Fig. 3. The ATPase activity of Hsp90 is abrogated by the slo^u45 mutation. (A) Hsp90a domain structure (amino acid numbers of domain boundaries are shown beneath; known functions of regions are indicated by black bars). The positions and consequences of mutations in three slo alleles, u45, tu44c and tm201, are also illustrated beneath. The equivalent residue locations of yeast Hsp90 as represented in B are shown above. (B) Pymol diagram showing critical binding interactions of ADP with yeast Hsp90. Dotted blue lines are hydrogen bonds; amino acid residues (corresponding residue numbers in zebrafish are in parentheses and are also indicated in A) involved are in green; water molecules are cyan balls; residues packed against the C- atom of Gly83 are shown in cyan. Illustrated residues are conserved between yeast and zebrafish, except Ser138/Thr149 and Ser140/His151 (grey), which are conservative substitutions unlikely to cause major structural changes. Binding of ATP/ADP to the Hsp90 N-terminal domain involves highly conserved interactions including the carboxylate side-chain of Asp79 and main-chain carboxyl of Leu34, via a tightly bound water molecule, to the exocyclic N6 of adenine. The same Asp79 also interacts via another tightly bound water to the N1 imino-nitrogen of the adenine. This same water is bound by an interaction with the side-chain hydroxyl of Thr171 and main-chain amide of Gly83. An aspartic acid residue substitution at Gly83 (mimicking the u45 mutation) would lead to steric clashes likely to disrupt critical hydrogen bonding interactions with ADP/ATP. (C) ATPase activity of yeast Hsp90, human HSP90 and their u45-mimic mutants. ATPase activity of the yeast G83D and human G91D mutants is negligible relative to corresponding wild type.