9LD4

The Apo-form structure of TkoKptA

Summary for 9LD4

• Entry DOI: 10.2210/pdb9ld4/pdb
• Descriptor: Probable RNA 2'-phosphotransferase (1 entity in total)
• Functional Keywords: apo-protein, kpta, transferase
• Biological source: Thermococcus kodakarensis (Pyrococcus kodakaraensis (strain KOD1))
• Total number of polymer chains: 4
• Total formula weight: 83913.04

Authors

Cao, X.L.,Yang, J.,Zhang, W.Z.,Gan, J.H. (deposition date: 2025-01-05, release date: 2025-11-12, Last modification date: 2026-05-27)

Primary citation

Cao, C.,Yang, J.,Zhang, W.,Chen, J.,Gao, Y.,Yao, Y.,Zhang, Y.,Li, H.,Li, L.,Luo, Z.,Wang, C.,Tang, G.,Cui, R.,Liu, H.,Wang, Q.,Huang, Z.,Ma, J.,Gan, J.
Crystal structures and snapshots along Tpt1-catalyzed phosphate transfer from nucleic acid to NAD.
Nat Commun, 16:10888-10888, 2025

PubMed Abstract: Tpt1/TRPT1/KptA family proteins are evolutionarily conserved in all three domains of life. In fungi and plants, Tpt1 transfers 2'-PO from tRNA splice junction to NAD, which is the final step of tRNA maturation and is critical for the function of tRNA. In mammals and bacteria, Tpt1-catalyzed reaction leads to 5'-end ADP ribosylation, a reversible chemical modification of nucleic acids. Based on in vivo and in vitro biochemical studies, a two-step catalytic mechanism has been established for Tpt1-catalyzed RNA 2'-PO transfer, including (i) the 2'-PO attacks NAD, releasing nicotinamide and forming a 2'-phospho-ADP-ribosylated RNA (2'-p-ADPR-RNA) intermediate; and (ii) transesterification of the ADP-ribose 2"-OH to RNA 2'-PO, displacing the 2'-OH RNA and producing ADP-ribose-1",2"-cyclic phosphate (Appr>P). However, neither 2'-p-ADPR-RNA intermediate nor Appr>P product has been captured in any reported Tpt1 structures. Here, we report a series of crystal structures of T. kodakarensis Tpt1 (TkoTpt1), capturing the key 2'-p-ADPR-RNA intermediate. In addition, our structures also capture the 5'-p-ADPR-DNA intermediate and Appr>P product. Structural analysis and in vitro catalytic assays revealed that TkoTpt1 utilizes similar mechanism in 2'-PO and 5'-PO transfer. In conclusion, our structures reaffirm the catalytic mechanism of Tpt1-catalyzed phosphate transfer.

PubMed: 41345401
DOI: 10.1038/s41467-025-65881-y

Experimental method

X-RAY DIFFRACTION (3.61 Å)