The AAA family proteins usually form a hexameric ring structure. The ATP-binding pocket, which is located at the interface of sub-units in the hexamer, consists of three functionally important motifs, the Walker A and B motifs, and the second region of homology (SRH). It is well known that Walker A and B motifs mediate ATP binding and hydrolysis, respectively. Highly conserved arginine residues in the SRH have been proposed to function as arginine fingers, which interact with the gamma-phosphate of bound ATP. To elucidate the mechanism of ATP hydrolysis, we prepared several mutants of the Caenorhabditis elegans fidgetin homologue FIGL-1 carrying a mutation in each of the above-mentioned three motifs. None of the constructed mutants showed ATPase activity. All the mutants except for K362A were able to bind ATP. A decrease in the ATPase activity by mixing wild-type and each mutant subunits was caused by the formation of hetero-hexamers. Mixtures of E416A and R471A, or N461A and R471A led to the formation of hetero-hexamers with partially restored ATPase activities, providing direct, firm evidence for the intersubunit catalysis model. In addition, based on the results obtained with mixtures of K362A with wild-type or R471A subunits, we propose that a conformational change upon ATP binding is required for proper orientation of the arginine fingers, which is essential for efficient hydrolysis of ATP bound to the neighboring subunit. (c) 2006 Elsevier Inc. All rights reserved.