Structural and mechanistic insights into guanylylation of RNA-splicing ligase RtcB joining RNA between 3 '-terminal phosphate and 5 '-OH

Abstract

The RtcB protein has recently been identified as a 3'-phosphate RNA ligase that directly joins an RNA strand ending with a 2', 3'-cyclic phosphate to the 5'-hydroxyl group of another RNA strand in a GTP/Mn2+-dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn2+ alone (RtcB/Mn2+) and together with a covalently bound GMP (RtcB-GMP/Mn2+). The RtcB/Mn2+ structure (at 1.6 angstrom resolution) shows two Mn2+ ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn2+ complex (at 2.3 angstrom resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn2+ ions, the four sulfates, and GMP are validated in extensive mutagenesis and biochemical experiments, which also provide a thorough characterization for the three steps of the RtcB ligation pathway: (i) guanylylation of the enzyme, (ii) guanylyl-transfer to the RNA substrate, and (iii) overall ligation. These results demonstrate that the enzyme's substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn2+ active center, which provides detailed insight into how the enzyme-bound GMP is tansferred to the 3'-phosphate of the RNA substrate for activation and subsequent nucleophilic attack by the 5'-hydroxyl of the second RNA substrate, resulting in the ligated product and release of GMP.