Structure-guided mutational analysis of T4 RNA ligase 1

Abstract

T4 RNA ligase 1 (Rnl1) is a tRNA repair enzyme that circumvents an RNA-damaging host antiviral response. Whereas the three-step reaction scheme of Rnl1 is well established, the structural basis for catalysis has only recently been appreciated as mutational and crystallographic approaches have converged. Here we performed a structure-guided alanine scan of nine conserved residues, including side chains that either contact the ATP substrate via adenine (Leu179, Val230), the 2'-OH (Glu159), or the gamma phosphate (Tyr37) or coordinate divalent metal ions at the ATP alpha phosphate (Glu159, Tyr246) or beta phosphate (Asp272, Asp273). We thereby identified Glu159 and Tyr246 as essential for RNA sealing activity in vitro and for tRNA repair in vivo. Structure-activity relationships at Glu159 and Tyr246 were clarified by conservative substitutions. Eliminating the phosphate-binding Tyr37, and the magnesium-binding Asp272 and Asp273 side chains had little impact on sealing activity in vitro or in vivo, signifying that not all atomic interactions in the active site are critical for function. Analysis of mutational effects on individual steps of the ligation pathway underscored how different functional groups come into play during the ligase-adenylylation reaction versus the subsequent steps of RNA-adenylylation and phosphodiester formation. Moreover, the requirements for sealing exogenous preformed RNA-adenylate are more stringent than are those for sealing the RNA-adenylate intermediate formed in situ during ligation of a 5'-PO4 RNA.

FIGURE 1.

Bacteriophage RNA ligase 1. The amino acid sequence of T4 Rnl1 is aligned to the sequences of the homologous proteins of coliphage RB69, vibriophage KVP40, and Aeromonas hydrophila phage Aeh1. The secondary structure elements of T4 Rnl1 are displayed above the sequence, with β-strands indicated by arrows and helices as cylinders. Nucleotidyl transferase motifs I, Ia, III, IIIa, IV, and V are highlighted in shaded boxes. T4 Rnl1 residues identified previously as essential are indicated by •. Nonessential residues are indicated by +. The nine amino acids mutated in the present study are denoted by “|.”

FIGURE 2.

Tertiary structure and active site of T4 Rnl1. (A) A stereo view of the Rnl1 fold (from PDB 2C5U) is shown with AMPCPP in the active site. The adenylyltransferase domain is colored green; the Rnl1-specific N-terminal module is colored blue; the unique all-helical C-terminal domain is colored beige. (B) A stereo view of the Rnl1 active site. The calcium ion at the α phosphate of AMPCPP is colored magenta; the magnesium ion at the β phosphate is colored cyan; waters are depicted as red spheres. Hydrogen bonding and ionic interactions are depicted as dashed lines. The image was prepared with Pymol.

FIGURE 3.

Rnl1-Ala mutants. (A) Aliquots (5 μg) of recombinant wild-type (WT) and mutated Rnl1 proteins were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the left. (B) RNA ligation reaction mixtures (10 μL) containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 2 mM DTT, 20 μM ATP, 1 pmol of pRNA substrate, and 400 ng of wild-type or mutant Rnl1 were incubated at 37°C for 30 min. The products were resolved by PAGE and visualized by autoradiography. A control reaction lacking Rnl1 is shown in lane “–.” The step 2 (RNA adenylation) and step 3 (phosphodiester bond formation) reactions of the ligation pathway are illustrated schematically at the bottom. (C) Adenylyltransferase specific activity was determined by label transfer from [α³²P]ATP to Rnl1 as described in Materials and Methods.

FIGURE 4.

Kinetics of RNA sealing by wild-type and mutant Rnl1 proteins. Ligation reaction mixtures containing labeled pRNA and Rnl1 as specified were incubated at 37°C. Aliquots were withdrawn and quenched at the times specified over the lanes. The products were resolved by PAGE and visualized by autoradiography. The yield of circular RNA product is plotted as a function of time at bottom.

FIGURE 5.

Effects of alanine mutations on ligation of a preadenylated RNA. Reactions mixtures contained radiolabeled AppRNA and Rnl1 as specified. The products were resolved by PAGE and visualized by autoradiography. Rnl1 was omitted from a control reaction (lane “–”).

FIGURE 6.

Effects of conservative mutations of Glu159 and Tyr246. (A) Reaction mixtures (10 μL) containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 2 mM DTT, 20 μM ATP, 1 pmol of pRNA substrate, and 400 ng of wild-type or mutant Rnl1 were incubated at 37°C for 30 min. A control reaction lacking Rnl1 is shown in lane “–.” (B) Kinetic analysis of single turnover pRNA ligation was performed as described in Materials and Methods. The product analysis is shown in the left panel; the extent of circularization is plotted as a function of time in the right panel.