Delineating specific regions of N- terminal domain of T3SS ATPase YsaN of governing its different oligomerization states.

Oligomerization of YsaN, a putative T3SS-ATPase is a necessary and crucial event for T3SS functioning in . Different oligomeric states have been proposed for similar ATPases, yet, the true nature of its activation and formation of different oligomers is still poorly understood. studies of YsaN reveal that its activation and oligomerization depend on its N-terminal region and occur as a result of active catalysis of ATP in an ATP concentration-dependent manner following two-step cooperative kinetics. Also, the N-terminal 83 amino acid residues of YsaN are crucial for higher-order oligomer formation while YsaN∆83 is capable of hexamer formation upon oligomerization. Enzyme kinetics study shows reduced ATPase activity of YsaN∆83 (3.19 ± 0.09 μmol/min/mg) in comparison to YsaN (9.076 ± 0.72 μmol/min/mg). Negative-TEM study of YsaN and YsaN∆83 oligomer suggests that the formation of higher-order oligomer (probably dodecamer) occurs by stacking of two hexamers through their N-terminal faces involving N-terminal 83 amino acid residues which have been further supported by the docking of two hexamers during the study. These results suggest that YsaN is an oligomerization-activated T3SS ATPase, where distinct regions of its N-terminal domain regulate its different oligomeric nature and is essential for its activation.