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Solution structures of the DNA-binding domains of immune-related zinc-finger protein ZFAT.

ABSTRACT: ZFAT is a transcriptional regulator, containing eighteen C2H2-type zinc-fingers and one AT-hook, involved in autoimmune thyroid disease, apoptosis, and immune-related cell survival. We determined the solution structures of the thirteen individual ZFAT zinc-fingers (ZF) and the tandemly arrayed zinc-fingers in the regions from ZF2 to ZF5, by NMR spectroscopy. ZFAT has eight uncommon bulged-out helix-containing zinc-fingers, and six of their structures (ZF4, ZF5, ZF6, ZF10, ZF11, and ZF13) were determined. The distribution patterns of the putative DNA-binding surface residues are different among the ZFAT zinc-fingers, suggesting the distinct DNA sequence preferences of the N-terminal and C-terminal zinc-fingers. Since ZFAT has three to five consecutive tandem zinc-fingers, which may cooperatively function as a unit, we also determined two tandemly arrayed zinc-finger structures, between ZF2 to ZF4 and ZF3 to ZF5. Our NMR spectroscopic analysis detected the interaction between ZF4 and ZF5, which are connected by an uncommon linker sequence, KKIK. The ZF4-ZF5 linker restrained the relative structural space between the two zinc-fingers in solution, unlike the other linker regions with determined structures, suggesting the involvement of the ZF4-ZF5 interfinger linker in the regulation of ZFAT function.

SUBMITTER: Tochio N

PROVIDER: S-EPMC4427657 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The RNA binding motif protein 5 (RBM5), also known as Luca15 or H37, is a component of prespliceosomal complexes that regulates the alternative splicing of several mRNAs, such as Fas and caspase-2. The RBM5 gene is located at the 2p21.3 chromosomal region, which is strongly associated with lung cancer and many other cancers. Both increased and decreased levels of RBM5 can play a role in tumor progression. In particular, downregulation of rbm5 is involved in lung cancer and other cancers upon Ras activation, and, also, represents a molecular signature associated with metastasis in various solid tumors. On the other hand, upregulation of RBM5 occurs in breast and ovarian cancer. Moreover, RBM5 was also found to be involved in the early stage of the HIV-1 viral cycle, representing a potential target for the treatment of the HIV-1 infection. While the molecular basis for RNA recognition and ubiquitin interaction has been structurally characterized, small molecules binding this zinc finger (ZF) domain that might contribute to characterizing their activity and to the development of potential therapeutic agents have not yet been reported. Using an NMR screening of a fragment library we identified several binders and the complex of the most promising one, compound 1, with the RBM5 ZF1 was structurally characterized in solution. Interestingly, the binding mechanism reveals that 1 occupies the RNA binding pocket and is therefore able to compete with the RNA to bind RBM5 RanBP2-type ZF domain, as indicated by NMR studies.

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Project description:The largest and most diverse class of eukaryotic transcription factors contain Cys2-His2 zinc fingers (C2H2-ZFs), each of which typically binds a DNA nucleotide triplet within a larger binding site. Frequent recombination and diversification of their DNA-contacting residues suggests that these zinc fingers play a prevalent role in adaptive evolution. Very little is known about the function and evolution of the vast majority of C2H2-ZFs, including whether they even bind DNA. Using the bacterial 1-hybrid (B1H) system, we determined DNA-binding motifs for thousands of individual natural C2H2-ZFs, and correlated them with C2H2-ZF specificity residues. The data reported here includes results of protein-binding microarray (PBM) assays for 146 of these natural C2H2-ZFs, performed in order to validate B1H assays and to explore the DNA-binding specificity of C2H2-ZFs. Protein binding microarray (PBM) experiments were performed for a set of 185 variants of mouse Egr1 in which the third zinc finger was replaced by different C2H2-ZFs from different organisms. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to two double-stranded 44K Agilent microarrays, each containing a different DeBruijn sequence design, in order to determine their sequence preferences. Details of the PBM protocol are described in Berger et al., Nature Biotechnology 2006. Among the 185 variants examined, 146 variants yielded motifs in PBMs, which are included here.

2015-02-17 | E-GEOD-52520 | biostudies-arrayexpress

Evaluation of DNA-binding preferences of C2H2 zinc finger domains by PBM

2015-02-17 | GSE52520 | GEO

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Project description:Individual zinc finger (ZF) domains that recognize DNA triplets with high specificity and affinity can be used to create designer transcription factors and nucleases that are specific for nearly any site in the genome. These domains can be treated as modular units and assembled to create a polydactyl protein that recognizes extended DNA sequences. Deter-mination of valid target sites and the subsequent design of ZF proteins (ZFPs) is error-prone and not trivial, however. As a result, the use of ZFPs have been restricted primarily to those labs with the appropriate expertise. To address these limitations, we have created a user-friendly utility called Zinc Finger Tools (ZF Tools) that can be accessed at the URL http://www.zincfingertools.org. User-supplied DNA sequences can be searched for target sites appropriate for either gene regulation or nuclease targeting. Using a database of experimentally characterized zinc finger domains, the amino acid sequence for a ZFP expected to bind to any chosen target site can be generated. A reverse engineering utility is provided to predict the binding site for a ZFP of known sequence.

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Project description:Introduction: DOF proteins are a family of plant-specific transcription factors with a conserved zinc finger (ZF) DNA-binding domain. Although several studies have demonstrated their specific DNA binding, quantitative affinity data is not available for the binding of DOF domains to their binding sites. Methods: ZF domains of DOF2.1, DOF3.4, and DOF5.8 from Arabidopsis thaliana were expressed and purified. Their DNA binding affinities were assessed using gel retardation assays and microscale thermophoresis with two different oligonucleotide probes containing one and two copies of recognition sequence AAAG. Results: DOF zinc finger domains (DOF-ZFs) were shown to form independently folded structures. Assessments using microscale thermophoresis demonstrated that DOF-ZFs interact more tightly (~ 100 fold) with double-motif probe than the single-motif probe. The overall Kd values for the DOF3.4-ZF and DOF5.8-ZF to the double-motif probe were ~2.3±1 and 2.5±1 µM, respectively. Conclusion: Studied DOF-ZF domains formed stable complexes with the double-motif probe. Although DOF3.4-ZF and DOF5.8-ZF do not dimerize with an appreciable affinity in the absence of DNA (judging from size-exclusion and multiangle laser light scattering data), it is possible that these ZFs form protein-protein contacts when bound to this oligonucleotide, consistent with previous reports that DOF proteins can homo- and hetero-dimerize.

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