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. 2018 Nov 2;46(19):10474-10488.
doi: 10.1093/nar/gky776.

T4 DNA Ligase Structure Reveals a Prototypical ATP-dependent Ligase With a Unique Mode of Sliding Clamp Interaction

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Free PMC article

T4 DNA Ligase Structure Reveals a Prototypical ATP-dependent Ligase With a Unique Mode of Sliding Clamp Interaction

Ke Shi et al. Nucleic Acids Res. .
Free PMC article

Abstract

DNA ligases play essential roles in DNA replication and repair. Bacteriophage T4 DNA ligase is the first ATP-dependent ligase enzyme to be discovered and is widely used in molecular biology, but its structure remained unknown. Our crystal structure of T4 DNA ligase bound to DNA shows a compact α-helical DNA-binding domain (DBD), nucleotidyl-transferase (NTase) domain, and OB-fold domain, which together fully encircle DNA. The DBD of T4 DNA ligase exhibits remarkable structural homology to the core DNA-binding helices of the larger DBDs from eukaryotic and archaeal DNA ligases, but it lacks additional structural components required for protein interactions. T4 DNA ligase instead has a flexible loop insertion within the NTase domain, which binds tightly to the T4 sliding clamp gp45 in a novel α-helical PIP-box conformation. Thus, T4 DNA ligase represents a prototype of the larger eukaryotic and archaeal DNA ligases, with a uniquely evolved mode of protein interaction that may be important for efficient DNA replication.

Figures

Figure 1.
Figure 1.
Overall structure of the T4 ligase–DNA complex. (A, B) Molecular surface (A) and cartoon representation (B) of the T4 ligase–DNA complex, with the three structural domains of ligase color-coded. A salt bridge between OB-fold domain residue Lys384 and DBD residues Asp112 completes the enveloping of DNA by the ligase. (C) Protein surface colored according to electrostatic potential (red, –5kT e−1, to blue, +5kT e−1). (D) Inter-strand base stacking (G12 and G32) across the nick stabilizing a large shift in the DNA helical axis.
Figure 2.
Figure 2.
Structural comparison between ATP-dependent DNA ligases. (A) Human Ligase 1 (hLig1, DBD in red) (19), T4 DNA ligase (DBD in cyan), and Chlorella virus ligase–DNA complex (25) structures shown in similar orientations, from left to right. (B, C) Side-by-side comparison (B) and superposition (C) of the DBDs from hLig1 and T4 DNA ligase. Color scheme follows that in (A).
Figure 3.
Figure 3.
DBD-DNA contacts. T4 DNA ligase DBD–DNA interactions, with details shown for two regions in magnified views. The yellow dotted lines highlight hydrogen bonding or salt bridge interactions.
Figure 4.
Figure 4.
T4 ligase–DNA interactions around the nick. (A) AMP interactions with the NTase domain residues. Red and green crosshairs represent water molecules and a putative magnesium ion, respectively. (B) Helix α11 from the NTase domain making DNA minor groove contacts. Viewed from the C-terminal end of α11. (C) A continuous minor groove interactions made by the N-terminal end of α11 from NTase domain and hydrophobic residues from OBD. (D) 2mFo-DFc electron density map contoured at 1.0σ shown for the region around the active site. (E) DNA backbone contacts by the NTase domain residues across and flanking the nick. The yellow dotted lines highlight hydrogen bonds or salt bridges.
Figure 5.
Figure 5.
OBD-DNA contacts. (A) Backbone contacts by OB-fold domain with the template (unnicked) DNA strand. A water molecule mediating protein-DNA interaction is shown by a small red sphere. (B) An overview of the engagement of the DNA minor groove by NTase-OBD ligase core. The disordered gp45-binding loop (residues 222–247) is shown by a pink dotted line, with the last ordered residues on either side of the loop highlighted by small pink spheres. (C) Contacts made by the OB-fold domain with the nicked DNA strand and base-pairs in the minor groove. Motif VI residues are shown in magenta.
Figure 6.
Figure 6.
T4 DNA ligase-gp45 interaction. (A) Size-exclusion chromatography profiles showing complex formation between the wild-type T4 DNA ligase and gp45 (upper panel), but not between the internally truncated ligase lacking residues 222–247 and gp45 (lower panel). (B) A representative ITC experiment wherein the T4 ligase loop peptide (525 μM) was titrated into gp45 (51 μM). The data were fit to an independent binding model implying a lack of cooperativity between the binding sites of the gp45 trimer. Five replicates gave an average Kd of 10.69 μM with a standard deviation of 3.16 μM.
Figure 7.
Figure 7.
Structure of the gp45-binding loop of T4 DNA ligase. (A) A gp45 trimer bound by three copies of the T4 ligase peptide. (B) Superposition of the three ligase-derived peptides showing their similar α-helical conformation and positioning of hydrophobic side chains. (C) Close-up views showing the α-helical conformation of the T4 ligase peptide with canonical hydrogen-bonds highlighted (left), and fitting within a hydrophobic pocket on the gp45 surface (right). (D) Human FEN1 PIP-box motif bound to PCNA (slate) (64), phage RB69 polymerase bound to gp45 (cyan) (63), and T4 ligase bound to gp45 (pink). hFEN-1 and RB69 have similar conformations with a 310 helix, whereas T4 ligase forms an α-helix with three consecutive residues fit in the pocket on gp45. The superpositions in the lower panels highlight the distinct α-helical conformation of the T4 ligase peptide.
Figure 8.
Figure 8.
A hypothetical model of the T4 ligase-gp45-DNA ternary complex. Molecular surface of a gp45 ring and ribbon model of T4 DNA ligase are shown. The color scheme for T4 DNA ligase follows that in Figure 1. The peptides occupying two remaining binding sites on the gp45 trimer are shown in pink. The modeling exercise suggests that the DBD/NTD might potentially interact with an inter-subunit cleft on the gp45 trimer.

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