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Crystal structure and RNA binding properties of the RNA recognition motif (RRM) and AlkB domains in human AlkB homolog 8 (ABH8), an enzyme catalyzing tRNA hypermodification.

ABSTRACT: Humans express nine paralogs of the bacterial DNA repair enzyme AlkB, an iron/2-oxoglutarate-dependent dioxygenase that reverses alkylation damage to nucleobases. The biochemical and physiological roles of these paralogs remain largely uncharacterized, hampering insight into the evolutionary expansion of the AlkB family. However, AlkB homolog 8 (ABH8), which contains RNA recognition motif (RRM) and methyltransferase domains flanking its AlkB domain, recently was demonstrated to hypermodify the anticodon loops in some tRNAs. To deepen understanding of this activity, we performed physiological and biophysical studies of ABH8. Using GFP fusions, we demonstrate that expression of the Caenorhabditis elegans ABH8 ortholog is widespread in larvae but restricted to a small number of neurons in adults, suggesting that its function becomes more specialized during development. In vitro RNA binding studies on several human ABH8 constructs indicate that binding affinity is enhanced by a basic ?-helix at the N terminus of the RRM domain. The 3.0-Å-resolution crystal structure of a construct comprising the RRM and AlkB domains shows disordered loops flanking the active site in the AlkB domain and a unique structural Zn(II)-binding site at its C terminus. Although the catalytic iron center is exposed to solvent, the 2-oxoglutarate co-substrate likely adopts an inactive conformation in the absence of tRNA substrate, which probably inhibits uncoupled free radical generation. A conformational change in the active site coupled to a disorder-to-order transition in the flanking protein segments likely controls ABH8 catalytic activity and tRNA binding specificity. These results provide insight into the functional and structural adaptations underlying evolutionary diversification of AlkB domains.

SUBMITTER: Pastore C

PROVIDER: S-EPMC3265892 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Crystal structure and RNA binding properties of the RNA recognition motif (RRM) and AlkB domains in human AlkB homolog 8 (ABH8), an enzyme catalyzing tRNA hypermodification.

Pastore Chiara C Topalidou Irini I Forouhar Farhad F Yan Amy C AC Levy Matthew M Hunt John F JF

The Journal of biological chemistry 20111107 3

Humans express nine paralogs of the bacterial DNA repair enzyme AlkB, an iron/2-oxoglutarate-dependent dioxygenase that reverses alkylation damage to nucleobases. The biochemical and physiological roles of these paralogs remain largely uncharacterized, hampering insight into the evolutionary expansion of the AlkB family. However, AlkB homolog 8 (ABH8), which contains RNA recognition motif (RRM) and methyltransferase domains flanking its AlkB domain, recently was demonstrated to hypermodify the a ...[more]

PMID: 22065580

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Project description:Systemic lupus erythematosus (SLE) is characterized by the production of grouped sets of autoantibodies targeting mainly the U1 ribonucleoprotein (RNP) and/or Ro/La RNP particles. Intraparticle diversification of the autoimmune response is believed to occur via epitope spreading. So far, it is not known how the autoimmune response "jumps" from one particle to another. To the extent that the majority of nuclear autoantigens in SLE are RNA binding proteins and major epitopes were previously mapped within their RRM (RNA recognition motifs), conserved sequences within RRM could be involved in the intermolecular and inter-particle diversification process of the autoimmune response. We investigated the potential of RRM of the La/SSB autoantigen to induce antibodies that cross-recognize components of the U1-RNP particle and therefore its capacity to produce interparticle epitope spreading. We immunized New Zealand white rabbits with a peptide corresponding to the epitope 145-164 of La/SSB (belonging to the RRM of La/SSB), attached in four copies on a scaffold carrier. Sera were drawn from 20 sera of patients with SLE and anti-U1-RNP antibodies and 26 sera of primary Sjögren syndrome patients with anti-La/SSB antibodies. All sera were evaluated for reactivity against the major epitope of La/SSB (pep349-364), the RNP antigen and the RRM-related epitope of La/SSB (pep145-164). Specific antibodies against pep145-164 were purified with immunoaffinity columns from selected sera. After the immunization of the animals with pep145-164, a specific IgG antibody response was detected, directed against the La/SSB autoantigen (wks 3-7), the immunizing peptide (wks 3-27), and the RNP autoantigen (wks 7-20). This response gradually decreased to low levels between postimmunization wks 27-42. Purified antibodies against pep145-164 recognized La/SSB and a 70-kD autoantigen in Western blot and exhibited significant reactivity in anti-U1-RNP ELISA. Depletion of anti-pep145-164 antibodies eliminated anti-U1-RNP reactivity from immunized rabbit sera but not from human sera. In addition, pep145-164 was recognized to a greater extent by autoimmune sera with anti-RNP reactivity compared with anti-La/SSB-positive sera, in contrast to pep349-364 of La/SSB, which was recognized almost exclusively by sera with anti-La/SSB reactivity. These data suggest that the RRM region of La/SSB can trigger interparticle B-cell diversification to U1-RNP-70 autoantigen via molecular mimicry. Identification of key sequences that trigger and perpetuate the autoimmune process is particularly important for understanding pathogenetic mechanisms in autoimmunity.

Dataset Information

Crystal structure and RNA binding properties of the RNA recognition motif (RRM) and AlkB domains in human AlkB homolog 8 (ABH8), an enzyme catalyzing tRNA hypermodification.

Publications

Crystal structure and RNA binding properties of the RNA recognition motif (RRM) and AlkB domains in human AlkB homolog 8 (ABH8), an enzyme catalyzing tRNA hypermodification.

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