Structural domains
T4 Rnl1 is a 374-amino acid polypeptide that consists of two structural domains.
- The N-terminal domain is responsible for the adenylyltransferase activity of the enzyme. It includes an a helix (a1), followed by antiparallel ß-sheets (ß1–ß4). It is the central ensemble of ß strands and loops that forms the nucleotide-binding pocket.
- The pocket is lined by six peptide motifs (I, Ia, III, IIIa, IV, and V). These motifs characterize the covalent nucleotidyltransferase super-family that includes DNA ligases, RNA ligases, and mRNA capping enzymes.
- The N-terminal polypeptide from from 1 to 254 amino acids suffices for the ligase–adenylylation reaction of T4 Rnl1.
- The C-terminal domain is an all-helical domain, specific to the Rnl1. This all-helical domain is not found in C-terminal domains of DNA ligases and RNA capping enzymes.
- The C domain confers the specificity for tRNA repair. The physiological substrate for Rnl1 is a tRNA containing a single break in the anticodon loop. Deleting the C domain abolishes the preference of the enzyme for this broken tRNA substrate, but does not abolish adenylyltransferase activity.
- A conserved Arg318–Lys319 dipeptide is a possible candidate that determines the tRNA specificity.
Ligands binding sites
- Divalent cation binding sites
Metal ions are essential for the nucleotidyltransferase catalysis by all T4 DNA and T4 RNA ligases, which use the same two-metal ions mechanism.
The true substrate in the adenylation reaction is the ATP-Mg2+ complex [Cherepanov, A. V., and de Vries, S. (2002) J. Biol. Chem. 277, 1695–1704], but nucleotidyltransferase enzymes cannot bind ATP-Mg2 directly. They bind ATP-Mg first, then a second Mg2+ ion. A Mg2+ ion interacts also with one phosphoryl oxygen in APC, three water molecules and two residues (Gly269 and Asp272), which both belong to the C-terminal domain.
The enzyme binds four Ca2+ ions. Two are coordinated to six water molecules. They do not directly interact with the enzyme, but via water molecules interacting with Glu227, Glu159, Lys99, Glu100, and Tyr246. They also interact with one phosphoryl oxygen of the APC. Two other are coordinated to six water molecules and interact with three enzyme residues(Asp212 in chain A and Ile211 and Asp212 in chain B).
