2Q0G

Terminal uridylyl transferase 4 from Trypanosoma brucei with bound UPU

Summary for 2Q0G

• Entry DOI: 10.2210/pdb2q0g/pdb
• Related: 2IKF 2NOM 2Q0C 2Q0d 2Q0e 2Q0f
• Descriptor: RNA uridylyl transferase, MAGNESIUM ION, CHLORIDE ION, ... (5 entities in total)
• Functional Keywords: tutase, trypanosoma, nucleotidyltransferase, utp-binding, rna-editing, transferase
• Biological source: Trypanosoma brucei
• Total number of polymer chains: 2
• Total formula weight: 80979.04

Authors

Stagno, J.,Luecke, H. (deposition date: 2007-05-21, release date: 2007-08-28, Last modification date: 2023-08-30)

Primary citation

Stagno, J.,Aphasizheva, I.,Aphasizhev, R.,Luecke, H.
Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases.
Proc.Natl.Acad.Sci.Usa, 104:14634-14639, 2007

PubMed Abstract: Terminal RNA uridylyltransferases (TUTases) catalyze template-independent UMP addition to the 3' hydroxyl of RNA. TUTases belong to the DNA polymerase beta superfamily of nucleotidyltransferases that share a conserved catalytic domain bearing three metal-binding carboxylate residues. We have previously determined crystal structures of the UTP-bound and apo forms of the minimal trypanosomal TUTase, TbTUT4, which is composed solely of the N-terminal catalytic and C-terminal base-recognition domains. Here we report crystal structures of TbTUT4 with bound CTP, GTP, and ATP, demonstrating nearly perfect superposition of the triphosphate moieties with that of the UTP substrate. Consequently, at physiological nucleoside 5'-triphosphate concentrations, the protein-uracil base interactions alone are not sufficient to confer UTP selectivity. To resolve this ambiguity, we determined the crystal structure of a prereaction ternary complex composed of UTP, TbTUT4, and UMP, which mimics an RNA substrate, and the postreaction complex of TbTUT4 with UpU dinucleotide. The UMP pyrimidine ring stacks against the uracil base of the bound UTP, which on its other face also stacks with an essential tyrosine. In contrast, the different orientation of the purine bases observed in cocrystals with ATP and GTP prevents this triple stacking, precluding productive binding of the RNA. The 3' hydroxyl of the bound UMP is poised for in-line nucleophilic attack while contributing to the formation of a binding site for a second catalytic metal ion. We propose a dual role for RNA substrates in TUTase-catalyzed reactions: contribution to selective incorporation of the cognate nucleoside and shaping of the catalytic metal binding site.

PubMed: 17785418
DOI: 10.1073/pnas.0704259104

Experimental method

X-RAY DIFFRACTION (2.3 Å)