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 α-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.

FIGURE 1.

ABH8 domain organization and catalytic activity. A, schematic diagram illustrating ABH8 domain organization and the human protein constructs used in this study. B, an unidentified enzyme or enzymes convert uridine at position 34 (U34) in the anticodon loop of tRNAs into cm⁵U, which is methylated by the MTase domain of ABH8 in complex with the Trm112 protein to generate mcm⁵U (15, 17). This product is stereoselectively hydroxylated by the AlkB domain of ABH8 to generate (S)-mchm⁵U (16, 18).

FIGURE 2.

Sequence-structure alignment for the ABH8 protein. A sequence alignment (59) of the RRM/AlkB domains of a set of metazoan ABH8 orthologs is combined with structure-based alignments of E. coli AlkB, human ABH2, human ABH3, the AlkB domain of human FTO, and the RRM domain of S. cerevisiae RNa15. The RNP1 consensus sequence in the RRM domain is indicated by asterisks (*), the invariant residues chelating Mn(II) and 2OG in the AlkB domain are indicated by pound signs (#), and the residues ligating Zn(II) in this domain are indicated by black dots (●). The black boxes indicate disordered regions in the crystal structure of the RRM/AlkB domains of human ABH8 (Table 2 and Fig. 6 below). Conserved and semiconserved residues, grouped according to their biophysical characteristics, are colored white on a red background and red on a white background, respectively. The secondary structural elements found in the crystal structure of the RRM/AlkB domains of human ABH8 are shown schematically above the sequence alignment, with α-helices represented as sine curves and β-strands as arrows. H., Homo; M., Mus; O., Ornithorhynchus; D., Drosophila; C., Caenorhabditis; S., Saccharomyces; E., Escherichia.

FIGURE 3.

Developmentally regulated expression of C14B1.10, the C. elegans ABH8 ortholog. A–D, P_C14B1.10gfp, a fusion of the C14B1.10 promoter to the GFP gene, shows expression in neuronal (smaller arrows) and non-neuronal (large arrow) cells. Expression is higher and more widely distributed at the early larval stage (A), reduced at the middle larvae stage (B), and almost exclusively detected in neuronal cells in the head (C) and tail (D) of adult animals. E and F, C14B1.10::GFP, the GFP protein fused to the C14B1.10 protein under its endogenous promoter control, is exclusively localized to the cytoplasm. Arrows indicate the positions of selected neurons in the head (E) and tail (F) of an adult animal. Scale bars, 5 μm.

FIGURE 4.

The C-terminal structural Zn(II)-binding site stabilizes the ABH8 RRM/AlkB double domain construct. A and B, thermal denaturation assays monitored using the fluorescent reporter SYPRO Orange demonstrate that the Zn(II)-binding site strongly stabilizes the folding of the AlkB domain. A shows thermal denaturation of the wild-type 1–354 construct in 150 mm NaCl, 50 mm Hepes, pH 7.5 either in the absence (black squares) or in the presence (gray diamonds) of 5 mm EDTA as well as thermal denaturation of the C341A/C349A double mutant of the same construct in the absence of EDTA (dark gray circles). B shows equivalent assays performed on the double mutant in the absence of EDTA with the addition of either 1 mm Mn(II) (light gray squares) or 1 mm Mn(II) plus 10 mm 2OG (gray triangles); the WT and double mutant denaturation curves in the absence of EDTA are shown here again for comparison. The cooperative action of Mn(II)/2OG in stabilizing the first of the two sequential transitions in the double mutant indicates that this transition corresponds to unfolding of the AlkB domain.

FIGURE 5.

RNA binding properties of ABH8 protein constructs. A and B, filter binding assays in which increasing concentrations of protein were titrated at room temperature onto 5 nm radiolabeled tRNA-Gly (A) or tRNA-Glu (B) in 150 mm NaCl, 1 mm MgCl₂, 20 mm HEPES, pH 7.5. The mean and standard deviation of the fraction of protein-bound RNA in triplicate assays are plotted for an RRM domain construct (1–133; black), an RRM/AlkB double domain construct (1–354; blue), and the entirety of ABH8 in complex with the Trm112 protein (1–633; red). C and D, fluorescence anisotropy assays in the same buffer in which increasing concentrations of protein were titrated at 25 °C onto 5′-fluorescein-labeled synthetic 17-mer step-loop matching the anticodon loop of tRNA-Gly (C) or aptamer ABH8-2.2 (D), which was selected in vitro to bind to the 1–354 RRM/AlkB double domain construct. Results from a single titration are plotted for an RRM domain construct (1–133; black), RRM/AlkB domain constructs either with (1–354; dark blue) or without (25–354; light blue) the first 24 N-terminal residues, an MTase domain construct in complex with Trm112 (MT; orange), and the entirety of ABH8 protein in complex with Trm112 (1–633; red).

FIGURE 6.

Crystal structure of the RRM/AlkB domains in human ABH8. A, stereo ribbon diagram of the structure in complex with Zn(II) and Mn(II)/2OG. The conserved and non-conserved regions in the RRM domain are colored red and orange, respectively, whereas the conserved and non-conserved regions in the AlkB domain are colored blue and magenta, respectively. The loop connecting the two domains is shown in yellow. Conserved residues in the RRM (Lys⁸⁰, Tyr⁸², and Phe⁸⁴) and AlkB (His²³⁸, Asp²⁴⁰, His²⁹², Arg³²⁸, and Arg³³⁴) domains as well as the 2OG bound to the AlkB domain are shown in stick representation (with carbon and oxygen colored white and red, respectively). The Mn(II) ion bound in the active site and the Zn(II) ion bound in the C-terminal structural Zn(II)-binding site are shown as purple and yellow spheres, respectively. The asterisk in A and B marks the location of a putative pyrimidine-binding pocket in the RRM domain (shown in C). The observed N terminus of the RRM domain at residue 29 and C terminus of the AlkB domain at residue 354 are labeled in black (“N29” and “C”, respectively). Residues 13–32 in ABH8, most of which were deleted to improve crystal quality, are shown as an α-helical wheel with basic amino acids colored blue. Assessment of structural conservation and the numbering of the secondary structural elements (supplemental Fig. S7A) in the AlkB domain are based on comparison with other AlkB family enzymes (–50) (rather than the Fe(II)/2OG dioxygenase superfamily as done in Yu et al. (35)). Residues 156–174 and 181–192 in the AlkB domain (dotted green lines), which are topologically equivalent to the segments forming the nucleotide-binding lid in E. coli AlkB, are disordered in ABH8. B, stereopair showing the electrostatic potential of the molecular surface of the domains oriented as in A. Fully saturated blue and red colors represent potentials of ±8 kT at 100 mm ionic strength as calculated by GRASP2 (60). C, two views of a putative pyrimidine-binding pocket formed in part by the RNP1 motif in the RRM domain. The entrance to this cavity, which can accommodate a pyrimidine base without steric clash, lies on the surface of the RRM domain below the active site in the AlkB domain. The entrance is marked by a black asterisk in A and B and in supplemental Fig. S7A. Residue Tyr⁸² is positioned at the base of the cavity (as shown in the right panel) where it could make a stacking interaction with a bound pyrimidine. The residues in the RNP1 motif are shown in stick representation colored according to atomic identity (carbon in gray, oxygen in red, and nitrogen in blue). The molecular surface is colored like the backbone except for the regions formed by the side chains of the residues in RNP1 motif, which are colored according to atomic identity. D, stereopair showing superposition of the active sites in ABH8 (blue) and E. coli AlkB (green; Protein Data Bank code 2FDH) with the invariant residues, the 2OG co-substrates, and the Mn(II) cofactors colored the same as the domains. Mn(II) is widely used in studies of Fe(II)/2OG dioxygenases as a catalytically inactive analog of Fe(II) that preserves active site stereochemistry. Although some high resolution crystal structures of Fe(II)/2OG dioxygenases have a weakly ordered H₂O molecule in the final ligation position on the Fe(II), Mn(II), or Co(II) ion occupying the catalytic site, this ligation site is empty in other structures (, , –50), as it is in the ABH8 active site shown here. However, other structures generally preserve octahedral coordination geometry for the ligating atoms as shown in D for E. coli AlkB, as opposed to the distorted geometry observed in all of the crystallographically independent views of the ABH8 active site in the structures reported in this study.

FIGURE 7.

Structural alignments of human ABH8 domains. A, structural alignment (51) of the RRM domains from ABH8 and S. cerevisiae Rna15 (56). The backbone of the ABH8 RRM domain is colored red and orange as in Fig. 6A, and the residues in its RNP1 motif are highlighted and shown in gray in stick representation (with black labels except for Tyr⁸², which is unlabeled). The N terminus of the ABH8 domain is indicated by the black label “N.” The backbone of Rna15 is colored cyan; its crystallographically observed side chains interacting with RNA (magenta with blue labels), the consensus residues in its RNP1 motif (light blue), and its bound RNA ligand (gray) are all shown in stick representation. Note that this RNA ligand binds to Rna15 on the ridge of the RRM domain proximal to the AlkB domain in the structure of ABH8. The residues in the RNP1 motif are solvent-exposed in Rna15 but partially buried by the N-terminal α-helical segment in ABH8. B, structural alignment of the ABH8 AlkB domain with E. coli AlkB (35). ABH8 is colored as in Fig. 6A with gray spheres added to highlight the termini of its disordered backbone segments. Its bound Mn(II) and Zn(II) cations are shown, respectively, as purple and yellow spheres, whereas its bound 2OG co-substrate is shown in purple in stick representation (adjacent to the Mn(II) cofactor). The core of E. coli AlkB is colored green, its nucleotide-recognition lid (NRL) is colored red, and its bound DNA substrate (TmAT) is colored orange. C, structural alignment of the AlkB domains from ABH8 and human FTO with the latter colored like E. coli AlkB in B (50). The secondary structure elements that differ between ABH8 and its homologs are colored in magenta in B and C.