Structure of 3'-PO4/5'-OH RNA ligase RtcB in complex with a 5'-OH oligonucleotide

Abstract

RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that join 2',3'-cyclic phosphate ends to 5'-OH ends via RtcB-(histidinyl-N)-GMP, RNA 3'-phosphate, and RNA3'pp5'G intermediates. RtcB can ligate either 5'-OH RNA or 5'-OH DNA strands in vitro. The nucleic acid contacts of RtcB are uncharted. Here we report a 2.7 Å crystal structure of Pyrococcus horikoshii RtcB in complex with a 6-mer 5'-OH DNA oligonucleotide _HOA¹pT²pG³pT⁴pC⁵pC⁶, which reveals enzymic contacts of Asn202 to the terminal 5'-OH nucleophile; Arg238 to the A¹pT² and T²pG³ phosphates; Arg190 and Gln194 to the T²pG³ phosphate; and an Arg190 π-cation interaction with the G3 nucleobase. The structural insights affirm functional studies of E. coli RtcB that implicated the conserved counterpart of Arg238 in engagement of the 5'-OH strand for ligation. The essential active site Cys98 that coordinates two manganese ions is oxidized to cysteine sulfonic acid in our structure, raising the prospect that RtcB activity might be sensitive to modulation during oxidative stress.

Keywords: RNA repair; cysteine sulfonic acid; tRNA splicing.

FIGURE 1.

RtcB active site occupancy by 5′-OH oligonucleotide. Stereo view of a simulated annealing difference density omit map of the RtcB active site (beige mesh), contoured at 2 σ with a carve radius of 3.6 Å. Strong electron density surrounding the Cys98 sulfur atom (shown in green) was modeled as cysteine sulfonic acid. A 6-mer DNA oligonucleotide, depicted as a stick model with gray carbons, was placed into density as shown. The nucleobases are labeled and numbered starting from the 5′-OH end. A sulfate anion (stick model) was also placed into density.

FIGURE 2.

Structure of RtcB in complex with 5′-OH oligonucleotide. (A) Stereo view of the RtcB fold, shown as a cartoon model with magenta β strands and cyan helices. The 5′-OH DNA oligonucleotide and sulfate are rendered as stick models. (B) Stereo view of a surface electrostatic model of RtcB (generated in Pymol) in the same orientation as in A, with the 5′-OH DNA and sulfate depicted as stick models.

FIGURE 3.

Structural consequences of Cys98 oxidation. Stereo view of the active site highlighting contacts to the cysteine sulfonic acid moiety (depicted as a stick model with green sulfur atom). Amino acids in the present structure that normally engage the two manganese ions are rendered with beige carbons. Hydrogen bond interactions with the cysteine sulfonic acid are indicated by dashed lines. The equivalent amino acids and the Mn1 and Mn2 manganese ions from PhoRtcB structure 4ISZ are superimposed and depicted as semitransparent stick models with gray carbons and semitransparent magenta spheres, respectively.

FIGURE 4.

RtcB interface with the 5′-OH strand. Stereo view of the active site highlighting atomic interactions with the 5′-OH DNA oligonucleotide. Hydrogen bonds are indicated by black dashed lines; van der Waals contacts by green dashed lines. The guanylate nucleotide from PhoRtcB structure 4IT0 is superimposed and depicted as a semitransparent stick model with yellow carbons. Putative hydrogen bonds from Asn202 to the guanylate are shown as yellow dashed lines.