Zinc finger (ZnF) proteins are a massive, diverse family of proteins that serve a wide variety of biological functions. Due to their diversity, it is difficult to come up with a simple definition of what unites all ZnF proteins; however, the most common approach is to define them as all small, functional domains that require coordination by at least one zinc ion (Laity et al., 2001). The zinc ion serves to stabilize the integration of the protein itself, and is generally not involved in binding targets. The “finger” refers to the secondary structures (α-helix and β-sheet) that are held together by the Zn ion. Zinc finger containing domains typically serve as interactors, binding DNA, RNA, proteins or small molecules (Laity et al., 2001).
ZnF Protein Families
Cys2His2 was the first domain discovered (also known as Krüppel-type). It was initially discovered as a repeating domain in the IIIA transcription factor in Xenopus laevis (Brown et al., 1985; Miller et al., 1985). IIIA has nine repeats of the 30 amino acids that make up the Cys2His2 domain. Each domain forms a left-handed ββα secondary structure, and coordinates a Zn ion between two cysteines on the β-sheet hairpin and two histidines in the α-helix, hence the name Cys2His2 (Lee et al., 1989). These resides are highly conserved, as well as a general hydrophobic core that allows the helix to form. The other residues can show great sequence diversity (Michael et al., 1992). Cys2His2 zinc fingers that bind DNA tend to have 2-4 tandem domains as part of a larger protein. The residues of the alpha helices form specific contacts with a specific DNA sequence motif by “reading” the nucleotides in major groove of DNA (Elrod-Erickson et al., 1996; Pavletich and Pabo, 1991). Cys2His2 proteins are the biggest group of transcription factors in most species. Non-DNA binding proteins can have much more flexible tertiary structure. Examples of Cys2His2 proteins include the Inhibitor of Apoptosis (IAP) family of proteins and the CTFC transcription factor.
Treble clef fingers are a very diverse group of ZnF protiens both in terms of structure and function. What makes them a family is a shared fold at their core that looks a little like a musical treble clef, especially if you squint (Grishin, 2001). Most treble clef finger motifs have a β hairpin, a variable loop region, a β hairpin, and an α helix. The “knuckle” of the β hairpin and the α helix contain the Cys-x-x-Cys sequence necessary to coordinate the Zn ion. Treble clef fingers often form the core of protein structures, for example the L24E and S14 ribosomal proteins and the RING finger family.
Zinc ribbons are a little less structurally complex than the other two major groups. Zinc ribbons contain two zinc knuckles, often β hairpins, coordinating a zinc ion via a two Cys residures separated by 2-4 other residues on one knuckle, and a Cys-x-x-Cys on the other (Hahn and Roberts, 2000). Examples of zinc ribbon-containing proteins include the basal transcription factors TFIIS and TFIIB that for a complex with RNAPII to bind DNA, and the Npl4 nuclear core protein that uses a zinc ribbon to bind ubiquitin (Alam et al., 2004). Cys2His2, treble clef fingers, and zinc ribbons form the majority of zinc fingers, but there are several other smaller groups that don’t fit neatly into these three.
Practical Uses for Zinc Finger Proteins
As soon as the specificity of ZnF proteins was understood, the idea of creating synthetic ZnF proteins became the focus of many biotech companies. Cys2His2 motifs each recognize a specific nucleotide triplet depending on the residues on their α helix. This was thought to form a simple code that could be used to recognize very specific DNA sequences by engineering specific ZnF motifs in tandem within a protein. Another domain of the protein could then serve some desired biological function once the ZnF bound the target sequence. For example, cutting at one specific point in the genome and inserting a transgenic element. But alas, it was not so simple. ZnF recognition residues also have cross-recognition to adjacent elements, thus each motif must be chosen in the context of those around it. These issues have now largely been addressed (Urnov et al., 2010). Custom ZnF proteins are now available for researchers to address their own questions. Weather this technology will become appealing enough to replace more trusted methods remains to be seen.
Zinc Finger Protein Additional Reading
Krishna, S.S., Majumdar, I., and Grishin, N.V. (2003). Structural classification of zinc fingers: survey and summary. Nucleic Acids Res. 31, 532-550.
This paper has laid the foundation for our current classification and understanding of ZnF structure. It was responsible for bringing together proteins that were not previously understood to be zinc fingers.
Wolfe, S.A., Nekludova, L., and Pabo, C.O. (2000). DNA recognition by Cys2His2 zinc finger proteins. Annu. Rev. Biophys. Biomol. Struct. 29, 183-212.
This is an older review, but it gives a good overview of the discovery and classification of ZnF proteins, especially Cys2His2.
Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S., and Gregory, P.D. (2010). Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636-646.
This review gives a lot of great info on how synthetic ZnF proteins can be generated and goes over their potential uses.
- Alam, S.L., Sun, J., Payne, M., Welch, B.D., Blake, B.K., Davis, D.R., Meyer, H.H., Emr, S.D., and Sundquist, W.I. (2004). Ubiquitin interactions of NZF zinc fingers. EMBO J. 23, 1411-1421.
- Brown, R.S., Sander, C., and Argos, P. (1985). The primary structure of transcription factor TFIIIA has 12 consecutive repeats. FEBS Lett. 186, 271-274.
- Elrod-Erickson, M., Rould, M.A., Nekludova, L., and Pabo, C.O. (1996). Zif268 protein-DNA complex refined at 1.6 A: a model system for understanding zinc finger-DNA interactions. Structure 4, 1171-1180.
- Grishin, N.V. (2001). Treble clef finger–a functionally diverse zinc-binding structural motif. Nucleic Acids Res. 29, 1703-1714.
- Hahn, S., and Roberts, S. (2000). The zinc ribbon domains of the general transcription factors TFIIB and Brf: conserved functional surfaces but different roles in transcription initiation. Genes Dev. 14, 719-730.
- Laity, J.H., Lee, B.M., and Wright, P.E. (2001). Zinc finger proteins: new insights into structural and functional diversity. Curr. Opin. Struct. Biol. 11, 39-46.
- Lee, M.S., Gippert, G.P., Soman, K.V., Case, D.A., and Wright, P.E. (1989). Three-dimensional solution structure of a single zinc finger DNA-binding domain. Science 245, 635-637.
- Michael, S.F., Kilfoil, V.J., Schmidt, M.H., Amann, B.T., and Berg, J.M. (1992). Metal binding and folding properties of a minimalist Cys2His2 zinc finger peptide. Proc. Natl. Acad. Sci. U. S. A. 89, 4796-4800.
- Miller, J., McLachlan, A.D., and Klug, A. (1985). Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 4, 1609-1614.
- Pavletich, N.P., and Pabo, C.O. (1991). Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science 252, 809-817.
- Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S., and Gregory, P.D. (2010). Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636-646.