39 Zinc finger proteins: new insights into structural and functional diversity John H Laity, Brian M Lee and Peter E Wright* Zinc finger proteins are among the most abundant proteins in to stabilize its structure. In this article, we review recent eukaryotic genomes. Their functions are extraordinarily diverse advances in the structural biology of zinc finger proteins. and include DNA recognition, RNA packaging, transcriptional We focus first on zinc fingers that function by binding activation, regulation of apoptosis, protein folding and nucleic acids or otherwise participate in transcriptional or assembly, and lipid binding. Zinc finger structures are as translational processes. However, for the sake of complete- diverse as their functions. Structures have recently been ness, we extend the discussion to encompass other novel reported for many new zinc finger domains with novel zinc finger domains whose structures have been elucidat- topologies, providing important insights into structure/function ed recently, even though their functions do not involve relationships. In addition, new structural studies of proteins interactions with nucleic acids. A notable omission from containing the classical Cys2His2 zinc finger motif have led to this review is the zinc fingers from nuclear hormone recep- novel insights into mechanisms of DNA binding and to a tors, which are reviewed in another article in this issue (see better understanding of their broader functions in the review by Rastinejad, pp 33–38). transcriptional regulation. DNA binding by Cys2His2 zinc fingers Addresses Proteins containing the classical Cys2His2 zinc finger are Department of Molecular Biology and Skaggs Institute for Chemical amongst the most abundant in eukaryotic genomes. Many Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, of these proteins are transcription factors that function by La Jolla, CA 92037, USA recognition of specific DNA sequences. Since the struc- *e-mail: [email protected] ture of a single zinc finger was first reported in 1989 [2] Current Opinion in Structural Biology 2001, 11:39–46 (Figure 1a), the structures of numerous complexes with 0959-440X/01/$ — see front matter DNA have been described, beginning with that of Zif268 © 2001 Elsevier Science Ltd. All rights reserved. [3]. These structures have established the invariance of the ββα framework of the Cys His zinc finger module and Abbreviations 2 2 Arf ADP-ribosylation factor have provided a sound basis for understanding the nature ATF-2 activating transcription factor 2 of the interactions that mediate DNA binding. Specificity BIR baculoviral IAP repeat is determined by sidechain–base interactions involving a CR cysteine-rich discrete set of residues located at the fingertip or on the CRE cyclic AMP response element surface of the helix. Additional interactions with the phos- FOG friend of GATA GAP GTPase-activating protein phate backbone and between adjacent zinc fingers IAP inhibitor of apoptosis contribute to binding affinity. The DNA-binding functions MRE metal-response element of zinc finger proteins have recently been the subject of an MTF-1 MRE-binding transcription factor 1 extensive review [4••]. NC nucleocapsid PDB Protein Data Bank PI(3)P phosphatidylinositol 3-phosphate Approximately half of the known Cys2His2 zinc finger pro- TFIIS transcription factor IIS teins contain a highly conserved linker of sequence TFIIIA transcription factor IIIA TGEKP that connects adjacent fingers [4••,5••]. A long- WT1 Wilms’ tumor suppressor protein standing question about zinc finger proteins has been whether the linkers play an active or passive role in DNA Introduction binding. Early mutagenesis studies suggested that the The zinc finger was first recognized 15 years ago as a linkers do play a role in DNA binding, with single site repeated zinc-binding motif, containing conserved cys- mutations reducing the binding affinity by as much as teine and histidine ligands, in Xenopus transcription factor 20-fold [6]. A recent comparison of the structures of IIIA (TFIIIA) [1]. Since that time, numerous other zinc- TGEKP linkers in the available X-ray and NMR structures binding motifs have been identified and designated as zinc of zinc finger proteins in complex with DNA revealed that fingers. These vary widely in structure, as well as in func- the linker conformation is remarkably conserved [5••]. tion, which ranges from DNA or RNA binding to Although it has long been known that one of the functions protein–protein interactions and membrane association. of the TGEKP linker is to cap the C terminus of the pre- We now recognize the classical Cys2His2 zinc finger as the ceding helix [3,7], it has only recently been recognized that first member of a rapidly expanding family of zinc-binding this C-capping interaction is not present in the free protein modules. For the purposes of this review, we define a zinc and is induced upon binding of the zinc finger protein to finger to be any small, functional, independently folded DNA [5••,8]. Dynamic NMR measurements on zinc finger domain that requires coordination of one or more zinc ions domains of TFIIIA and of the Wilms’ tumor suppressor 40 Protein–nucleic acid interactions protein (WT1) [9,10••] revealed that the linker is dynami- will be discussed only briefly. One of the current challenges cally disordered in the free protein, but adopts a in zinc finger protein engineering is in finding methods to well-defined structure with restricted backbone flexibility link subsets of zinc fingers together to achieve the high upon binding to DNA. binding affinity and exquisite specificity that would be required for applications in gene therapy. The binding A survey of zinc finger protein sequences in the SWISS- affinity of Cys2His2 zinc finger proteins increases approxi- PROT database shows that more than 70% of the linkers mately 1000-fold for each additional finger connected by can potentially form a C-capping motif, suggesting that canonical TGEKP linkers. However, only modest improve- DNA-induced helix capping plays an important and gen- ments in affinity occur when proteins with more than three eral role in zinc finger protein function [5••]. A model for fingers are connected by TGEKP linkers; much effort has DNA binding has been proposed in which it is assumed therefore been focused on the identification of suitable that the intrinsic flexibility of the linkers will facilitate dif- linkers for connecting three-finger modules (reviewed by fusion along the DNA as a zinc finger protein searches for Choo and Isalan [13], and Wolfe et al. [4••]). An alternative its cognate binding site. Once the correct DNA sequence approach is to introduce dimerization motifs to facilitate the is encountered, structure is induced in the linkers, which assembly of multiple zinc fingers on DNA. The utility of then act as inducible ‘snaplocks’, holding the fingers in the this approach has been demonstrated very recently by the proper orientation for optimal interactions in the major construction of a fusion protein containing the GCN4 groove [5••]. Recognition of the important functional role leucine zipper dimerization motif and fingers two and three played by conserved capping motifs in linker sequences from Zif268 [14••]. This four-finger chimera binds DNA may facilitate the identification of subsets of DNA-binding with similar affinity and specificity as the best six-finger fingers from the genome sequences. construct designed to date. The limitation of this chimeric protein is the relatively small size of the DNA site recog- Modification of linker sequences in vivo can have profound nized (10 base pairs), but additional fingers can presumably functional and physiological consequences. An excellent be incorporated to produce a protein that recognizes longer example of this is WT1, in which alternate splicing results DNA sequences. in the insertion (+KTS) or omission (–KTS) of the tripep- tide Lys-Thr-Ser in the canonical TGEKP linker between Zinc-sensing proteins the third and fourth zinc fingers. The relative abundance Regulatory mechanisms have evolved in all organisms to of these isoforms is tightly regulated in the cell and varia- control the homeostasis of metal ions. The response of tions are associated with human disease: for example, mammalian cells to surplus zinc appears to be largely Frasier syndrome is caused by an intronic mutation that mediated by metallothionein, which acts to chelate and prevents production of the +KTS isoform [11]. The pres- sequester the excess metal from the cellular environment. ence or absence of the KTS insert modulates both the Metallothionein synthesis is induced by zinc excess and is DNA-binding affinity and the functional distribution of regulated through the metal-response element (MRE) by WT1 within the nucleus. The –KTS isoform binds DNA the transcription factor MTF-1 (MRE-binding transcrip- with high affinity and regulates transcription; in contrast, tion factor 1), which contains six Cys2His2 zinc fingers. In the +KTS variant binds DNA only weakly and associates yeast, the cellular response to zinc deficiency is regulated preferentially with the splicing machinery, where it may by the metal-sensing transcriptional activator Zap1, which interact with RNA [12]. A recent NMR study of the +KTS contains seven Cys2His2 zinc fingers. Recent papers have and –KTS isoforms bound to DNA has revealed the mole- begun to provide insights into the structural basis of zinc cular basis by which alternate splicing mediates the sensing by MTF-1 [15,16•] and Zap1 [17]. It appears that biological function of WT1 [10••]. The KTS insertion both proteins contain a core DNA-binding domain com- increases the flexibility of the linker between fingers three prising a subset of the zinc fingers. In Zap1, the and four, and abrogates binding of the fourth zinc finger to DNA-binding and zinc-responsive domains overlap and its cognate site in the DNA major groove, significantly low- map to a set of five zinc fingers at the C-terminal end of ering the DNA-binding affinity.