PDBsum entry 2q0e

Transferase

PDB id: 2q0e

Contents

Protein chains

331 a.a.

308 a.a.

Ligands

GTP

Metals

_MG ×2

Waters ×226

References listed in PDB file

Key reference

Title

Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases.

Authors

J.Stagno, I.Aphasizheva, R.Aphasizhev, H.Luecke.

Ref.

Proc Natl Acad Sci U S A, 2007, 104, 14634-14639. [DOI no: 10.1073/pnas.0704259104]

PubMed id

17785418

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.

Figure 3.
Fig. 3. The prereaction complex of UTP and UMP ("RNA") in the active site of TbTUT4. Mg^2+ ions (black) are labeled Mg1 and Mg2, where Mg1 is the binding site previously observed in the TbTUT4:UTP structure (8). (A) Triphosphate coordination by Mg1 and formation of a binding site for a second metal ion (Mg2) upon UMP binding. (B) Direct protein–UMP hydrogen bond contacts. (C) Hydrogen bond contacts with UMP ("RNA") at the terminal (gray) and modeled penultimate (green) UMP residues.

Figure 4.
Fig. 4. Triple-stacking interaction is required for productive RNA binding. Stereo views of the superposition of TbTUT4:ATP and TbTUT4:UTP:UMP, respectively, illustrate the various degrees of stacking of the aromatic rings of Y189, the NTP base, and UMP (RNA) for purine NTPs (adenine, shown in green) versus pyrimidine NTPs (uracil, shown in yellow). Upon superposition, there is virtually no base stacking observed between the pyrimidine ring of UMP and the purine ring of ATP.