Structure and mechanism of tRNA splicing enzymes Trl1 and Tpt1: therapeutic targets for fungal infection.
Structural Biology
Final Report Abstract
In yeast the healing and sealing pathways during tRNA splicing utilizes trifunctional tRNA ligase, Trl1 and 2ʹ-phosphotransferase, Tpt1, that are essential for growth and are potential antifungal therapeutic targets for invasive fungal infections e.g., candidiasis and aspergillosis. Lack of atomic-level insights into the reaction mechanism was the main bottleneck for rational drug-design. To this end we have deciphered structure and mechanism of Trl1 and Tpt1 that paved a path for developing novel antifungal drug targeting the fungal tRNA splicing enzymes. Trl1 has three separable functional domains, 2ʹ,3ʹ-cyclic phosphodiesterase (CPD), GTP-dependent kinase, and ATP-dependent ligase. We obtained the crystal structure of the all the domains. The structure of the ligase domain of Trl1 from thermophilic filamentous fungi Chaetomium thermophilum, in a covalent lysyl–adenylate intermediate and ATP•Mg2+ Michaelis complex of the adenylation reaction that described a two-metal ion reaction mechanism of the ligase reaction. The crystal structure of the Kinase-CPDase domain fusion of the pathogenic fungi Candida albicans is the first atomic description of the fungal tRNA ligase 2H family CPD module that adapted an extended conformation when fused to the Kinase domain with an unstructured linker. A series of crystal structures of the Kinase module as apoenzyme, bound to GTP and other ligands described the conformational switch in the G-loops (that specifically recognizes the guanine base) and the lid-loop (that anticipate with the nucleotide phosphate binding). The 1.4 Å resolution crystal structure of the Clostridium thermocellum Tpt1 represent the step 2 product mimetic complex with ADP-ribose-1ʹʹ-phosphate (ADPR-1ʹʹ-P) in the C-terminal NAD+ binding site and pAp at the N-terminal RNA site, that was acquired and preoccupied in the protein during the recombinant protein production in E. coli. This structure described how catalytic tetrad (R-H-R-R) specifically involves in i) recognizing the 2ʹ-Phosphate of an RNA and ii) the mechanism of the RNA-phospho-ADP-ribosylation intermediate formation followed by step 1. This study opened up a new avenue as we for the first time discovered ADPR-1ʹʹ-P in bacteria (E. coli) and the question arises: what was the ʹnative substrateʹ in E. coli that C. thermocellum Tpt1 acted on and produced ADPR-1ʹʹ-P? The NMR solution structures of Runella slithyformis Tpt1 homolog as apoenzyme and NAD+ bound state that described the structural changes upon NAD+ binding. The structure thoroughly highlights the step 1 catalytic process that might proceed via an oxocarbenium transition-state. Alongside, we found out that a subset of Tpt1 homologs are adept to install a novel 5ʹ-Phsopho-ADP-ribose nucleic acids cap. The physiological relevance is unclear. In addition to Trl1 and Tpt1 atomic-level mechanistic insights we were able to trap a 3ʹ-P/5ʹ- OH DNA oligonucleotide bound complex of RtcB. RtcB is adept to successfully ligate 3ʹ-P and 5ʹ-OH DNA ends. We have solved the first structure of a binary complex of Pyrococcus horikoshii RtcB with a 6-mer DNA oligonucleotide HOA1pT2pG3pT4pC5pC6p. This revealed how RtcB recognize the 5ʹ-OH end and RtcB mainly interacts with the phosphodiester backbone which explain the versatility of RtcB mediated ligation.
Publications
- Atomic structures of the RNA endhealing 5′-OH kinase and 2′,3′-cyclic phosphodiesterase domains of fungal tRNA ligase: Conformational switches in the kinase upon binding of the GTP phosphate donor. Nucleic Acids Res. 47, 11826–11838 (2019)
Banerjee, A., Goldgur, Y., Schwer, B. & Shuman, S.
(See online at https://doi.org/10.1093/nar/gkz1049) - NMR solution structures of Runella slithyformis RNA 2′-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity. Nucleic Acids Res. (2021)
Alphonse, S., Banerjee, A., Dantuluri, S., Shuman, S. & Ghose, R.
(See online at https://doi.org/10.1093/nar/gkab241) - Structure of 3′-PO 4 /5′-OH RNA ligase RtcB in complex with a 5′-OH oligonucleotide. RNA 27, 584–590 (2021)
Banerjee, A., Goldgur, Y. & Shuman, S.
(See online at https://doi.org/10.1261/rna.078692.121)
