A 6 bp Z‐DNA hairpin binds two Zα domains from the human RNA editing enzyme ADAR1

Schade, Markus; Behlke, Joachim; Lowenhaupt, Ky; Herbert, Alan; Rich, Alexander; Oschkinat, Hartmut

doi:10.1016/s0014-5793(99)01119-9

Cited by 31 publications

(42 citation statements)

References 20 publications

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“…Residues 1-78 of the vaccinia virus gene E3L (GenBank no. AAA02759), comprising the Z␣ E3L domain, with four additional vector-encoded residues at the N terminus, were expressed as a fusion protein with an N-terminal (His) 6 tag from a pET-28 vector (Novagen) in Escherichia coli strain BL21(DE3). Alternatively, residues 5-70 of E3L were expressed similarly for DNA interaction assays.…”

Section: Methodsmentioning

confidence: 99%

“…The supernatant was applied on a Ni-NTA column (Qiagen, Hilden, Germany). After washing with 50 mM NaH 2 PO 4 (pH 8.0), 300 mM NaCl, 20 mM imidazole, the (His) 6 tag protein attached to the Ni 2ϩ matrix was eluted by thrombin digestion overnight at room temperature in PBS. The cleaved protein was loaded on a Resource Q column (Amersham Biosciences), and the bound protein was eluted with a gradient of 0-1 M NaCl (20 mM NaH 2 PO 4 , pH 6.5).…”

Section: Methodsmentioning

confidence: 99%

“…Alternatively, bacteria were lysed with Bugbuster (Novagen) following the manufacturer's protocol, and protein was purified as described (4). Briefly, the (His) 6 -fusion protein was purified by using HisBind resin (Novagen). Bound protein was stepped off with 300 mM imidazole in 500 mM NaCl, 20 mM Tris (pH 8.0).…”

Section: Methodsmentioning

confidence: 99%

“…Bound protein was stepped off with 300 mM imidazole in 500 mM NaCl, 20 mM Tris (pH 8.0). The (His) 6 tag was removed by thrombin digestion for 3 h at room temperature in 20 mM Tris⅐HCl (pH 8.0), 150 mM NaCl, 2.5 mM CaCl 2 , 1 mM EDTA. Z␣ E3L protein was further purified by using a HiTrap Q column (Amersham Biosciences) and a 25-500 mM NaCl gradient.…”

Section: Methodsmentioning

confidence: 99%

“…The structurally defined Z␣ domains are 62-residue (␣ plus ␤) helix-turn-helix proteins with an additional ␤-sheet that bind to left-handed Z-DNA with medium nanomolar affinity in vitro (5,6). The 3D structures of the human Z␣ domains of the RNAediting enzyme ADAR1 (Z␣ ADAR1 ) and of the tumor-related protein DLM1 (Z␣ DLM1 ) were solved complexed with Z-DNA (7,8).…”

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The solution structure of the N-terminal domain of E3L shows a tyrosine conformation that may explain its reduced affinity to Z-DNA in vitro

Kahmann

Wecking

Pütter

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The N-terminal domain of the vaccinia virus protein E3L (Z␣E3L) is essential for full viral pathogenicity in mice. It has sequence similarity to the high-affinity human Z-DNA-binding domains Z␣ADAR1 and Z␣DLM1. Here, we report the solution structure of Z␣E3L and the chemical shift map of its interaction surface with Z-DNA. The global structure and the Z-DNA interaction surface of Z␣ E3L are very similar to the high-affinity Z-DNA-binding domains Z␣ ADAR1 and Z␣DLM1. However, the key Z-DNA contacting residue Y48 of Z␣ E3L adopts a different side chain conformation in unbound Z␣E3L, which requires rearrangement for binding to Z-DNA. This difference suggests a molecular basis for the significantly lower in vitro affinity of Z␣ E3L to Z-DNA compared with its homologues.V accinia virus is a member of the large double-stranded DNA family of poxviruses. It has been used globally as a vaccine to eradicate smallpox, a devastating disease caused by variola virus that is presently an important threat of bioterrorism (1). The vaccinia virus protein E3L, which is conserved in variola and related viruses, plays a key role in circumventing the IFNmediated defense of host cells (2). E3L contains two domains, of which the C-terminal double-stranded RNA-binding domain is essential and sufficient for evading IFN host defense in cultured cells. In animal models, however, full pathogenesis requires the N-terminal domain of E3L (2), which has sequence homology to the family of Z-DNA-binding protein domains (Z␣) but shows only comparatively low affinity to Z-DNA in vitro (3). When the Z␣ E3L domain is removed from vaccinia virus and is replaced by either Z␣ ADAR1 or Z␣ DLM1 , the virus retains full pathogenicity in the mouse model. Mutational studies show that these domains bind to Z-DNA (4).The structurally defined Z␣ domains are 62-residue (␣ plus ␤) helix-turn-helix proteins with an additional ␤-sheet that bind to left-handed Z-DNA with medium nanomolar affinity in vitro (5, 6). The 3D structures of the human Z␣ domains of the RNAediting enzyme ADAR1 (Z␣ ADAR1 ) and of the tumor-related protein DLM1 (Z␣ DLM1 ) were solved complexed with Z-DNA (7, 8). Further, the solution structure of Z␣ ADAR1 was determined in the unbound state (9), and residues essential for binding to Z-DNA were identified by alanine-scanning mutagenesis (5). Single-point mutations in two such residues, which strongly reduce the affinity of Z␣ ADAR1 to Z-DNA in vitro, were recently shown to abrogate vaccinia virus pathogenicity in a mouse model when introduced in homologous positions in the N-terminal domain of E3L (Z␣ E3L ) (4). Considering this close correlation in the function of such residues between Z␣ ADAR1 and Z␣ E3L the lack of correlation in their in vitro affinity to Z-DNA is intriguing.Here, we report the solution structure of Z␣ E3L and a chemical shift map of its interaction surface with Z-DNA. The 3D structure and interaction surface of Z␣ E3L is grossly very similar to Z␣ ADAR1 and Z␣ DLM1 but differs in the side chain conformation of a pivotal Z-...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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The solution structure of the N-terminal domain of E3L shows a tyrosine conformation that may explain its reduced affinity to Z-DNA in vitro

Kahmann

Wecking

Pütter

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Survey of the 1999 surface plasmon resonance biosensor literature

2000

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The application of surface plasmon resonance biosensors in life sciences and pharmaceutical research continues to increase. This review provides a comprehensive list of the commercial 1999 SPR biosensor literature and highlights emerging applications that are of general interest to users of the technology. Given the variability in the quality of published biosensor data, we present some general guidelines to help increase confidence in the results reported from biosensor analyses. Copyright © 2000 John Wiley & Sons, Ltd.

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Specific Induced Circular Dichroism and Enhanced B to Z Transitions of Duplexes Stabilized by Chromophore‐Linked Alkynylnucleoside Residues

Fujimoto

Aizawa

Oota

et al. 2010

Chemistry A European J

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We have developed an induced circular dichroism (ICD) probe with a chromophore-linked alkynyldeoxyribose skeleton for analyzing higher-order structures of DNA duplexes in the visible-light region. When CG-repeated oligonucleotides (ODNs) with the probe at their 5' ends adopted Z-form duplexes at a high NaCl concentration, strong ICD signals were observed at the absorptive region of the chromophore. On the other hand, their B-form duplexes, formed at a low NaCl concentration, produced a faint ICD signal. The specific ICD for the Z-form duplexes was found to appear only when the chromophores were attached at the 5' ends of each of the ODNs. Furthermore, the chromophoric alkynylnucleoside residues effectively promoted the B to Z transition of the ODN.

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A 6 bp Z‐DNA hairpin binds two Zα domains from the human RNA editing enzyme ADAR1

Cited by 31 publications

References 20 publications

The solution structure of the N-terminal domain of E3L shows a tyrosine conformation that may explain its reduced affinity to Z-DNA in vitro

The solution structure of the N-terminal domain of E3L shows a tyrosine conformation that may explain its reduced affinity to Z-DNA in vitro

Survey of the 1999 surface plasmon resonance biosensor literature

Specific Induced Circular Dichroism and Enhanced B to Z Transitions of Duplexes Stabilized by Chromophore‐Linked Alkynylnucleoside Residues

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