Structures of the Noncanonical RNA Ligase RtcB Reveal the Mechanism of Histidine Guanylylation
详细信息 查看全文
- 作者：Kevin K. Desai ; Craig A. Bingman ; George N. Phillips ; Jr. ; Ronald T. Raines
- 刊名：Biochemistry
- 出版年：2013
- 出版时间：April 16, 2013
- 年：2013
- 卷：52
- 期：15
- 页码：2518-2525
- 全文大小：467K
- 年卷期：v.52,no.15(April 16, 2013)
- ISSN：1520-4995
文摘
RtcB is an atypical RNA ligase that joins either 2鈥?3鈥?cyclic phosphate or 3鈥?phosphate termini to 5鈥?hydroxyl termini. In contrast to typical RNA ligases, which rely on ATP and Mg(II), catalysis by RtcB is dependent on GTP and Mn(II) with ligation proceeding through a covalent RtcB鈥揾istidine鈥揋MP intermediate. Here, we present three structures of Pyrococcus horikoshii RtcB complexes that capture snapshots along the entire guanylylation pathway. These structures show that prior to binding GTP, a single manganese ion (Mn1) is bound to RtcB. To capture the step immediately preceding RtcB guanylylation, we determined a structure of RtcB in complex with Mn(II) and the unreactive GTP analogue guanosine 5鈥?(伪-thio)triphosphate (GTP伪S). This structure shows that Mn1 is poised to stabilize the pentavalent transition state of guanylylation while a second manganese ion (Mn2) is coordinated to a nonbridging oxygen of the 纬-phosphoryl group. The pyrophosphate leaving group of GTP伪S is oriented apically to His404 with the 蔚-nitrogen poised for in-line attack on the 伪-phosphorus atom. The structure of RtcB in complex with GTP伪S also reveals the network of hydrogen bonds that recognize GTP and illuminates the significant conformational changes that accompany the binding of this cofactor. Finally, a structure of the enzymic histidine鈥揋MP intermediate depicts the end of the guanylylation pathway. The ensuing molecular description of the RtcB guanylylation pathway shows that RtcB and classical ATP- and Mg(II)-dependent nucleic acid ligases have converged upon a similar two-metal mechanism for formation of the nucleotidylated enzyme intermediate.