title: 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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Markus
authors.initials: M
authors.lastname: Englert
authors.forename: Shuangluo
authors.initials: S
authors.lastname: Xia
authors.forename: Chiaki
authors.initials: C
authors.lastname: Okada
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Nakamura
authors.forename: Ved
authors.initials: V
authors.lastname: Tanavde
authors.forename: Min
authors.initials: M
authors.lastname: Yao
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Eom
authors.forename: William H
authors.initials: WH
authors.lastname: Konigsberg
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.forename: Jimin
authors.initials: J
authors.lastname: Wang
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D006152:Cyclic GMP / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry; D007477:Ions; D008345:Manganese / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D011118:Polynucleotide Ligases / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D043902:Pyrococcus horikoshii / Q000378:metabolism*; D012326:RNA Splicing; D012343:RNA, Transfer / Q000737:chemistry; D013379:Substrate Specificity; D013431:Sulfates / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D007477:Ions; D013431:Sulfates; D008345:Manganese; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; C561935:RtcB protein, E coli; D011118:Polynucleotide Ligases; D006152:Cyclic GMP