Structures of the RNA end-healing 5`-OH kinase and 2`,3`-cyclic phosphodiesterase domains of fungal tRNA ligase: conformational switches in the kinase upon binding of GTP

Fungal tRNA ligase (Trl1) repairs RNA breaks with 2`,3`-cyclic-PO₄and 5`-OH ends incurred during tRNA splicing. Trl1 is composed of C-terminal cyclic phosphodiesterase (CPD) and central polynucleotide kinase (KIN) domains that heal the broken ends to generate 3`-OH,2`-PO<sub>4</sub>and 5`-PO₄termini required for sealing by an N-terminal ligase domain. Trl1 enzymes present in all human fungal pathogens are promising targets for antifungal drug discovery. Here we report a 1.9 Å crystal structure of Trl1 KIN–CPD from the pathogenic fungus Candida albicans, which adopts an extended conformation in which KIN and CPD domains are far apart and connected by an unstructured linker. CPD belongs to the 2H phosphotransferase superfamily by dint of its conserved central concave bsheet and interactions of its dual HxT motif histidines and threonines with phosphate in the active site. Additional active site motifs conserved among the fungal CPD clade of 2H enzymes are identified. We present structures of the Candida Trl1 KIN domain at 1.5 to 2.0 Å resolution – as apoenzyme and in complexes with GTP•Mg²⁺, IDP•PO₄, and dGDP•PO₄– that highlight conformational switches in the G-loop (which recognizes the guanine base) and lid-loop (poised over the nucleotide phosphates) that accompany nucleotide binding.