Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.

Soluble oligomers of the β-amyloid peptide, Aβ, are associated with the progression of Alzheimer's disease. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aβ oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aβ. Binding changes the color of the dyes from purple to blue, and causes them to fluoresce red when irradiated with green light. Job plot and analytical ultracentrifugation experiments reveal that two trimers complex with one dye molecule. Studies with several triphenylmethane dyes reveal that three N, N-dialkylamino substituents are required for complexation. Several mutant trimers, in which Phe, Phe, and Ile were mutated to cyclohexylalanine, valine, and cyclohexylglycine, were prepared to probe the triphenylmethane dye binding site. Size exclusion chromatography, SDS-PAGE, and X-ray crystallographic studies demonstrate that these mutations do not impact the structure or assembly of the triangular trimer. Fluorescence spectroscopy and analytical ultracentrifugation experiments reveal that the dye packs against an aromatic surface formed by the Phe side chains and is clasped by the Ile side chains. Docking and molecular modeling provide a working model of the complex in which the triphenylmethane dye is sandwiched between two triangular trimers. Collectively, these findings demonstrate that the X-ray crystallographic structures of triangular trimers derived from Aβ can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aβ.

A key challenge in studying the biological and biophysical properties of amyloid-forming peptides is that they assemble to form heterogeneous mixtures of soluble oligomers and insoluble fibrils. Photolabile protecting groups have emerged as tools to control the properties of biomolecules with light. Blocking intermolecular hydrogen bonds that stabilize amyloid oligomers provides a general strategy to control the biological and biophysical properties of amyloid-forming peptides. In this paper we describe the design, synthesis, and characterization of macrocyclic β-hairpin peptides that are derived from amyloidogenic peptides and contain the N-2-nitrobenzyl photolabile protecting group. Each peptide contains two heptapeptide segments from Aβ or Aβ constrained into β-hairpins. The N-2-nitrobenzyl group is appended to the amide backbone of Gly to disrupt the oligomerization of the peptides by disrupting intermolecular hydrogen bonds. X-ray crystallography reveals that N-2-nitrobenzyl groups can either block assembly into discrete oligomers or permit formation of trimers, hexamers, and dodecamers. Photolysis of the N-2-nitrobenzyl groups with long-wave UV light unmasks the amide backbone and alters the assembly and the biological properties of the macrocyclic β-hairpin peptides. SDS-PAGE studies show that removing the N-2-nitrobenzyl groups alters the assembly of the peptides. MTT conversion and LDH release assays show that decaging the peptides induces cytotoxicity. Circular dichroism studies and dye leakage assays with liposomes reveal that decaging modulates interactions of the peptides with lipid bilayers. Collectively, these studies demonstrate that incorporating N-2-nitrobenzyl groups into macrocyclic β-hairpin peptides provides a new strategy to probe the structures and the biological properties of amyloid oligomers.

RNA undergoes many different types of modifications and edits after being transcribed by RNA polymerase. These post-transcriptional alterations regulate many biological processes and aberrant RNA modification is linked to many phenotypes and diseases. One class of modification is called RNA editing, which changes the transcript's genomically-encoded sequence by inserting, deleting, or chemically altering nucleotides. The most abundant type of RNA editing is the enzymatically catalyzed deamination of Adenosine to Inosine (A-to-I), which is observed in millions of human RNA transcripts. Because Inosine base-pairs with Cytosine, the cellular machinery interprets this base as Guanosine. These A-to-I edits therefore can alter RNA secondary structure, splicing events, and re-code specific mRNA codons.Adenosine deaminases acting on RNA (ADARs) are editing enzymes that catalyze the hydrolytic deamination of the 6-amino group of Adenosine to generate Inosine. ADAR proteins are modular with two or three N-terminal doublestranded RNA (dsRNA)-binding domains (dsRBDs) and a C-terminal catalytic or deamination domain comprising roughly 400 amino acids, which regulates much of the RNA sequence specificity. ADARs can only edit adenosines in regions of dsRNA. We recently determined the X-ray crystal structures of various human ADAR2 constructs bound to different dsRNA substrates with natural and unnatural nucleotide analogs to better understand the ADAR reaction mechanism, the origin of editing-site selectivity, and the effect of mutations on diseases. These structures, together with structure-guided mutagenesis experiments, explain the basis of the ADAR deaminase domain's dsRNA specificity, its targeted base-flipping mechanism, and its nearest-neighbor preferences. In addition, the structures revealed that ADARs can bind to dsRNA substrates as an asymmetric dimer, uncovering a previously unknown dimerization interface, which was shown to be biologically significant.

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