=11340073. 
  19. ^ Bae KH, Kwon YD, Shin HC, et al. (March 2003). "Human zinc fingers as building blocks in the construction of artificial transcription factors". Nat. Biotechnol. 21 (3): 275–80. doi:10.1038/nbt796. PMID 12592413. 
  20. ^ C.L. Ramirez; J.E. Foley; D.A. Wright; F. Muller-Lerch; S.H. Rahman; T.I. Cornu; R.J. Winfrey; J.D. Sander; F. Fu; J.A. Townsend; T. Cathomen; D.F. Voytas; J.K. Joung (2009). "Unexpected failure rates for modular assembly of engineered zinc fingers". Nature Methods 5 (5): 374–375. doi:10.1038/nmeth0508-374. PMID 18446154. http://www.nature.com/doifinder/10.1038/nmeth0508-374. 
  21. ^ Kim HJ, Lee HJ, Kim H, Cho SW, Kim JS (July 2009). "Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly". Genome Res. 19 (7): 1279–88. doi:10.1101/gr.089417.108. PMC 2704428. PMID 19470664. http://www.genome.org/cgi/pmidlookup?view=long&pmid=19470664. 
  22. ^ Sander, Jeffry D; Dahlborg, Elizabeth J; Goodwin, Mathew J; Cade, Lindsay; Zhang, Feng; Cifuentes, Daniel; Curtin, Shaun J; Blackburn, Jessica S et al (2010). "Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)". Nature Methods 8 (1): 67–69. doi:10.1038/nmeth.1542. PMC 3018472. PMID 21151135. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3018472.  edit
  23. ^ Greisman HA, Pabo CO (January 1997). "A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites". Science 275 (5300): 657–61. doi:10.1126/science.275.5300.657. PMID 9005850. http://www.sciencemag.org/cgi/pmidlookup?view=long&pmid=9005850. 
  24. ^ M.L. Maeder et al. (September 2008). "Rapid "Open-Source" Engineering of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification". Mol. Cell 31 (2): 294–301. doi:10.1016/j.molcel.2008.06.016. PMC 2535758. PMID 18657511. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2535758. 
  25. ^ A.C. Jamieson; J.C. Miller; C.O. Pabo (May 2003). "Drug Discovery with Engineered zinc-finger proteins". Nat. Rev. Drug Discov. 2 (5): 361–8. doi:10.1038/nrd1087. PMID 12750739. http://www.nature.com/nrd/journal/v2/n5/abs/nrd1087.html. 
  26. ^ Moscou MJ, Bogdanove AJ (December 2009). "A simple cipher governs DNA recognition by TAL effectors". Science 326 (5959): 1501. Bibcode 2009Sci...326.1501M. doi:10.1126/science.1178817. PMID 19933106. 
  27. ^ Boch J, Scholze H, Schornack S, et al. (December 2009). "Breaking the code of DNA binding specificity of TAL-type III effectors". Science 326 (5959): 1509–12. doi:10.1126/science.1178811. PMID 19933107. 
  28. ^ Christian M, Cermak T, Doyle EL, et al. (July 2010). "TAL Effector Nucleases Create Targeted DNA Double-strand Breaks". Genetics 186 (2): 757–761. doi:10.1534/genetics.110.120717. PMC 2942870. PMID 20660643. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2942870. 
  29. ^ Li T, Huang S, Jiang WZ, et al. (August 2010). "TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain". Nucleic Acids Res 39 (1): 359–372. doi:10.1093/nar/gkq704. PMC 3017587. PMID 20699274. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=3017587. 
  30. ^ S.C. Ekker (2008). "Zinc finger-based knockout punches for zebrafish genes". Zebrafish 5 (2): 1121–3. doi:10.1089/zeb.2008.9988. PMC 2849655. PMID 18554175. http://www.liebertonline.com/doi/abs/10.1089/zeb.2008.9988. 
  31. ^ D. Carroll (2008). "Progress and prospects: Zinc-finger nucleases as gene therapy agents". Gene Therapy 15 (22): 1463–1468. doi:10.1038/gt.2008.145. PMC 2747807. PMID 18784746. http://www.nature.com/gt/journal/v15/n22/abs/gt2008145a.html. 
  32. ^ Geurts AM, Cost GJ, Freyvert Y, et al. (July 2009). "Knockout rats via embryo microinjection of zinc-finger nucleases". Science 325 (5939): 433. doi:10.1126/science.1172447. PMC 2831805. PMID 19628861. http://www.sciencemag.org/cgi/pmidlookup?view=long&pmid=19628861. 
  33. ^ Tebas, Pablo et al. (February 2009). "Autologous T-Cells Genetically Modified at the CCR5 Gene by Zinc Finger Nucleases SB-728 for HIV (Zinc-Finger)". ClinicalTrials.gov. http://clinicaltrials.gov/ct2/show/NCT00842634. 

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