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/Mn(2+)-dependent reaction. Here, we report two crystal structures of Pyrococcus horikoshii RNA-splicing ligase RtcB in complex with Mn(2+) alone (RtcB/ Mn(2+)) and together with a covalently bound GMP (RtcB-GMP/Mn(2+)). The RtcB/ Mn(2+) structure (at 1.6 Å resolution) shows two Mn(2+) 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/Mn(2+) complex (at 2.3 Å resolution) reveals the detailed geometry of guanylylation of histidine 404. The critical roles of the key residues involved in the binding of the two Mn(2+) 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 Mn(2+) 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.

Fig. 1.

An overview of structure-derived reaction mechanism of the 3′-P RNA ligation. (A) Composite features of the two PH RtcB structures reported here with emphasis on the relationship of four sulfates and two Mn²⁺ ions with guanylylated H404 in the catalytic site in ribbons representation along with other highlighted residues, namely D65 and N202. (B) A summary of the RtcB-catalyzed pathway in three individual steps (described in the main text and schematized in C–E) as deduced from our structures and biochemistry. (C) The RtcB residues that specifically recognize the substrate GTP are shown in green. The putative PPi site coincides with SO₄-4 (orange). The carboxylate group of D65 is hydrogen bonded to H404 Nδ1-H, thus enhancing the electron negativity of H404 Nε2 for nucleophilic attack on GTP’s α-phosphate that is in contact with the Mn²⁺ in octahedral geometry. (D) Coordination of tetrahedral Mn²⁺ allows binding the RNA strand ending with the 2′,3′-cyclic phosphate (RNA>p) superimposed with the last two phosphates at the SO₄-1 and SO₄-2 sites. (E) Positioning the ^HORNA strand with its first phosphate at the SO₄-3 allows the optimal geometry of the 5′-hydroxyl for nucleophilic attack on the 3′-P activated terminus to form the internucleotide phosphodiester bond with the release of GMP.

Fig. 2.

The Mn²⁺-bound RtcB complex. (A) Overview of the secondary structures of RtcB in rainbow colors from the N-to-C terminus in stereo. Active site residues, metal ions, four sulfate molecules, and (1,5-anhydro-d-glucose)-α1β2-(2,5-anhydro-l-fructose) (labeled as dGlF) are shown in balls-and-sticks. (B) Close-up view on the RtcB/Mn²⁺ active site. Residues cordinating the two Mn²⁺ ions in octahedral and tetrahedral geometry are highlighted. SO₄-2 and three ordered water molecules serve as additional ligands.

Fig. 3.

GTP substrate recognition revealed from the RtcB-GMP/Mn²⁺ structure. RtcB has reacted with GTP to covalently bind GMP to H404. Annotated PPi (Fig. S2) interacts with R408 and R412. Residues specifically recognizing the guanine base and the ribose are indicated.

Fig. 4.

Induced GTP-binding pocket as revealed from structural comparison. Superpositon of the RtcB/Mn²⁺ (blue) and RtcB-GMP/Mn²⁺ (turquois) structure shows opening of the cleft for the guanine base. The peptide backbone of A406 and G407 reorientate to allow amide interactions with the hydroxyl groups of the ribose.

Fig. 5.

Activity assays for PH RtcB. (A) The 21-nt RNA with 5′-hydroxyl and 2′,3′-cyclic phosphate temini is incubated with WT or mutant RtcB proteins and separated on PAGE for PhosphorImager analysis. WT RtcB allows not only formation of the faster migrating circular RNA, but also cumulation of a slower migrating RNA. The same nonradioactive RNA incubated with α[³²P]GTP identifies this species as guanylylated RNA intermediate. (B) WT and three RtcB variants are incubated with α[³²P]GTP and separated on SDS PAGE for PhosphorImager analysis indicating protein guanylylation.