Characterisation and engineering of a thermophilic RNA ligase from Palaeococcus pacificus

Abstract

RNA ligases are important enzymes in molecular biology and are highly useful for the manipulation and analysis of nucleic acids, including adapter ligation in next-generation sequencing of microRNAs. Thermophilic RNA ligases belonging to the RNA ligase 3 family are gaining attention for their use in molecular biology, for example a thermophilic RNA ligase from Methanobacterium thermoautotrophicum is commercially available for the adenylation of nucleic acids. Here we extensively characterise a newly identified RNA ligase from the thermophilic archaeon Palaeococcus pacificus (PpaRnl). PpaRnl exhibited significant substrate adenylation activity but low ligation activity across a range of oligonucleotide substrates. Mutation of Lys92 in motif I to alanine, resulted in an enzyme that lacked adenylation activity, but demonstrated improved ligation activity with pre-adenylated substrates (ATP-independent ligation). Subsequent structural characterisation revealed that in this mutant enzyme Lys238 was found in two alternate positions for coordination of the phosphate tail of ATP. In contrast mutation of Lys238 in motif V to glycine via structure-guided engineering enhanced ATP-dependent ligation activity via an arginine residue compensating for the absence of Lys238. Ligation activity for both mutations was higher than the wild-type, with activity observed across a range of oligonucleotide substrates with varying sequence and secondary structure.

Graphical Abstract

Figure 1.

Characterisation of PpaRnl ligation and adenylation activity. (A) PpaRnl activity with RNA Oligonucleotide 1 over a temperature gradient (30 - 85°C) in the presence of 15 μM ATP and 1 mM Mg²⁺. The graph represents ligation and adenylation activity. (B) RNA Oligonucleotide 1 substrate adenylation and ligation activity with varying ATP concentrations (0 to 1 mM) in the presence of 1 mM Mg²⁺; C, no ligase control. Adenylation and end-joining activities depicted in the line graph. (C) Substrate ligation and adenylation activity across a test panel of nine-oligonucleotides varying in sequence, secondary structure and T_m in the presence of 70 μM ATP. Adenylation, circularisation and end-joining ligation activity is quantified in the histogram. (D) PpaRnl activity with a single-stranded DNA oligonucleotide substrate (DNA Oligonucleotide 1) in the presence of 70 μM ATP. Reaction products and the input substrate are indicated accordingly; pRNA (input RNA), AppRNA (5′-PO₄ adenylated RNA), RNA circle (circularised RNA) and end to end ligation (end-joining). Control reactions included no PpaRnl enzyme in the reaction mix as indicated. Assays were performed in triplicate, and a representative of the gel image is shown. Graphs represent averages of the triplicate with error bars, representing standard error.

Figure 2.

Crystal structure of the PpaRnl wild-type enzyme with AMP covalently bound and complexed with Mg²⁺. Two views of the dimer rendered as (A) surface and (B) cartoon models, coloured as shown in panel A. (C) Stereoview of the PpaRnl wild-type AMP-Mg²⁺(H₂O)₅ complex and the second Mg²⁺ binding site with 2Fo – Fc electron density map for AMP (grey). Amino acids and AMP are shown as stick models; the AMP is coloured light pink with phosphorus atoms coloured orange; amino acids in the active site are coloured yellow. The Mg²⁺ ions and interacting waters are depicted as green and red spheres, respectively. Atomic contacts are indicated by dashed lines.

Figure 3.

Effects of mutations on catalytic activity and active site structure. (A) Ligation efficiencies of PpaRnl(K238G) across a panel of nine oligonucleotides varying in sequence, secondary structure and T_m (Supplementary Table S1) in the presence of ATP. The experiments were performed in triplicate, and a representative of the gel image is shown. The different reaction products and the input substrate are marked accordingly; pRNA (input RNA), AppRNA (adenylated RNA). Superimposition of the wild-type active site (yellow) residues with the (B) K92A ATP-bound Michaelis complex (light green) and (C) covalent AMP-bound K238G structure (light blue), with Fo-Fc electron density map for AMP and ATP nucleotides (grey). Amino acids, AMP and ATP nucleotides (pink) are rendered as stick models. The Mg²⁺ ions (green) and waters (red) are depicted spheres. Panel B highlights the coordination and binding of ATP in active sites. Panel C highlights the difference and coordination of Arg70, hypothesised to compensate for the mutated Lys238 in step 1 and step 2 adenylation reactions.

Figure 4.

Surface representation with electrostatic potential of the PpaRnl NTase domain and a portion of the dimerisation domain (residues 64–250) (A) without and (B) with residues involved in coordinating Mg²⁺-B. Mg²⁺ ions at the active site are depicted as green spheres. The nucleotide binding site (indicated with arrows) is marked with positive potential (blue), with the AMP moiety (pink) bound in a highly negatively charged pocket (red). The AMP phosphate tail is coordinated by a catalytic Mg²⁺ ion (Mg²⁺-A) in negative charged region. The putative Mg²⁺-B binding site is positioned in the same negatively charged binding pocket as the catalytic Mg²⁺-A, indicated by arrows.

Figure 5.

Ligation efficiency of PpaRnl mutants in ATP-independent reactions. (A & B) 3′-blocked SR1/SR1-S RNA, 3′-blocked SR1/SR1-S DNA and blocked/unblocked oligonucleotide 1 for (A) K92A and (B) K238G mutants. (C & D) 3′-blocked SR1 RNA ligation across an oligonucleotide panel varying in sequence, secondary structures, and T_m (Supplementary Table S1) for (C) K92A and (D) K238G mutants. All assay reactions include a no enzyme control (as indicated). The different reaction products and the input substrate are marked accordingly; pRNA/pDNA (input RNA or DNA), AppSR1-RNA or AppSR1-DNA (adenylated SR1 RNA or DNA). Assays performed in triplicate, and a representative of the gel image is shown. Graphs represent the average of the triplicate with errors representative of standard error.