Structure of the error-prone DNA ligase of African swine fever virus identifies critical active site residues

Abstract

African swine fever virus (ASFV) is contagious and can cause highly lethal disease in pigs. ASFV DNA ligase (AsfvLIG) is one of the most error-prone ligases identified to date; it catalyzes DNA joining reaction during DNA repair process of ASFV and plays important roles in mutagenesis of the viral genome. Here, we report four AsfvLIG:DNA complex structures and demonstrate that AsfvLIG has a unique N-terminal domain (NTD) that plays critical roles in substrate binding and catalytic complex assembly. In combination with mutagenesis, in vitro binding and catalytic assays, our study reveals that four unique active site residues (Asn153 and Leu211 of the AD domain; Leu402 and Gln403 of the OB domain) are crucial for the catalytic efficiency of AsfvLIG. These unique structural features can serve as potential targets for small molecule design, which could impair genome repair in ASFV and help combat this virus in the future.

Fig. 1

Domain architecture and ATP recognition pattern of AsfvLIG. a Schematic view comparing the domain compositions of AsfvLIG and HsLIG1. b In vitro DNA ligation catalyzed by wild-type AsfvLIG. Data represent the mean of three independent experiments, the standard deviation (SD) values are indicated by error bars. c Overall fold of the non-catalytic AsfvLIG:DNA complex. AsfvLIG is shown as surface with the NTD, AD, and OB domains colored in magenta, yellow, and green, respectively. DNA and ATP are shown as cartoons and spheres in white and atomic colors (C, light-blue; N, blue; O, red; P, orange), respectively. d Cartoon-and-stick view showing ATP recognition by the AsfvLIG AD domain. ATP is shown as sticks and outlined with simulated annealing 2F_o-F_c omit map, which is contoured at the 1.5 sigma level

Fig. 2

The catalytic AsfvLIG:DNA complex structures. a The overall structure of AsfvLIG:CT1 complex demonstrating the arrangement of individual domains in the catalytic form complex. AsfvLIG and DNA are shown as surface and cartoon, respectively. b Comparison of the catalytic form complexes of AsfvLIG and HsLIG1 (PDB_ID: 1 × 9 N). DNAs are colored in white, whereas the ligase NTD (or DBD), AD and OB domains are colored in magenta, yellow and green, respectively. In the HsLIG1 structure, the AMP that pyrophosphate linked to the 5′-P downstream of the DNA is shown as red spheres

Fig. 3

The interaction between DNA and AsfvLIG NTD. a Cartoon-and-surface view showing the relative orientation between AsfvLIG NTD and DNA. b–f Detailed interactions between AsfvLIG NTD and DNA residues. AsfvLIG NTD residues are shown as sticks in atomic colors (C, green; N, blue; O, red); the DNAs are also shown as sticks, the C-atoms of the template strand, upstream and downstream of the broken strand are colored in white, yellow, and pink, respectively. g Nucleotide-residue contact map showing individual nucleotide-residues interactions for the preferred binding site. Small and large markers on each nucleotide represent the major and minor groove contacts, respectively. Filled-in pink markers highlight which nucleotides are contacted by at least one residue in the minor groove

Fig. 4

Impacts of AsfvLIG NTD on DNA binding and ligation. a Relative orientation between the NTD and OB domains in the catalytic form AsfvLIG-DNA complex. b–d Relative orientation and detailed interactions between the NTD and AD domains in the catalytic form structure. e Comparison of nick and duplex DNA-CG binding by WT AsfvLIG and AsfvLIG NTD. All data points from three independent experiments are shown with the median expressed as bars. The standard deviation (±SD) values are indicated by error bars. f In vitro DNA-CG ligation catalyzed by WT AsfvLIG and AsfvLIG with NTD deletion (for AsfvLIG ΔN). The substrate and product bands are labeled as S and P, respectively. Uncropped gels are shown in Supplementary Fig. 7

Fig. 5

Structural basis for C:T and C:G base pair recognition by AsfvLIG. a, b Local conformations of the C:T and C:G pairs captured in the AsfvLIG:CT1 and AsfvLIG:CG structures, respectively. c Superposition of the nick site base pairs in AsfvLIG:CT1 and AsfvLIG:CG. d Local conformations of the C:T pair captured in the AsfvLIG:CT2 structure. e Superposition of the nick site base pairs in AsfvLIG:CT1 and AsfvLIG:CT2. f Detailed conformations of the active site residues Leu402 and Gln403, based on the AsfvLIG:CT1 structure. g, h In vitro DNA ligation catalyzed by L402R and Q403F mutants, respectively. The data represent the mean of three independent experiments, the standard deviation (±SD) values are indicated by error bars. In panels (a–e), the C-atoms of protein and DNA residues are colored in green, yellow, and magenta for the AsfvLIG:CT1, AsfvLIG:CT2, and AsfvLIG:CG structures, respectively

Fig. 6

Unique ligase residues in proximity of the nick in the substrate DNA of AsfvLIG. a, b Cartoon representation showing the DNA-binding surface formed by the AD and OB domains of AsfvLIG and HsLIG1, respectively. c Superposition of the ligase residues in proximity of the nick in the substrate DNA. In (a–c), all the ligase residues are shown as sticks. Identical colors are utilized for the N-atoms (blue) and O-atoms (red) in both structures, but the C-atoms are colored in yellow and green in AsfvLIG and HsLIG1, respectively. d Comparison of the in vitro DNA-CT binding by WT AsfvLIG, and L402R and Q403F mutants. In the right panel, WT AsfvLIG, and L402R and Q403F mutants are colored in red, green, and blue, respectively. The complexes with protein:DNA molecular ratio of 2:1 are colored in light-green or light-blue. All data points from three independent experiments are shown with the median expressed as bars. The standard deviation (±SD) values are indicated by error bars. Uncropped gels are shown in Supplementary Fig. 13. e In vitro DNA ligation catalyzed by WT HsLIG1. f In vitro DNA ligation catalyzed by HsLIG1-d mutant. HsLIG1-d and HsLIG1-q stand for R871L/F872Q double mutant and D570N/F635L/R871L/F872Q quadruple mutant, respectively. The data represent the mean of three independent experiments, and the standard deviation (±SD) values are indicated by error bars

Fig. 7

Comparison of ATP and DNA binding by AsfvLIG and HsLIG1. a The conserved catalytic mechanism of ATP-dependent DNA ligation. b Superposition showing the similar ATP binding observed in AsfvLIG and HsLIG1 structures. AsfvLIG of the non-catalytic AsfvLIG:DNA complex is shown as cartoon in yellow, and ATP and ATP-interacting residues are shown as sticks in atomic colors (C, yellow; N, blue; O, red; P, orange). HsLIG1 in the complex with adenylated DNA (PDB_ID: 1 × 9 N) is shown as cyan cartoon. DNA, AMP, and AMP-interacting residues of HsLIG1 are shown as sticks in atomic colors (C, cyan; N, blue; O, red; P, orange). c In vitro DNA binding by AsfvLIG1. d In vitro DNA binding by HsLIG1. All data points from three independent experiments are shown with the median expressed as bars. The standard deviation (±SD) values are indicated by error bars