NORMAL GENOMIC

• Bedard M, Maltais L, Beaulieu ME, Bilodeau J, Bernard D, Lavigne P
• NMR structure note: solution structure of human Miz-1 zinc fingers 8 to 10.
• J Biomol NMR. 2012; 54: 317-23

• Murakami K et al.
• Tfb6, a previously unidentified subunit of the general transcription factor TFIIH, facilitates dissociation of Ssl2 helicase after transcription initiation.
• Proc Natl Acad Sci U S A. 2012; 109: 4816-21
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• General transcription factor TFIIH, previously described as a 10-subunit complex, is essential for transcription and DNA repair. An eleventh subunit now identified, termed Tfb6, exhibits 45% sequence similarity to human nuclear mRNA export factor 5. Tfb6 dissociates from TFIIH as a heterodimer with the Ssl2 subunit, a DNA helicase that drives promoter melting for the initiation of transcription. Tfb6 does not, however, dissociate Ssl2 from TFIIH in the context of a fully assembled transcription preinitiation complex. Our findings suggest a dynamic state of Ssl2, allowing its engagement in multiple cellular processes.

• Briknarova K, Atwater DZ, Glicken JM, Maynard SJ, Ness TE
• The PR/SET domain in PRDM4 is preceded by a zinc knuckle.
• Proteins. 2011; 79: 2341-5

• Kainov DE et al.
• Structure determination of the minimal complex between Tfb5 and Tfb2, two subunits of the yeast transcription/DNA-repair factor TFIIH: a retrospective study.
• Acta Crystallogr D Biol Crystallogr. 2010; 66: 745-55
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• Tfb5 interacts with the Tfb2 subunit of the general transcription factor TFIIH to ensure efficient nucleotide-excision repair in eukaryotes. The crystal structure of the complex between Tfb5 and the C-terminal region of Tfb2 (Tfb2C) from Saccharomyces cerevisiae has recently been reported. Here, the structure-determination process is described as a case study. Although crystals were obtained readily, it was not possible to determine experimental phases from a first crystal form (Tfb2(412-513)-Tfb5(2-72)) that diffracted to 2.6 A resolution. Shortening of the Tfb2C from its N-terminus was decisive and modified the crystal packing, leading to a second crystal form (Tfb2(435-513)-Tfb5(2-72)). These crystals diffracted to 1.7 A resolution with excellent mosaicity and allowed structure determination by conventional approaches using heavy atoms. The refined structure from the second crystal form was used to solve the structure of the first crystal form by molecular replacement. Comparison of the two structures revealed that the N-terminal region of Tfb2C and (to a lesser extent) the C-terminal region of Tfb5 contributed to the crystal packing. A detailed analysis illustrates how variation in domain boundaries influences crystal packing and quality.

• Bessiere D et al.
• Structure-function analysis of the THAP zinc finger of THAP1, a large C2CH DNA-binding module linked to Rb/E2F pathways.
• J Biol Chem. 2008; 283: 4352-63
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• THAP1, the founding member of a previously uncharacterized large family of cellular proteins (THAP proteins), is a sequence-specific DNA-binding factor that has recently been shown to regulate cell proliferation through modulation of pRb/E2F cell cycle target genes. THAP1 shares its DNA-binding THAP zinc finger domain with Drosophila P element transposase, zebrafish E2F6, and several nematode proteins interacting genetically with the retinoblastoma protein pRb. In this study, we report the three-dimensional structure and structure-function relationships of the THAP zinc finger of human THAP1. Deletion mutagenesis and multidimensional NMR spectroscopy revealed that the THAP domain of THAP1 is an atypical zinc finger of approximately 80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel beta-sheet interspersed by a long loop-helix-loop insertion. Alanine scanning mutagenesis of this loop-helix-loop motif resulted in the identification of a number of critical residues for DNA recognition. NMR chemical shift perturbation analysis was used to further characterize the residues involved in DNA binding. The combination of the mutagenesis and NMR data allowed the mapping of the DNA binding interface of the THAP zinc finger to a highly positively charged area harboring multiple lysine and arginine residues. Together, these data represent the first structure-function analysis of a functional THAP domain, with demonstrated sequence-specific DNA binding activity. They also provide a structural framework for understanding DNA recognition by this atypical zinc finger, which defines a novel family of cellular factors linked to cell proliferation and pRb/E2F cell cycle pathways in humans, fish, and nematodes.

• Di Lello P et al.
• p53 and TFIIEalpha share a common binding site on the Tfb1/p62 subunit of TFIIH.
• Proc Natl Acad Sci U S A. 2008; 105: 106-11
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• The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the alpha-subunit of the general transcription factor IIE (TFIIEalpha). We have determined that the acidic carboxyl terminus of TFIIEalpha (TFIIEalpha(336-439)) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIEalpha binding site on p62/Tfb1 is identical to the binding site for the second transactivation domain of p53 (p53 TAD2). In addition, we demonstrate that TFIIEalpha(336-439) is capable of competing with p53 for a common binding site on p62/Tfb1 and that TFIIEalpha(336-439) and the diphosphorylated form (pS46/pT55) of p53 TAD2 have similar binding constants. NMR structural studies reveal that TFIIEalpha(336-439) contains a small domain (residues 395-433) folded in a novel betabetaalphaalphaalpha topology. NMR mapping studies demonstrate that two unstructured regions (residues 377-393 and residues 433-439) located on either side of the folded domain appear to be required for TFIIEalpha(336-439) binding to p62/Tfb1 and that these two unstructured regions are held close to each other in three-dimensional space by the novel structured domain. We also demonstrate that, like p53, TFIIEalpha(336-439) can activate transcription in vivo. These results point to an important interplay between the general transcription factor TFIIEalpha and the tumor suppressor protein p53 in regulating transcriptional activation that may be modulated by the phosphorylation status of p53.

• Argentaro A et al.
• Structural consequences of disease-causing mutations in the ATRX-DNMT3-DNMT3L (ADD) domain of the chromatin-associated protein ATRX.
• Proc Natl Acad Sci U S A. 2007; 104: 11939-44
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• The chromatin-associated protein ATRX was originally identified because mutations in the ATRX gene cause a severe form of syndromal X-linked mental retardation associated with alpha-thalassemia. Half of all of the disease-associated missense mutations cluster in a cysteine-rich region in the N terminus of ATRX. This region was named the ATRX-DNMT3-DNMT3L (ADD) domain, based on sequence homology with a family of DNA methyltransferases. Here, we report the solution structure of the ADD domain of ATRX, which consists of an N-terminal GATA-like zinc finger, a plant homeodomain finger, and a long C-terminal alpha-helix that pack together to form a single globular domain. Interestingly, the alpha-helix of the GATA-like finger is exposed and highly basic, suggesting a DNA-binding function for ATRX. The disease-causing mutations fall into two groups: the majority affect buried residues and hence affect the structural integrity of the ADD domain; another group affects a cluster of surface residues, and these are likely to perturb a potential protein interaction site. The effects of individual point mutations on the folding state and stability of the ADD domain correlate well with the levels of mutant ATRX protein in patients, providing insights into the molecular pathophysiology of ATR-X syndrome.

• Vitorino M et al.
• Solution structure and self-association properties of the p8 TFIIH subunit responsible for trichothiodystrophy.
• J Mol Biol. 2007; 368: 473-80
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• Trichothiodystrophy (TTD) is a rare hereditary multi-system disorder associated with defects in nucleotide excision repair (NER) and transcription as consequences of mutations in XPB, XPD and p8/TTD-A subunits of transcription factor IIH (TFIIH). Here, we report the solution structure of the p8/TTD-A protein, a small alpha/beta protein built around an antiparallel beta-sheet that forms a homodimer with an extended interface. In order to characterize the dimer interface, we have introduced a mutation at position 44, which destabilizes the dimeric form of the protein. We have shown that this mutation has no effect on the intrinsic ability of p8/TTD-A to stimulate NER in vitro, but affects the capacity of p8/TTD-A to restore TFIIH concentration in TTD-A fibroblasts. Point mutations found in TTD-A patients are discussed on the basis of the present structure.

• Lecordier L et al.
• Characterization of a TFIIH homologue from Trypanosoma brucei.
• Mol Microbiol. 2007; 64: 1164-81
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• Trypanosomes are protozoans showing unique transcription characteristics. We describe in Trypanosoma brucei a complex homologous to TFIIH, a multisubunit transcription factor involved in the control of transcription by RNA Pol I and RNA Pol II, but also in DNA repair and cell cycle control. Bioinformatics analyses allowed the detection of five genes encoding four putative core TFIIH subunits (TbXPD, TbXPB, Tbp44, Tbp52), including a novel XPB variant, TbXPBz. In all cases sequences known to be important for TFIIH functions were conserved. We performed a molecular analysis of this core complex, focusing on the two subunits endowed with a known enzymatic (helicase) activity, XPD and XPB. The involvement of these T. brucei proteins in a bona fide TFIIH core complex was supported by (i) colocalization by immunofluorescence in the nucleus, (ii) direct physical interaction of TbXPD and its interacting regulatory subunit Tbp44 as determined by double-hybrid assay and tandem affinity purification of the core TFIIH, (iii) involvement of the core proteins in a high molecular weight complex and (iv) occurrence of transcription, cell cycle and DNA repair phenotypes upon either RNAi knock-down or overexpression of essential subunits.

• Carlier L et al.
• Solution structure of the region 51-160 of human KIN17 reveals an atypical winged helix domain.
• Protein Sci. 2007; 16: 2750-5
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• Human KIN17 is a 45-kDa eukaryotic DNA- and RNA-binding protein that plays an important role in nuclear metabolism and in particular in the general response to genotoxics. Its amino acids sequence contains a zinc finger motif (residues 28-50) within a 30-kDa N-terminal region conserved from yeast to human, and a 15-kDa C-terminal tandem of SH3-like subdomains (residues 268-393) only found in higher eukaryotes. Here we report the solution structure of the region 51-160 of human KIN17. We show that this fragment folds into a three-alpha-helix bundle packed against a three-stranded beta-sheet. It belongs to the winged helix (WH) family. Structural comparison with analogous WH domains reveals that KIN17 WH module presents an additional and highly conserved 3(10)-helix. Moreover, KIN17 WH helix H3 is not positively charged as in classical DNA-binding WH domains. Thus, human KIN17 region 51-160 might rather be involved in protein-protein interaction through its conserved surface centered on the 3(10)-helix.

• Pugh DJ, Ab E, Faro A, Lutya PT, Hoffmann E, Rees DJ
• DWNN, a novel ubiquitin-like domain, implicates RBBP6 in mRNA processing and ubiquitin-like pathways.
• BMC Struct Biol. 2006; 6: 1-1
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• BACKGROUND: RBBP6 is a 250 kDa splicing-associated protein that has been identified as an E3 ligase due to the presence of a RING finger domain. In humans and mice it interacts with both p53 and Rb, and plays a role in the induction of apoptosis and regulation of the cell cycle. RBBP6 has recently been shown to be highly up-regulated in oesophageal cancer, and to be a promising target for immunotherapy against the disease. RESULTS: We show here using heteronuclear NMR that the N-terminal 81 amino acids of RBBP6 constitute a novel ubiquitin-like domain, which we have called the DWNN domain. The domain lacks conserved equivalents of K48 and K63, although the equivalents of K6 and K29 are highly, although not absolutely, conserved. The di-glycine motif that is characteristic of proteins involved in ubiquitination is found in the human and mouse form of the domain, although it is not present in all organisms. It forms part of a three-domain form of RBBP6 containing the DWNN domain, a zinc knuckle and a RING finger domain, which is found in all eukaryotic genomes so far examined, in the majority of cases at single copy number. The domain is also independently expressed in vertebrates as a single domain protein. CONCLUSION: DWNN is a novel ubiquitin-like domain found only at the N-terminus of the RBBP6 family of splicing-associated proteins. The ubiquitin-like structure of the domain greatly increases the likelihood that RBBP6 functions through some form of ubiquitin-like modification. Furthermore, the fact that the DWNN domain is independently expressed in higher vertebrates leads us to propose that the domain may itself function as a novel ubiquitin-like modifier of other proteins.

• Di Lello P et al.
• Structure of the Tfb1/p53 complex: Insights into the interaction between the p62/Tfb1 subunit of TFIIH and the activation domain of p53.
• Mol Cell. 2006; 22: 731-40
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• The interaction between the amino-terminal transactivation domain (TAD) of p53 and TFIIH is directly correlated with the ability of p53 to activate both transcription initiation and elongation. We have identified a region within the p53 TAD that specifically interacts with the pleckstrin homology (PH) domain of the p62 and Tfb1 subunits of human and yeast TFIIH. We have solved the 3D structure of a complex between the p53 TAD and the PH domain of Tfb1 by NMR spectroscopy. Our structure reveals that p53 forms a nine residue amphipathic alpha helix (residues 47-55) upon binding to Tfb1. In addition, we demonstrate that diphosphorylation of p53 at Ser46 and Thr55 leads to a significant enhancement in p53 binding to p62 and Tfb1. These results indicate that a phosphorylation cascade involving Ser46 and Thr55 of p53 could play an important role in the regulation of select p53 target genes.

• Di Lello P et al.
• NMR structure of the amino-terminal domain from the Tfb1 subunit of TFIIH and characterization of its phosphoinositide and VP16 binding sites.
• Biochemistry. 2005; 44: 7678-86
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• General transcription factor IIH (TFIIH) is recruited to the preinitiation complex (PIC) through direct interactions between its p62 (Tfb1) subunit and the carboxyl-terminal domain of TFIIEalpha. TFIIH has also been shown to interact with a number of transcriptional activator proteins through interactions with the same p62 (Tfb1) subunit. We have determined the NMR solution structure of the amino-terminal domain from the Tfb1 subunit of yeast TFIIH (Tfb1(1-115)). Like the corresponding domain from the human p62 protein, Tfb1(1-115) contains a PH domain fold despite a low level of sequence identity between the two functionally homologous proteins. In addition, we have performed in vitro binding studies that demonstrate that the PH domains of Tfb1 and p62 specifically bind to monophosphorylated inositides [PtdIns(5)P and PtdIns(3)P]. NMR chemical shift mapping demonstrated that the PtdIns(5)P binding site on Tfb1 (p62) is located in the basic pocket formed by beta-strands beta5-beta7 of the PH domain fold. Interestingly, the structural composition of the PtdIns(5)P binding site is different from the composition of the binding sites for phosphoinositides on prototypic PH domains. We have also determined that the PH domains from Tfb1 and p62 are sufficient for binding to the activation domain of VP16. NMR chemical shift mapping demonstrated that the VP16 binding site within the PH domain of Tfb1 (p62) overlaps with the PtdIns(5)P binding site on Tfb1 (p62). These results provide new information about the recognition of phosphoinositides by PH domains, and point to a potential role for phosphoinositides in VP16 regulation.

• Potter BM et al.
• The six zinc fingers of metal-responsive element binding transcription factor-1 form stable and quasi-ordered structures with relatively small differences in zinc affinities.
• J Biol Chem. 2005; 280: 28529-40
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• Six Cys(2)His(2) zinc fingers (F1-6) comprise the DNA binding domain of metal-responsive element binding transcription factor-1 (MTF-1). F1-6 is necessary for basal and zinc-induced expression of metallothionein genes. Analysis of NMR structural and dynamic data for an F1-6 protein construct demonstrates that each zinc finger adopts a stable betabetaalpha fold in the presence of stoichiometric Zn(II), provided that all cysteine ligands are in a reduced state. Parallel studies of protein constructs spanning the four N-terminal core DNA binding fingers (F1-4) and two C-terminal low DNA affinity fingers (F5-6) reveal similar stable zinc finger structures. In both the F1-6 and F5-6 proteins, the finger 5 cysteines were found to readily oxidize at neutral pH. Detailed spectral density and hydrodynamic analysis of (15)N relaxation data revealed quasi-ordered anisotropic rotational diffusion properties of the six F1-6 zinc fingers that could influence MTF-1 DNA binding function. A more general effect on the rotational diffusion properties of Cys(2)His(2) zinc fingers was also uncovered that is dependent upon the position of each finger within multifinger domains. Analysis of NMR (1)H-(15)N-heteronuclear single quantum coherence spectral peak intensities measured as a function of added Zn(II) in conjunction with Zn(II) binding modeling studies indicated that the Zn(II) affinities of all MTF-1 zinc fingers are within approximately 10-50-fold. These analyses further suggested that metal sensing by MTF-1 in eukaryotic cells involves multiple zinc fingers and occurs over a 100-fold or less range of accessible Zn(II) concentration.

• Liu JL, Rigolet P, Dou SX, Wang PY, Xi XG
• The zinc finger motif of Escherichia coli RecQ is implicated in both DNA binding and protein folding.
• J Biol Chem. 2004; 279: 42794-802
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• The RecQ family of DNA helicases has been shown to be important for the maintenance of genomic integrity. Mutations in human RecQ genes lead to genomic instability and cancer. Several RecQ family of helicases contain a putative zinc finger motif of the C4 type at the C terminus that has been identified in the crystalline structure of RecQ helicase from Escherichia coli. To better understand the role of this motif in helicase from E. coli, we constructed a series of single mutations altering the conserved cysteines as well as other highly conserved residues. All of the resulting mutant proteins exhibited a high level of susceptibility to degradation, making functional analysis impossible. In contrast, a double mutant protein in which both cysteine residues Cys397 and Cys400 in the zinc finger motif were replaced by asparagine residues was purified to homogeneity. Slight local conformational changes were detected, but the rest of the mutant protein has a well defined tertiary structure. Furthermore, the mutant enzyme displayed ATP binding affinity similar to the wild-type enzyme but was severely impaired in DNA binding and in subsequent ATPase and helicase activities. These results revealed that the zinc finger binding motif is involved in maintaining the integrity of the whole protein as well as DNA binding. We also showed that the zinc atom is not essential to enzymatic activity.

• Yamasaki K et al.
• A novel zinc-binding motif revealed by solution structures of DNA-binding domains of Arabidopsis SBP-family transcription factors.
• J Mol Biol. 2004; 337: 49-63
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• SQUAMOSA promoter binding proteins (SBPs) form a major family of plant-specific transcription factors related to flower development. Although SBPs are heterogeneous in primary structure, they share a highly conserved DNA-binding domain (DBD) that has been suggested to be zinc binding. Here we report the NMR solution structures of DBDs of two SBPs of Arabidopsis thaliana, SPL4 and SPL7. The two share essentially the same structural features. Each structure contains two zinc-binding sites consisting of eight Cys or His residues in a Cys3HisCys2HisCys or Cys6HisCys sequence motif in which the first four residues coordinate to one zinc and the last four coordinate to the other. These structures are dissimilar to other known zinc-binding structures, and thus represent a novel type of zinc-binding motif. The electrostatic profile on the surface suggested that a continuous region, including all the conserved basic residues, is involved in the DNA binding, the mode of which is likely to be novel as well.

• Jawhari A et al.
• Domain architecture of the p62 subunit from the human transcription/repair factor TFIIH deduced by limited proteolysis and mass spectrometry analysis.
• Biochemistry. 2004; 43: 14420-30
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• TFIIH is a multiprotein complex that plays a central role in both transcription and DNA repair. The subunit p62 is a structural component of the TFIIH core that is known to interact with VP16, p53, Eralpha, and E2F1 in the context of activated transcription, as well as with the endonuclease XPG in DNA repair. We used limited proteolysis experiments coupled to mass spectrometry to define structural domains within the conserved N-terminal part of the molecule. The first domain identified resulted from spontaneous proteolysis and corresponds to residues 1-108. The second domain encompasses residues 186-240, and biophysical characterization by fluorescence studies and NMR analysis indicated that it is at least partially folded and thus may correspond to a structural entity. This module contains a region of high sequence conservation with an invariant FWxxPhiPhi motif (Phi representing either tyrosine or phenylalanine), which was also found in other protein families and could play a key role as a protein-protein recognition module within TFIIH. The approach used in this study is general and can be straightforwardly applied to other multidomain proteins and/or multiprotein assemblies.

• Jauch R et al.
• The zinc finger-associated domain of the Drosophila transcription factor grauzone is a novel zinc-coordinating protein-protein interaction module.
• Structure. 2003; 11: 1393-402
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• About one-third of the more than 300 C2H2 zinc finger proteins of Drosophila contain a conserved sequence motif, the zinc finger-associated domain (ZAD). Genes that encode ZAD proteins are specific for and expanded in the genomes of insects. Only three ZAD-encoding gene functions are established, and the role of ZAD is unknown. Here we present the crystal structure of the ZAD of Grauzone (ZAD(Grau)), a Drosophila transcription factor that specifically controls the maternal Cdc20-like APC subunit Cortex. ZAD forms an atypical treble-clef-like zinc-coordinating fold. Head-to-tail arrangement of two ZAD(Grau) molecules in the crystals suggests dimer formation, an observation supported by crosslinking and dynamic light scattering. The results indicate that ZAD provides a novel protein-protein interaction module that characterizes a large family of insect transcription factors.

• Peng H, Zheng L, Lee WH, Rux JJ, Rauscher FJ 3rd
• A common DNA-binding site for SZF1 and the BRCA1-associated zinc finger protein, ZBRK1.
• Cancer Res. 2002; 62: 3773-81
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• More than 220 Kruppel-associated box-zinc finger protein (KRAB-ZFP) genes are encoded in the human genome. KRAB-ZFPs function as transcriptionalrepressors by binding DNA through their tandem zinc finger motifs.Gene silencing is mediated by the highly conserved KRAB domain, which recruits histone deacetylase complexes, histone methylases, and heterochromatin proteins. However, little is known of the biological programs regulated by KRAB-ZFPs, in large part because of the difficulty in identifying DNA-binding sites recognized by long arrays of zinc fingers. In an attempt to identify the natural target genes for a KRAB-ZFP, we chose SZF1, a hematopoietic progenitor-restricted, KRAB-ZFP that contains only four C(2)H(2) zinc finger motifs. Using recombinant SZF1 protein and a PCR-based binding site selection strategy, we identified a 15-bp consensus DNA sequence recognized by SZF1. Remarkably, this sequence is similar to the core DNA-binding site described recently for ZBRK1, a KRAB-ZFP that binds to BRCA1 and is involved in coordinating the cellular DNA damage response. The SZF1 and ZBRK1 proteins bind to both the experimentally derived SZF1 site and the canonical ZBRK1 site. The KRAB domain from SZF1 bound directly to the KAP-1 corepressor and displayed intrinsic silencing activity. Moreover, full-length SZF1 repressed a promoter containing ZBRK1 recognition sequences. Thus, SZF1 and ZBRK1 may regulate a common set of target genes in vivo.

• Whittaker CA, Hynes RO
• Distribution and evolution of von Willebrand/integrin A domains: widely dispersed domains with roles in cell adhesion and elsewhere.
• Mol Biol Cell. 2002; 13: 3369-87
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• The von Willebrand A (VWA) domain is a well-studied domain involved in cell adhesion, in extracellular matrix proteins, and in integrin receptors. A number of human diseases arise from mutations in VWA domains. We have analyzed the phylogenetic distribution of this domain and the relationships among approximately 500 proteins containing this domain. Although the majority of VWA-containing proteins are extracellular, the most ancient ones, present in all eukaryotes, are all intracellular proteins involved in functions such as transcription, DNA repair, ribosomal and membrane transport, and the proteasome. A common feature seems to be involvement in multiprotein complexes. Subsequent evolution involved deployment of VWA domains by Metazoa in extracellular proteins involved in cell adhesion such as integrin beta subunits (all Metazoa). Nematodes and chordates separately expanded their complements of extracellular matrix proteins containing VWA domains, whereas plants expanded their intracellular complement. Chordates developed VWA-containing integrin alpha subunits, collagens, and other extracellular matrix proteins (e.g., matrilins, cochlin/vitrin, and von Willebrand factor). Consideration of the known properties of VWA domains in integrins and extracellular matrix proteins allows insights into their involvement in protein-protein interactions and the roles of bound divalent cations and conformational changes. These allow inferences about similar functions in novel situations such as protease regulators (e.g., complement factors and trypsin inhibitors) and intracellular proteins (e.g., helicases, chelatases, and copines).

• Grishin NV
• Treble clef finger--a functionally diverse zinc-binding structural motif.
• Nucleic Acids Res. 2001; 29: 1703-14
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• Detection of similarity is particularly difficult for small proteins and thus connections between many of them remain unnoticed. Structure and sequence analysis of several metal-binding proteins reveals unexpected similarities in structural domains classified as different protein folds in SCOP and suggests unification of seven folds that belong to two protein classes. The common motif, termed treble clef finger in this study, forms the protein structural core and is 25-45 residues long. The treble clef motif is assembled around the central zinc ion and consists of a zinc knuckle, loop, beta-hairpin and an alpha-helix. The knuckle and the first turn of the helix each incorporate two zinc ligands. Treble clef domains constitute the core of many structures such as ribosomal proteins L24E and S14, RING fingers, protein kinase cysteine-rich domains, nuclear receptor-like fingers, LIM domains, phosphatidylinositol-3-phosphate-binding domains and His-Me finger endonucleases. The treble clef finger is a uniquely versatile motif adaptable for various functions. This small domain with a 25 residue structural core can accommodate eight different metal-binding sites and can have many types of functions from binding of nucleic acids, proteins and small molecules, to catalysis of phosphodiester bond hydrolysis. Treble clef motifs are frequently incorporated in larger structures or occur in doublets. Present analysis suggests that the treble clef motif defines a distinct structural fold found in proteins with diverse functional properties and forms one of the major zinc finger groups.

• Hiroaki H, Ago T, Ito T, Sumimoto H, Kohda D
• Solution structure of the PX domain, a target of the SH3 domain.
• Nat Struct Biol. 2001; 8: 526-30
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• The phox homology (PX) domain is a novel protein module containing a conserved proline-rich motif. We have shown that the PX domain isolated from the human p47phox protein, a soluble subunit of phagocyte NADPH oxidase, binds specifically to the C-terminal SH3 domain derived from the same protein. The solution structure of p47 PX has an alpha + beta structure with a novel folding motif topology and reveals that the proline-rich motif is presented on the molecular surface for easy recognition by the SH3 domain. The proline-rich motif of p47 PX in the free state adopts a distorted left-handed polyproline type II helix conformation.

• Snoek M, van Kooij M, de Groot K, van Vugt H
• The mouse p52 subunit of the transcription/DNA repair factor TFIIH is not located in the class III region of the H2 complex, but resides next to a G7a/Bat6 homologue in the telomeric part of the major histocompatibility complex.
• Immunogenetics. 2000; 51: 164-7

• Muda M, Manning ER, Orth K, Dixon JE
• Identification of the human YVH1 protein-tyrosine phosphatase orthologue reveals a novel zinc binding domain essential for in vivo function.
• J Biol Chem. 1999; 274: 23991-5
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• A human orthologue of the Saccharomyces cerevisiae YVH1 protein-tyrosine phosphatase is able to rescue the slow growth defect caused by the disruption of the S. cerevisiae YVH1 gene. The human YVH1 gene is located on chromosome 1q21-q22, which falls in a region amplified in human liposarcomas. The evolutionary conserved COOH-terminal noncatalytic domain of human YVH1 is essential for in vivo function. The cysteine-rich COOH-terminal domain is capable of coordinating 2 mol of zinc/mol of protein, defining it as a novel zinc finger domain. Human YVH1 is the first protein-tyrosine phosphatase that contains and is regulated by a zinc finger domain.

• Boggon TJ, Shan WS, Santagata S, Myers SC, Shapiro L
• Implication of tubby proteins as transcription factors by structure-based functional analysis.
• Science. 1999; 286: 2119-25
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• Tubby-like proteins (TULPs) are found in a broad range of multicellular organisms. In mammals, genetic mutation of tubby or other TULPs can result in one or more of three disease phenotypes: obesity (from which the name "tubby" is derived), retinal degeneration, and hearing loss. These disease phenotypes indicate a vital role for tubby proteins; however, no biochemical function has yet been ascribed to any member of this protein family. A structure-directed approach was employed to investigate the biological function of these proteins. The crystal structure of the core domain from mouse tubby was determined at a resolution of 1.9 angstroms. From primarily structural clues, experiments were devised, the results of which suggest that TULPs are a unique family of bipartite transcription factors.

• Aravind L, Ponting CP
• Homologues of 26S proteasome subunits are regulators of transcription and translation.
• Protein Sci. 1998; 7: 1250-4
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• Single copies of an alpha-helical-rich motif are demonstrated to be present within subunits of the large multiprotein 26S proteasome and eukaryotic initiation factor-3 (eIF3) complexes, and within proteins involved in transcriptional regulation. In addition, p40 and p47 subunits of eIF3 are shown to be homologues of the proteasome subunit Mov34, and transcriptional regulators JAB1/pad1. Finally, the proteasome subunit S5a and the p44 subunit of the basal transcription factor IIH (TFIIH) are identified as homologues. The presence of homologous, and sometimes identical, proteins in contrasting functional contexts suggests that the large multisubunit complexes of the 26S proteasome, eIF3 and TFIIH perform overlapping cellular roles.

• Turner RB, Smith DL, Zawrotny ME, Summers MF, Posewitz MC, Winge DR
• Solution structure of a zinc domain conserved in yeast copper-regulated transcription factors.
• Nat Struct Biol. 1998; 5: 551-5
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• The three dimensional structure of the N-terminal domain (residues 1-42) of the copper-responsive transcription factor Amtl from Candida glabrata has been determined by two-dimensional 1H-correlated nuclear magnetic resonance (NMR) methods. The domain contains an array of zinc-binding residues (Cys-X2-Cys-X8-Cys-X-His) that is conserved among a family of Cu-responsive transcription factors. The structure is unlike those of previously characterized zinc finger motifs, and consists of a three-stranded antiparallel beta-sheet with two short helical segments that project from one end of the beta-sheet. Conserved residues at positions 16, 18 and 19 form a basic patch that may be important for DNA binding.

• Mackay JP, Crossley M
• Zinc fingers are sticking together.
• Trends Biochem Sci. 1998; 23: 1-4

• Konrat R, Weiskirchen R, Krautler B, Bister K
• Solution structure of the carboxyl-terminal LIM domain from quail cysteine-rich protein CRP2.
• J Biol Chem. 1997; 272: 12001-7
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• Proteins of the cysteine-rich protein (CRP) family (CRP1, CRP2, and CRP3) are implicated in diverse processes linked to cellular differentiation and growth control. CRP proteins contain two LIM domains, each formed by two zinc-binding modules of the CCHC and CCCC type, respectively. The solution structure of the carboxyl-terminal LIM domain (LIM2) from recombinant quail CRP2 was determined by multidimensional homo- and heteronuclear magnetic resonance spectroscopy. The folding topology retains both independent zinc binding modules (CCHC and CCCC). Each module consists of two orthogonally arranged antiparallel beta-sheets, and the carboxyl-terminal CCCC module is terminated by an alpha-helix. 15N magnetic relaxation data indicate that the modules differ in terms of conformational flexibility. They pack together via a hydrophobic core region. In addition, Arg122 in the CCHC module and Glu155 in the CCCC module are linked by an intermodular hydrogen bond and/or salt bridge. These residues are absolutely conserved in the CRP family of LIM proteins, and their interaction might contribute to the relative orientation of the two zinc-binding modules in CRP LIM2 domains. The global fold of quail CRP2 LIM2 is very similar to that of the carboxyl-terminal LIM domain of the related but functionally distinct CRP family member CRP1, analyzed recently. The carboxyl-terminal CCCC module is structurally related to the DNA-binding domain of the erythroid transcription factor GATA-1. In the two zinc-binding modules of quail CRP2 LIM2, flexible loop regions made up of conserved amino acid residues are located on the same side of the LIM2 domain and may cooperate in macromolecular recognition.

• Perez-Alvarado GC et al.
• Structure of the carboxy-terminal LIM domain from the cysteine rich protein CRP.
• Nat Struct Biol. 1994; 1: 388-98
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• The three dimensional solution structure of the carboxy terminal LIM domain of the avian Cysteine Rich Protein (CRP) has been determined by nuclear magnetic resonance spectroscopy. The domain contains two zinc atoms bound independently in CCHC (C = Cys, H = His) and CCCC modules. Both modules contain two orthogonally-arranged antiparallel beta-sheets, and the CCCC module contains an alpha-helix at its C terminus. The modules pack due to hydrophobic interactions forming a novel global fold. The structure of the C-terminal CCCC module is essentially identical to that observed for the DNA-interactive CCCC modules of the GATA-1 and steroid hormone receptor DNA binding domains, raising the possibility that the LIM motif may have a DNA binding function.

• Humbert S, van Vuuren H, Lutz Y, Hoeijmakers JH, Egly JM, Moncollin V
• p44 and p34 subunits of the BTF2/TFIIH transcription factor have homologies with SSL1, a yeast protein involved in DNA repair.
• EMBO J. 1994; 13: 2393-8
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• The human BTF2 (TFIIH) transcription factor is a multisubunit protein involved in transcription initiation by RNA polymerase II (B) as well as in DNA repair. In addition to the previously characterized p62 and p89/ERCC3 subunits, we have cloned two other subunits of BTF2, p44 and p34. The gene encoding p44 appeared to be the human counterpart of SSL1, a gene involved in translation and UV resistance in yeast. Interestingly, the p34 subunit also has homology with a domain of SSL1, suggesting that it corresponds to an as yet unidentified protein involved in DNA repair. Both p44 and p34 possess zinc finger domains that may mediate BTF2 binding to nucleic acids.

• Qian X, Gozani SN, Yoon H, Jeon CJ, Agarwal K, Weiss MA
• Novel zinc finger motif in the basal transcriptional machinery: three-dimensional NMR studies of the nucleic acid binding domain of transcriptional elongation factor TFIIS.
• Biochemistry. 1993; 32: 9944-59
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• Transcriptional elongation provides a key control point in the regulation of eukaryotic gene expression. Here we describe homonuclear and 15N-heteronuclear 3D NMR studies of the nucleic acid binding domain of human transcriptional elongation factor TFIIS. This domain contains a Cys4 Zn(2+)-binding site with no homology to previously characterized Cys4, Cys6, or Cys2-His2 Zn fingers. Complete 1H and 15N NMR resonance assignment of a 50-residue TFIIS peptide-Zn2+ complex is obtained. Its solution structure, as determined by distance geometry/simulated annealing (DG/SA) calculations, exhibits a novel three-stranded antiparallel beta-sheet (designated the Zn ribbon). Analogous sequence motifs occur in a wide class of proteins involved in RNA or DNA transactions, including human basal transcriptional initiation factor TFIIE. A three-dimensional model of the TFIIE Cys4 domain is obtained by DG-based homology modeling. The role of the TFIIS Zn ribbon in the control of eukaryotic transcriptional elongation is discussed.

• Malviya AN, Block C
• A bipartite nuclear targeting motif in protein kinase C?
• Trends Biochem Sci. 1992; 17: 176-176

• Hoffman M
• New role found for a common protein "motif".
• Science. 1991; 253: 742-742