From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase

Figure 9

Crystal structures of ATP-dependent DNA ligases. (a) The viral and bacterial ATP-dependent DNA ligases from Chlorella virus (left, blue) and bacteriophage T7 (right, yellow) are two-domain ligases. These ligases revealed the common structures of two distinct domains, designated as the adenylylation domain (AdD) and the oligonucleotide/oligosaccharide-binding-fold domain (OBD), which are jointly called the catalytic core domains. In these two structures, the catalytic core domains adopted an open form. The three-domain structure of DNA ligase is characteristic of the archaeal (Sulfolobus solfataricus: SsoLig (orange, left) and Pyrococcus furiosus: PfuLig (blue, right)) and eukaryotic (human: hLigI (green, center)) DNA ligases. The AdD contains most of the catalytic residues and is assisted by two flanking domains that also bind to DNA, the N-terminal DNA-binding domain (DBD) and the C-terminal OBD. Each C-terminal helix is highlighted in yellow (SsoLig), magenta (hLigI), and red (PfuLig). The figure was prepared using Chimera [105]. (b) Schematic diagram of the structure-based ligation mechanism. Before and after the ligation reaction, DNA ligase adopts the extended conformation, as expected from the SsoLig crystal structure. In step 1, the closed conformation of the catalytic core domains at the carboxyl terminus in PfuLig creates a small compartment, which holds a covalently bound AMP molecule. In step 2, the interactions with the substrate DNA must be stabilized for the enzyme to adopt a compact conformation, as seen in the crystal structure of hLig1 bound to the nicked-DNA. Finally, the ligation of the DNA strands and the subsequent release of AMP and DNA strands from DNA ligase occur during step 3.

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