Atomic 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 the GTP phosphate donor

Abstract

Fungal tRNA ligase (Trl1) rectifies RNA breaks with 2',3'-cyclic-PO4 and 5'-OH termini. Trl1 consists of three catalytic modules: an N-terminal ligase (LIG) domain; a central polynucleotide kinase (KIN) domain; and a C-terminal cyclic phosphodiesterase (CPD) domain. Trl1 enzymes found in all human fungal pathogens are untapped 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 separate KIN and CPD domains are connected by an unstructured linker. CPD belongs to the 2H phosphotransferase superfamily by dint of its conserved central concave β sheet 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.

Figure 1.

Structure of Candida Trl1 KIN-CPD. The tertiary structure is shown as a cartoon model with the KIN domain in magenta, CPD domain in cyan, and the interdomain linker in beige. GDP and magnesium in the KIN active site are depicted as a stick model and a green sphere, respectively. Phosphate anion in the CPD active site is shown as a stick model.

Figure 2.

Structure of the CPD domain. (A) Stereo view of the CPD tertiary structure, depicted as a cartoon model with magenta β strands, cyan α helices (numbered sequentially), and blue 3₁₀ helices. The phosphate anion in the active site and the histidine and threonine side chains that coordinate the phosphate are rendered as stick models. (B) Secondary structure elements (colored as in panel A) are displayed above the CPD primary structure. The histidine and threonine residues in the signature HxT motifs are denoted by red dots.

Figure 3.

CPD active site. (A) Stereo view of the CPD active site highlighting CPD interactions with the phosphate anion. Amino acids are shown as stick models with beige carbons. Waters are denoted by red spheres. Atomic contacts are indicated by dashed lines. (B) Conservation of three active site motifs among Trl1 CPD domains from six species of human pathogenic fungi: Candida albicans, Candida auris, Aspergillus fumigatus, Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis. Positions of amino acid side chain identity or similarity in all six proteins are indicated by dots above the C. albicans sequence. The phosphate-binding histidines and threonines are highlighted in gold shading. Amino acids making water-bridged contacts to the phosphate are in green shading. Two aromatic residues imputed to interact with the terminal nucleoside are shaded gray. The corresponding motifs in S. cerevisiae Trl1 CPD are shown at bottom.

Figure 4.

Structures of the KIN•GTP•Mg²⁺ complex and KIN apoenzyme. (A) Stereo view of the superimposed tertiary structures of the KIN•GTP•Mg²⁺ complex (colored gold) and the KIN apoenzyme (colored cyan). The seven α helices flanking the central β sheet are numbered sequentially. GDP is depicted as a stick model with yellow phosphorus atoms. Magnesium is depicted as a magenta sphere. The phosphate anion in the KIN apoenzyme is shown as a stick model with a green phosphorus atom. (B) Stereo view of the KIN active site highlighting interactions with GTP•Mg²⁺. Amino acids are shown as stick models with beige carbons. Magnesium and waters are denoted by magenta and red spheres, respectively. Atomic contacts are indicated by dashed lines. (C) Stereo view of the equivalent active site region of the KIN apoenzyme, highlighting a conformational switch in the G-loop segment.

Figure 5.

Structures of KIN in complexes with IDP and dGDP. The atomic interactions with IDP (panel A; KIN domain colored green) and dGDP (panel B; KIN domain colored cyan) and phosphate anions near the nucleotide β phosphates are indicated by dashed lines. The lid-loop is visible in both structures.

Figure 6.

Effects of KIN and CPD mutations on Candida Trl1 activity in vivo. S. cerevisiae trl1Δ p[CEN LEU2 CalTRL1] strains with CalTRL1 alleles as specified on the left were grown in YPD liquid culture at 30°C and adjusted to the same A₆₀₀. Serial 10-fold dilutions were spotted to YPD agar and incubated at the 30°C and 37°C. Photographs of the plates are shown. Lethal CalTRL1-Ala alleles that failed to complement trl1Δ are indicated at the bottom.