Molecular mechanism of substrate recognition and specificity of tRNA^His guanylyltransferase during nucleotide addition in the 3′–5′ direction

Structural comparison between the active sites of Thg1 and TLP. (A) Multiple sequence alignment of Thg1 and TLP enzymes. Secondary structures of CaThg1 and M. acetivorans TLP (MaTLP) are indicated at the top (α, α-helix; β, β-strand; η, 3₁₀-turn). The additional N-terminal helix is highlighted with a pink box. The species aligned are as follows: C. albicans, CaThg1; S. cerevisiae, ScThg1; Human, HsThg1; D. discoideum, DdiThg1; M. acetivorans, MaTLP; M. thermautotrophicus, MtTLP; and M. kandleri, MkTLP. Each protein sequence was aligned by CLUSTALW (Thompson et al. 1994) and ESPript (Robert and Gouet 2014) was used to prepare the figure. (B) Superposition of CaThg1-tRNA complex without a tRNA molecule (PDB ID: 3WC2, cyan and magenta) and MaTLP-tRNA-GDPNP complex (PDB ID: 5AXN, green), where the tRNA molecule bound to MaTLP is shown as a blue ribbon model. GDPNP and the opposite C₇₂ with Watson–Crick hydrogen bonds (black dashed lines) in the tertiary complex structure of MaTLP are shown as yellow sticks, and Mg²⁺ ions are indicated as black spheres. The residues around the active site of CaThg1 and MaTLP are shown as stick models. The N-terminal helix and the eukaryotic Thg1-specifc HINNLYN sequence are indicated in magenta. Possible interactions between CaThg1 and incoming GTP are indicated as magenta dashed lines. Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC) was used to generate the figure.