PDBsum entry 2q0c

Transferase

PDB id: 2q0c

Contents

Protein chains

327 a.a. *

311 a.a. *

Ligands

CTP

Metals

_MG

Waters ×230

* Residue conservation analysis

PDB id:

2q0c

Name:

Transferase

Title:

Terminal uridylyl transferase 4 from trypanosoma brucei with bound ctp

Structure:

RNA uridylyl transferase. Chain: a, b. Synonym: RNA uridylyltransferase 4. Engineered: yes

Source:

Trypanosoma brucei. Organism_taxid: 5691. Gene: tut4. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.20Å R-factor: 0.234 R-free: 0.281

Authors:

J.Stagno,H.Luecke

Key ref:

J.Stagno et al. (2007). Dual role of the RNA substrate in selectivity and catalysis by terminal uridylyl transferases. Proc Natl Acad Sci U S A, 104, 14634-14639. PubMed id: 17785418 DOI: 10.1073/pnas.0704259104

Date:

21-May-07 Release date: 28-Aug-07

Protein chain

Q381M1  (Q381M1_TRYB2) -  Terminal uridylyltransferase 4 from Trypanosoma brucei brucei (strain 927/4 GUTat10.1)

Seq: 333 a.a.

Struc: 327 a.a.

Protein chain

Q381M1  (Q381M1_TRYB2) -  Terminal uridylyltransferase 4 from Trypanosoma brucei brucei (strain 927/4 GUTat10.1)

Seq: 333 a.a.

Struc: 311 a.a.

Enzyme reactions

• Enzyme class: Chains A, B: E.C.2.7.7.52 - Rna uridylyltransferase.
• Reaction: RNA(n) + UTP = RNA(n)-3'-uridine ribonucleotide + diphosphate

RNA(n) + UTP (Bound ligand (Het Group name = CTP) matches with 93.33% similarity) = RNA(n)-3'-uridine ribonucleotide + diphosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1073/pnas.0704259104

Proc Natl Acad Sci U S A 104:14634-14639 (2007)

PubMed id: 17785418

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

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

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.

Selected figure(s)

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.

Figures were selected by an automated process.