Characterization of the ATPase FlaI of the motor complex of the Pyrococcus furiosus archaellum and its interactions between the ATP-binding protein FlaH

The archaellum, the rotating motility structure of archaea, is best studied in the crenarchaeon Sulfolobus acidocaldarius. To better understand how assembly and rotation of this structure is driven, two ATP-binding proteins, FlaI and FlaH of the motor complex of the archaellum of the euryarchaeon Pyrococcus furiosus, were overexpressed, purified and studied. Contrary to the FlaI ATPase of S. acidocaldarius, which only forms a hexamer after binding of nucleotides, FlaI of P. furiosus formed a hexamer in a nucleotide independent manner. In this hexamer only 2 of the ATP binding sites were available for binding of the fluorescent ATP-analog MANT-ATP, suggesting a twofold symmetry in the hexamer. P. furiosus FlaI showed a 250-fold higher ATPase activity than S. acidocaldarius FlaI. Interaction studies between the isolated N- and C-terminal domains of FlaI showed interactions between the N- and C-terminal domains and strong interactions between the N-terminal domains not previously observed for ATPases involved in archaellum assembly. These interactions played a role in oligomerization and activity, suggesting a conformational state of the hexamer not observed before. Further interaction studies show that the C-terminal domain of PfFlaI interacts with the nucleotide binding protein FlaH. This interaction stimulates the ATPase activity of FlaI optimally at a 1:1 stoichiometry, suggesting that hexameric PfFlaI interacts with hexameric PfFlaH. These data help to further understand the complex interactions that are required to energize the archaellar motor.