Recently, Deng Zengqin’s team from the Chinese Academy of Sciences Wuhan Virus Research Institute/Antiviral Research Center completed the latest research achievement entitled “Structure-function analysis of the ATPase domain of African swine fever virus topoisomerase”, which was published online on mBio.
DNA topoisomerases are widely present in archaea, prokaryotes, eukaryotes, and some nuclear-cytoplasmic large DNA viruses, playing important roles in gene replication, transcription, recombination, and chromosome separation processes. DNA topoisomerase is a target for various anti-tumor and antibacterial drugs. Type II topoisomerase utilizes the energy generated by the hydrolysis of ATP to catalyze the breaking and binding of DNA double strands, thereby regulating the topological state of DNA. Although the amino acid sequence homology is low, almost all type II topoisomerases have two conserved functional domains – the ATPase domain located at the N-terminus of the protein and the DNA binding and cleavage domain immediately following. Research has shown that type II topoisomerases can control multiple catalytic steps, including DNA binding, ATP hydrolysis, and DNA strand transfer, through conformational regulation between structural domains. However, the molecular coupling mechanism between structural domains is still unclear.
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African swine fever virus (ASFV) is a highly pathogenic pathogen that can cause African swine fever (ASF) with a high mortality rate. There is currently no effective vaccine or specific therapeutic drug for African swine fever. ASFV specifically encodes type II topoisomerase, which plays a crucial role in viral genome replication and transcription, and is a good drug target for anti-ASF. In the early stage, Deng Zengqin’s team analyzed the cryo-electron microscopy structure of ASFV topoisomerase under two different functional states for the first time, revealing its unique structural characteristics and enhancing our understanding of the structure and function of virus topoisomerase. However, perhaps due to the greater flexibility of the connecting regions between ASFV topoisomerase domains, their ATPase domains have not been resolved in cryo-electron microscopy structures.
Recently, the team utilized X-ray crystallography techniques to analyze the high-resolution crystal structure of the ASFV topoisomerase ATPase domain and AMPPNP complex, revealing three characteristic structural regions – the intramolecular interface composed of ATP lid, QTK loop, and a9 helix, the K-loop loop that may participate in DNA binding, and the antennae like a helix pair located at the top of the ATPase domain. The mutations of key amino acids in the above three regions reduced the ATPase activity stimulated by the base and DNA, and inhibited the activity of the relaxed negative supercoiled DNA of topoisomerase, indicating that these characteristic structures are important for maintaining topoisomerase activity. The results showed that mutations in the three regions had parallel effects on ATPase activity and whole enzyme relaxation activity. This indicates that the ATP hydrolysis, DNA binding, and chain transfer processes of type II topoisomerase are highly coupled and jointly completed by synergistic interactions between structural domains. The above achievements have deepened scientists’ understanding of the regulatory mechanism of type II topoisomerase activity and laid a structural foundation for the research of ASFV antiviral drugs targeting the ATPase domain.
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Reference
Kuang W, Zhao Y, Li J, Deng Z. 0. Structure-function analysis of the ATPase domain of African swine fever virus topoisomerase. mBio 0:e03086-23. https://doi.org/10.1128/mbio.03086-23