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 RNA_3′pp_5′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.