Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase

Abstract

ATP-dependent RNA ligases are agents of RNA repair that join 3′-OH and 5′-PO 4 RNA ends. Naegleria gruberi RNA ligase (NgrRnl) exemplifies a family of RNA nick-sealing enzymes found in bacteria, viruses, and eukarya. Crystal structures of NgrRnl at three discrete steps along the reaction pathway-covalent ligase-(lysyl- 2+ complexhighlight a two-metal mechanism of nucleotidyl transfer, whereby (i) an enzyme-bound "catalytic" metal coordination complex lowers the pK a of the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; and (ii) a second metal coordination complex bridges the β-and γ-phosphates. The NgrRnl N domain is a distinctively embellished oligonucleotide-binding (OB) fold that engages the γ-phosphate and associated metal complex and orients the pyrophosphate leaving group for in-line catalysis with stereochemical inversion at the AMP phosphate. The unique domain architecture of NgrRnl fortifies the theme that RNA ligases have evolved many times, and independently, by fusions of a shared nucleotidyltransferase domain to structurally diverse flanking modules. The mechanistic insights to lysine adenylylation gained from the NgrRnl structures are likely to apply broadly to the covalent nucleotidyltransferase superfamily of RNA ligases, DNA ligases, and RNA capping enzymes.NζRNA repair | covalent nucleotidyltransferase | lysyl-AMP B iochemically diverse RNA repair systems rely on RNA ligases to maintain or manipulate RNA structure in response to purposeful RNA breakage events (1-5). RNA breaks destined for repair are inflicted by sequence-specific or structure-specific endoribonucleases during physiological RNA processing (e.g., tRNA splicing; kinetoplast mRNA editing) and under conditions of cellular stress (e.g., virus infection, starvation, unfolded protein response). RNA cleavage can occur either: (i) by a transesterification mechanism (generally metal-independent) that yields 2′,3′-cyclic-PO 4 and 5′-OH ends; or (ii) via a hydrolytic mechanism (typically metaldependent) that leaves 3′-OH and 5′-PO 4 ends. RNA repair enzymes capable of sealing 2′,3′-cyclic-PO 4 /5′-OH breaks or 3′-OH/5′-PO 4 breaks are distributed widely in all phylogenetic domains of life.ATP-dependent RNA ligases join 3′-OH and 5′-PO 4 RNA termini via a series of three nucleotidyl transfer steps similar to those of DNA ligases (6). In step 1, RNA ligase reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate plus pyrophosphate. In step 2, AMP is transferred from ligase-adenylate to the 5′-PO 4 RNA end to form an RNA-adenylate intermediate, AppRNA. In step 3, ligase catalyzes attack by an RNA 3′-OH on the RNA-adenylate to seal the two ends via a phosphodiester bond and release AMP.The autoadenylylation reaction of RNA ligases is performed by a nucleotidyltransferase (NTase) domain that is conserved in ATP-dependent and NAD + -dependent DNA ligases and GTPdependent mRNA capping enzymes (6, 7). The NTase domain includes six peptide motifs (I, Ia, III, IIIa, IV, and V) that form the nucleotide-bin...

High-throughput sequencing reveals circular substrates for an archaeal RNA ligase

“…Pab1020 is the founding member of the conserved Rnl3 family of RNA ligases 28 that are predominantly found in hyperthermophiles (archaea, bacteria) and halophiles. Each Pab1020 monomer consists of 4 domains: the amino-terminal (N-term), the catalytic nucleotide transferase (NTase), the dimerization (Dim) and the C-terminal (C-term) domains [ 22 , Fig.…”

“…[24][25][26][27] Here using a combination of biochemical, RNA-seq and computational analyses, we have investigated the molecular function of the Pab1020 RNA ligase family. 24,28 Our genomewide RNA co-immunoprecipitation studies, using affinity purified Pab1020 antibodies, led to the discovery of a large number of circular RNAs that specifically interact with Pab1020 RNA ligase in P. abyssi cells and were indeed efficiently circularized by the Pab1020 in vitro. Our studies also indicate a widespread importance of circular RNAs in prokaryotes and suggest a functional speciation of an archaeal polynucleotide ligase toward RNA circularization activity in many thermophilic archaea and bacteria.…”

Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis

“…The N-terminal ligase domain (LIG) includes three landmark motifs that form the active site for adenylate transfer by all known clades of ATP-dependent polynucleotide ligases (Unciuleac et al 2015). Motif I (KENG) contains the lysine nucleophile that forms the covalent LIG-AMP intermediate; motif IV (EGFV[V/I]R) includes a glutamate that coordinates a catalytic divalent cation; motif V (FFKYK) contains two lysines that coordinate the ATP phosphates and the 5 ′ -PO 4 of the polynucleotide substrate.…”

Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD + -dependent polynucleotide ligases

“…A metal-driven mechanism was revealed by the recent crystal structure of Naegleria gruberi RNA ligase (NgrRnl) as a step 1 Michaelis complex with ATP and manganese (its preferred metal cofactor) (7). The key to capturing the Michaelis-like complex was the replacement of the lysine nucleophile by an isosteric methionine.…”

“…The sixth ligand site in the catalytic metal complex was occupied by an ATP α phosphate oxygen, indicative of a role for the metal in stabilizing the transition state of the autoadenylylation reaction. A key insight, fortified by superposition of the Michaelis complex on the structure of the covalent NgrRnl-(Lys-Nζ)-AMP intermediate, concerned the role of the catalytic metal complex in stabilizing the unprotonated state of the lysine nucleophile before catalysis, via local positive charge and atomic contact of Lys-Nζ to one of the metal-bound waters (7).…”