Structure of MPXV DNA ligase complex reveals a virus-specific pocket for selective inhibition

Mpox, caused by monkeypox virus (MPXV), remains a public health concern, yet the structural basis of poxvirus DNA ligase function has been poorly defined despite strong genetic and biochemical evidence supporting its essential role in viral DNA replication and repair. Here, we report the crystal structure of MPXV DNA ligase (MpxvLig) in complex with nicked DNA at 2.04 Å resolution. MpxvLig adopts the canonical three-domain architecture of ATP-dependent DNA ligases, comprising an N-terminal DNA-binding domain (DBD), a nucleotidyltransferase domain, and a C-terminal oligonucleotide-binding fold domain (OBD), which together form a clamp-like configuration that embraces nicked DNA for catalysis. The structure captures MpxvLig in an active catalytic state corresponding to the adenylylation step, in which Lys233 forms a covalent ligase-AMP intermediate. Comparative structural and biochemical analyses with the catalytic core of human DNA ligase III reveal substantial differences in domain arrangement and DNA engagement, consistent with the stronger DNA-binding capacity and higher ligation activity of MpxvLig in vitro. Small-angle X-ray scattering (SAXS) further shows that apo-MpxvLig adopts an extended flashlight-like conformation in solution. Notably, we identify a virus-specific small-molecule-binding pocket within the DNA-binding domain. Mitoxantrone binds directly to this pocket and inhibits MpxvLig activity at micromolar concentrations, while exerting minimal effects on the human ligase. Mutations in pocket-defining residues markedly attenuate this inhibition, supporting an on-target mechanism. Together, these findings establish the structural basis of poxvirus DNA ligation and provide a framework for the rational development of selective antiviral inhibitors targeting MpxvLig.