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Write on zinc fingers
A zinc finger is a small protein structural motif that is characterized by the coordination of one or
more zinc ions in order to stabilize the fold. Originally coined to describe the finger-like
appearance of a hypothesized structure from Xenopus laevis transcription factor IIIA, the zinc
finger name has now come to encompass a wide variety of differing protein structures. (Klug and
Rhodes, 1987) Xenopus laevis TFIIIA was originally demonstrated to contain zinc and require
the metal for function in 1983, the first such reported zinc requirement for a gene regulatory
protein (Hanas et al., 1983; Berg, 1990).
Proteins that contain zinc fingers (zinc finger proteins) are classified into several different
structural families. Unlike many other clearly defined super secondary structures such as Greek
keys or β hairpins, there are a number of types of zinc fingers, each with a unique threedimensional architecture. A particular zinc finger protein's class is determined by this threedimensional structure, but it can also be recognized based on the primary structure of the protein
or the identity of the ligands coordinating the zinc ion. In spite of the large variety of these
proteins, however, the vast majority typically functions as interaction modules that bind DNA,
RNA, proteins, or other small, useful molecules, and variations in structure serve primarily to
alter the binding specificity of a particular protein.
Since the original discovery and the elucidation of their structure, these interaction modules have
proven ubiquitous in the biological world. In addition, zinc fingers have become extremely
useful in various therapeutic and research capacities. Engineering zinc fingers to have an affinity
for a specific sequence is an area of active research, and zinc finger nucleases and zinc finger
transcription factors are two of the most important applications of this to be realized to date.
History
Zinc fingers were first identified in a study of transcription in the African clawed frog, Xenopus
laevis. A study of the transcription of a particular RNA sequence revealed that the binding
strength of a small transcription factor (transcription factor IIIA; TFIIIA) was due to the presence
of zinc-coordinating finger-like structures. Amino acid sequencing of TFIIIA revealed nine
tandem sequences of 30 amino acids, including two invariant pairs of cysteine and histidine
residues. Extended x-ray absorption fine structure confirmed the identity of the zinc ligands: two
cysteines and two histidines (Klug, 2010). The DNA-binding loop formed by the coordination of
these ligands by zinc were thought to resemble fingers, hence the name(Klug and Rhodes, 1987).
More recent work in the characterization of proteins in various organisms has revealed the
importance of zinc ions in polypeptide stabilization (Miller et al, 2010; Low et al., 2002).
Domain
Zinc finger (Znf) domains are relatively small protein motifs that contain multiple finger-like
protrusions that make tandem contacts with their target molecule. Some of these domains bind
zinc, but many do not, instead binding other metals such as iron or no metal at all. For example,
some family members form salt bridges to stabilize the finger-like folds. They were first
identified as a DNA-binding motif in transcription factor TFIIIA from Xenopus laevis (African
clawed frog), however they are now recognized to bind DNA, RNA, protein, and/or lipid
substrates ( Klug, 1999; Brown, 2005, Gamsjaeger et al., 2007). Their binding properties depend
on the amino acid sequence of the finger domains and on the linker between fingers, as well as
on the higher-order structures and the number of fingers. Zinc finger domains are often found in
clusters, where fingers can have different binding specificities. There are many superfamilies of
Zinc finger motifs, varying in both sequence and structure. They display considerable versatility
in binding modes, even between members of the same class (e.g., some bind DNA, others
protein), suggesting that Zinc finger motifs are stable scaffolds that have evolved specialized
functions. For example, Zinc finger-containing proteins function in gene transcription,
translation, mRNA trafficking, cytoskeleton organization, epithelial development, cell adhesion,
protein folding, chromatin remodeling, and zinc sensing, to name but a few (Laity et al., 2001).
Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon
binding their target.
Classes
Initially, the term zinc finger was used solely to describe DNA-binding motif found in Xenopus
laevis; however, it is now used to refer to any number of structures related by their coordination
of a zinc ion. In general, zinc fingers coordinate zinc ions with a combination of cysteine and
histidine residues. Originally, the number and order of these residues was used to classify
different types of zinc fingers (e.g.Cys2His2, Cys4, and Cys6). More recently, a more systematic
method has been used to classify zinc finger proteins instead. This method classifies zinc finger
proteins into "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 (Krishna et al., 2003).
The following table shows the different structures and their key features:
Fold
Group
Representative
structure
Ligand placement
Cys2His2
Two ligands form a
knuckle and two more
form the c terminus of
a helix.
Gag
knuckle
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
Two ligands each
form two knuckles.
Zn2/Cys6
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.
Cys2His2
The Cys2His2-like fold group is by far the best-characterized class of zinc fingers and is
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
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 3bp 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 Cterminal 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.
Zinc ribbon
The zinc ribbon fold is characterized by two beta-hairpins forming two structurally similar zincbinding sub-sites.
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.
Applications
Various protein engineering techniques can be used to alter the DNA-binding specificity of zinc
fingers and tandem repeats of such engineered zinc fingers can be used to target desired genomic
DNA sequences (Jamieson et al., 2003). Fusing a second protein domain such as a
transcriptional activator or repressor to an array of engineered zinc fingers that bind near the
promoter of a given gene can be used to alter the transcription of that gene (Jamieson et al.,
2003). Fusions between engineered zinc finger arrays and protein domains that cleave or
otherwise modify DNA can also be used to target those activities to desired genomic loci
(Jamieson et al., 2003). The most common applications for engineered zinc finger arrays include
zinc finger transcription factors and zinc finger nucleases, but other applications have also been
described. 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.
Zinc finger nucleases
Engineered zinc finger arrays are often fused to a DNA cleavage domain (usually the cleavage
domain of FokI) to generate zinc finger nucleases. Such zinc finger-FokI fusions have become
useful reagents for manipulating genomes of many higher organisms including Drosophila
melanogaster, Caenorhabditis elegans, tobacco, corn (Shukla et al., 2009), zebrafish (Reynolds
and Miller, 1988), various types of mammalian cells (Carroll, 2008), and rats (Geurts et al.,
2009). Targeting a double-strand break to a desired genomic locus can be used to introduce
frame-shift mutations into the coding sequence of a gene due to the error-prone nature of the
non-homologous DNA repair pathway. If a homologous DNA "donor sequence" is also used
then the genomic locus can be converted to a defined sequence via the homology directed repair
pathway. 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.
Methods of engineering zinc finger arrays
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 (Christy and Nathans, 1989). The structure of this protein
bound to DNA was solved in 1991 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 (Rebar and Pabo, 1994; Jamieson et al., 1994; Cho and Klug,
1994). 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 (Kim et al., 2010)
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. 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 6finger 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. The Barbas Laboratory of The Scripps Research Institute used phage display to
develop and characterize zinc finger domains that recognize most DNA triplet sequences (Segal
et al., 1999; Dreier et al., 2005; Dreier et al.,2001) while another group isolated and
characterized individual fingers from the human genome (Bae et al., 2003). 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.
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 (Kim et
al., 2009). 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.
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 process that generated
a completely optimized 3-finger array by adding and optimizing a single zinc finger at a time
was developed (Greisman and Pabo, 1997). 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 (Maeder et al., 2008). 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.
Examples
This entry represents the CysCysHisCys (C2HC) type zinc finger domain found in eukaryotes.
Proteins containing these domains include:



MYST family histone acetyltransferases (Smith et al., 2005; Akhtar and Becker, 2001)
Myelin transcription factor Myt1 (Kim et al., 1997)
Suppressor of tumourigenicity protein 18 (ST18) (Jandrig et al., 2004)
References
1. Akhtar, A. and Becker, P.B. (2001). The histone H4 acetyltransferase MOF uses a C2HC
zinc finger for substrate recognition. EMBO Reports. 2 (2): 113–118.
2. Bae, K.H., Kwon, Y.D., Shin, .H.C, Hwang, M.S., Ryu, E.H., Park, K.S., Yang, H.Y.,
Lee, D.K., Lee, Y., Park, J., Kwon, H.S., Kim, H.W., Yeh, B.I., Lee, H.W., Sohn, S.H.,
Yoon, J., Seol, W., Kim, J.S. ( 2003). Human zinc fingers as building blocks in the
construction of artificial transcription factors. Nature Biotechnology. 21 (3): 275–280.
3. Berg, J., (1990). Zinc fingers and other metal-binding domains. Elements for interactions
between macromolecules. Journal of Biological Chemistry. 265 (12): 6513–6516.
4. Brown, R.S., (2005). Zinc finger proteins: getting a grip on RNA. Current Opinion in
Structural Biology. 15 (1): 94–8.
5. Carroll, D., (2008). Progress and prospects: zinc-finger nucleases as gene therapy agents.
Gene Therapy. 15 (22): 1463–1468.
6. Christy, B., Nathans, D., (1989). DNA binding site of the growth factor-inducible protein
Zif268. Proceedings of the National Academy of Sciences of the United States of
America. 86 (22): 8737–8741.
7. Choo, Y., Klug, A. (1994). Toward a code for the interactions of zinc fingers with DNA:
selection of randomized fingers displayed on phage. Proceedings of the National
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8. Dreier, B., Beerli, R.R., Segal, D.J., Flippin, .J.D, Barbas, C.F., (2001). Development of
zinc finger domains for recognition of the 5'-ANN-3' family of DNA sequences and their
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Barbas, C.F., (2005). Development of zinc finger domains for recognition of the 5'-CNN3' family DNA sequences and their use in the construction of artificial transcription
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structural and functional diversity. Current Opinion in Structural Biology. 11 (1): 39–46
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Voytas, D.F., Joung, J.K. (2008). Rapid open-source engineering of customized zincfinger nucleases for highly efficient gene modification. Molecular Cell. 31 (2): 294–301