Crystal structure of NAD(+)-dependent DNA ligase: modular architecture and functional implications

Abstract

DNA ligases catalyze the crucial step of joining the breaks in duplex DNA during DNA replication, repair and recombination, utilizing either ATP or NAD(+) as a cofactor. Despite the difference in cofactor specificity and limited overall sequence similarity, the two classes of DNA ligase share basically the same catalytic mechanism. In this study, the crystal structure of an NAD(+)-dependent DNA ligase from Thermus filiformis, a 667 residue multidomain protein, has been determined by the multiwavelength anomalous diffraction (MAD) method. It reveals highly modular architecture and a unique circular arrangement of its four distinct domains. It also provides clues for protein flexibility and DNA-binding sites. A model for the multidomain ligase action involving large conformational changes is proposed.

Fig. 1. The structure of Tfi DNA ligase. (A) Domains and conserved sequence motifs of Tfi DNA ligase. Domains are in different colors: subdomain 1a, blue; subdomain 1b, cyan; domain 2, green; subdomain 3a, yellow; subdomain 3b, orange; domain 4, light pink. Residues in red are more strongly conserved than others. (B) Stereo ribbon diagram of Tfi DNA ligase. Drawn with MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Bacon, 1997). Domain colors are the same as in (A). The covalently bound AMP moiety (stick model in purple) and a zinc ion (a cyan ball) are shown. Secondary structures were defined by PROCHECK (Laskowski et al., 1993). (C) Electron density map calculated using MAD solvent-flattened phases around the covalently bound AMP group. Atoms are yellow for carbon, red for oxygen, blue for nitrogen and cyan for phosphorus. (D) Residues around the AMP moiety. Atom colors are the same as above except black for carbon. Hydrogen bonds between the AMP and protein residues are represented by green dotted lines. Drawn with LIGPLOT (Wallace et al., 1995).

Fig. 2. Stereo C_α superposition of Tfi DNA ligase. (A) One of the two crystallographically independent ligase molecules in the native structure takes a more closed conformation (gray) than the other (black), and its BRCT domain is visible in the electron density map. Superposition is made for domain 1. (B) Subdomain 1a of Bst ligase (gray) takes a very different orientation from that of Tfi ligase (black). Superposition is made for subdomain 1b.

Fig. 3. Comparison of OB-fold domains, zinc fingers and HhH motifs. (A) OB-fold domains of Tfi ligase, human replication protein A subunit (RPA14), yeast aspartyl tRNA synthetase and E.coli translation initiation factor 1 (IF 1) are shown in similar orientations. (B) Cys₄-type zinc fingers of Tfi ligase, human estrogen receptor DNA-binding domain (ER DBD), rat glucocorticoid receptor DBD (GR DBD) and chicken erythroid transcription factor (GATA–1) are shown in similar orientations. Cysteines that coordinate the zinc ion are labeled. (C) HhH motifs of Tfi ligase (top row), human DNA polymerase β, Mycobacterium leprae RuvA, E.coli endonuclease III and E.coli AlkA (bottom row) are shown in similar orientations. Conserved glycine residues are indicated.

Fig. 4. Possible interactions between a duplex DNA and Tfi ligase in the observed adenylated structure. (A) Stereo view of the electrostatic potential surface (Nicholls et al., 1991) of Tfi ligase with a duplex DNA interacting with the two putative binding sites. The surface is color-coded according to the potential: red, –15 kT; white, 0 kT; blue, +10 kT. The covalently bound AMP is indicated in a ball-and-stick model. The BRCT domain is in gray because its model lacks side chains. The arrow indicates the highly negatively charged site near Lys116 that is formed by Asp118, Glu281 and Asp283. This view shows the ‘catalytic’ DNA-binding site between domains 1 and 2. (B) This was obtained by rotating the view in (A) by 90° around the vertical axis. In order to show the ‘non-catalytic’ DNA-binding site, the BRCT domain has been omitted from this figure.

Fig. 5. Schematic model proposed for the Tfi ligase active site. Residues that are likely to participate in binding metal ions and the 5′–phosphate end of the nick are indicated.

Fig. 6. Model for Tfi ligase action. Domains are color-coded: domain 1, blue; domain 2, green; domain 3, orange; domain 4, gray. DNA is in red and the bound AMP is in cyan. Apo enzyme (A) is self-adenylated (B) and a duplex DNA is bound to the ‘non-catalytic’ DNA-binding site (C). Tfi ligase slides along DNA and recognizes a nick (D). The duplex DNA is kinked at the nick (D) and the kinked DNA is bound to both the ‘catalytic’ and ‘non-catalytic’ DNA-binding sites, triggering a major domain rearrangement (E). The AMP is de-adenylated from Lys116 and is transferred to the 5′–phosphate of the nicked site, and magnesium ions are bound (E). Nick closure occurs and the ligated duplex DNA is now detached from the ‘catalytic’ DNA-binding site, and the second domain movement restores the ligase conformation (F). The duplex DNA is released to continue another reaction cycle.