title: Mutational analysis of the functional motifs in the ATPase domain of Caenorhabditis elegans fidgetin homologue FIGL-1: firm evidence for an intersubunit catalysis mechanism of ATP hydrolysis by AAA ATPases.
abstract: The AAA family proteins usually form a hexameric ring structure. The ATP-binding pocket, which is located at the interface of subunits 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.
authors.affiliation: Division of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan.
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Yakushiji
authors.forename: Shingo
authors.initials: S
authors.lastname: Nishikori
authors.forename: Kunitoshi
authors.initials: K
authors.lastname: Yamanaka
authors.forename: Teru
authors.initials: T
authors.lastname: Ogura
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D006868:Hydrolysis; D008869:Microtubule-Associated Proteins; D008954:Models, Biological; D008958:Models, Molecular; D009687:Nuclear Proteins; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology
chemical_list: D029742:Caenorhabditis elegans Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D001120:Arginine; D000251:Adenosine Triphosphatases; C496225:figl-1 protein, C elegans; D000074183:ATPases Associated with Diverse Cellular Activities; C498542:Fign protein, mouse