Through AlphaFold2 artificial intelligence program, the key active sites of the thermophilic bacteria Themus oshimai Pif1 (ToPif1) helicase involved in unwinding of the special nucleic acid secondary structure G quadruplex (G4) were predicted and verified at atomic level. The thermophilic properties of ToPif1 helicase were determined by variable temperature CD and unwinding experiment. The interaction amino acid sites of ToPif1 with G4 structures were predicted by AlphaFold2, and molecular biology tools were used to mutate the interaction sites into alanine respectively. The single-point mutant proteins of ToPif1 were obtained by prokaryotic expression system and the basic activities of the mutated proteins were verified to be not impaired by ATP hydrolysis assay. The binding activities and unwinding activities of ToPif1 wild-type and mutants on G4 DNA with different configurations were compared by fluorescence anisotropy test and fluorescence resonance energy transfer coupled fast-stop technique, and the key amino acid sites of ToPif1 binding and unwinding G4 were confirmed. The results showed that ToPif1 helicase still maintained protein conformation and unwinding activity at high temperature. Prediction results showed that there were eight potential sites on ToPif1 interacted with G4, and single-point mutations of these sites would not destroy the ATP hydrolysis activity of ToPif1. R355 was the key amino acid site binding ToPif1 to G4CEB and G4Tel, while R135 and R355 were the sites that significantly affected G4 unwinding for ToPif1. This study clarifies the key residues that ToPif1 binds and unwinds G4 DNA, and provides a reference for understanding the mechanism on G4 DNA recognition and unwinding of thermophilic Pif1 helicase.

helicase；active sites；AlphaFold2 structure prediction；G quadruplex；Pif1 family