Structure and two-metal mechanism of fungal tRNA ligase

Fungal tRNA ligase (Trl1) is an essential enzyme that repairs RNA breaks with 2′,3′-cyclic-PO(4) and 5′-OH ends inflicted during tRNA splicing and non-canonical mRNA splicing in the fungal unfolded protein response. Trl1 is composed of C-terminal cyclic phosphodiesterase (CPD) and central GTP-dependent polynucleotide kinase (KIN) domains that heal the broken ends to generate the 3′-OH,2′-PO(4) and 5′-PO(4) termini required for sealing by an N-terminal ATP-dependent ligase domain (LIG). Here we report crystal structures of the Trl1-LIG domain from Chaetomium thermophilum at two discrete steps along the reaction pathway: the covalent LIG-(lysyl-Nζ)–AMP•Mn(2+) intermediate and a LIG•ATP•(Mn(2+))(2) Michaelis complex. The structures highlight a two-metal mechanism whereby a penta-hydrated metal complex stabilizes the transition state of the ATP α phosphate and a second metal bridges the β and γ phosphates to help orient the pyrophosphate leaving group. A LIG-bound sulfate anion is a plausible mimetic of the essential RNA terminal 2′-PO(4). Trl1-LIG has a distinctive C-terminal domain that instates fungal Trl1 as the founder of an Rnl6 clade of ATP-dependent RNA ligase. We discuss how the Trl1-LIG structure rationalizes the large body of in vivo structure–function data for Saccharomyces cerevisiae Trl1.