4DWQ

RNA ligase RtcB-GMP/Mn(2+) complex

Summary for 4DWQ

• Entry DOI: 10.2210/pdb4dwq/pdb
• Related: 4DWR
• Descriptor: tRNA-splicing ligase RtcB, GUANOSINE-5'-MONOPHOSPHATE, MANGANESE (II) ION, ... (8 entities in total)
• Functional Keywords: rtcb, guanylylation, rna ligase, rtcb-gmp, mn2+, ligase
• Biological source: Pyrococcus horikoshii; More
• Total number of polymer chains: 2
• Total formula weight: 109142.29

Authors

Okada, C.,Xia, S.,Englert, M.,Yao, M.,Soll, D.,Wang, J. (deposition date: 2012-02-26, release date: 2012-09-05, Last modification date: 2024-11-20)

Primary citation

Englert, M.,Xia, S.,Okada, C.,Nakamura, A.,Tanavde, V.,Yao, M.,Eom, S.H.,Konigsberg, W.H.,Soll, D.,Wang, J.
Structural and mechanistic insights into guanylylation of RNA-splicing ligase RtcB joining RNA between 3'-terminal phosphate and 5'-OH.
Proc.Natl.Acad.Sci.USA, 109:15235-15240, 2012

PubMed 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.

PubMed: 22949672
DOI: 10.1073/pnas.1213795109

Experimental method

X-RAY DIFFRACTION (2.25 Å)