The solution structure of the Zα domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA

Abstract

Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Z␣. Here we report the solution structure of free Z␣ and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Z␣) 2͞Z-DNA complex shows that most Z-DNA contacting residues in free Z␣ are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (␣؉␤)helix-turn-helix͞B-DNA complexes suggests that binding of Z␣ to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B-and Z-DNA. RNA editing in mammals alters codons in mRNA through site-specific deamination of adenosines and cytosines, leading to proteins with modified function. Adenosine to inosine (A 3 I) editing modulates the calcium permeability of neural glutamate receptors (1) and reduces the G-protein coupling efficacy of serotonin 2C receptors (2). Double-stranded RNA deaminases I and II (ADAR1͞2) catalyze these A 3 I conversions, but unknown auxiliary factors are thought to be involved in the control of editing efficiency in vivo (3). ADAR1, but not ADAR2, has two left-handed Z-DNA binding domains, Z␣ and Z␤, at its N terminus. These domains may contribute to the control of ADAR1-mediated editing in vivo (4). Z-DNA formation in vivo has been shown to be transcription dependent in prokaryotes and eukaryotes (5). Z-DNA can be generated transiently 5Ј to a moving RNA polymerase in alternating purine͞pyrimidine sequences (5), thereby providing a transient binding site for Z␣ and Z␤. Thus, Z-DNA binding may ensure that the catalytic activity of ADAR1 is targeted to sites where nascent pre-mRNA substrates emerge (5).Here we have determined the solution structure of free Z␣ and mapped the interaction surface between Z␣ and a 6-bp d(CG) substrate DNA by two-dimensional (2D) 15 N-heteronuclear single quantum correlation (HSQC) NMR spectroscopy. Z␣ binds this substrate with high affinity (K d ϭ 30 nM) and a stoichiometry of 2:1 (protein͞DNA) (6-10). The map of the interaction surface in solution agrees well with the crystal structure of Z␣ complexed with Z-DNA (7). Further the structure of Z␣ free in solution demonstrates that there are only minor conformational changes upon binding Z-DNA. Not only is the overall structure the same, but unexpectedly, most Z-DNA contacting residues are prepositioned in free Z␣ to fit Z-DNA. This study also examines why Z␣ preferentially binds Z-DNA rather than B-DNA despite its high structural homology to (␣ϩ␤) helix-turn-helix (␣ϩ␤HTH) B-DNA binding proteins. Materials and MethodsProtein Preparation. The Z␣ domain, comprising residues 119-200 of human ADAR1 (GenBank accession no. U10439), has been described (8). Z␣ was expressed as a fusion protein with a N-terminal (His) 6 -tag from a pET-21a vector (Novagen) in Escherichia coli strain HM174(DE3). For isotope labeling, bacteria were grown in M9 medium containing 1 g͞liter 15 NH 4 Cl and 1.5 g͞liter 13 C-gl...

Seven novel mutations of theADAR gene in Chinese families and sporadic patients with dyschromatosis symmetrica hereditaria (DSH)

“…The ADAR gene spans 30 kb and contains 15 exons (Wang et al, 1995). Two Z-alpha domains, three dsRNAbinding domains and the putative deaminase domain are located in exon 2, exons 2-7 and exons 9-15, respectively (Wang et al, 1995;Schade et al, 1999). The heterozygosity for the ADAR knockout causes embryonic lethality in mice (Wang et al, 2000), whereas patients with DSH have a good prognosis of dyschromatosis, which is localized specifically on the backs of hands and on tops of the feet.…”

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 side chains of the other residues in this loop (D60, D61, I62, and R65) are flexible in the structural ensemble, giving this loop the shape of a solvent accessible finger with a rigid backbone. Mutational (5) and structural studies (7,9) have demonstrated that this distinctly conserved feature is essential for selective interaction of the homologous Z␣ ADAR1 domain with Z-DNA. In the co-crystal structure of Z␣ ADAR1 and Z-DNA, the protein makes several important van der Waals contacts to Z-DNA.…”

“…Alternating d(CG) n oligomers have been successfully used to study the interaction of the Z-DNA-binding domains Z␣ ADAR1 (7,9) and Z␣ DLM1 (8) with Z-DNA. In contrast to these high-affinity Z-DNA-binding homologues, Z␣ E3L does not f lip the B-DNA conformation of these substrates into the Zconformation when incubated with each other under physiological buffer conditions at micromolar concentrations (3).…”

“…contrast, the Y48 Ϫ W66 distance in bound (9) and unbound Z␣ ADAR1 (7) measures only 3.9 and 4.5 Å, respectively. The experimental foundation of this marked difference is the observation of several long-range NOEs between the aromatic rings of Y48 and W66 in the NMR structure of unbound Z␣ ADAR1 but none of such NOEs in the 13 C-edited NOESY spectra of Z␣ E3L .…”

“…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).…”