Dissecting the role of a conserved motif (the second region of homology) in the AAA family of ATPases - Site-directed mutagenesis of the ATP-dependent protease FtsH

Escherichia coli FtsH is an ATP-dependent protease that belongs to the AAA protein family. The second region of homology (SRH) is a highly conserved m otif among AAA family members and distinguishes these proteins in part from the wider family of Walker-type ATPases. Despite its conservation across t he AAA family of proteins, very little is known concerning the function of the SRH. To address this question, we introduced point mutations systematic ally into the SRH of FtsH and studied the activities of the mutant proteins . Highly conserved amino acid residues within the SRH were found to be crit ical for the function of FtsH, with mutations at these positions leading to decreased or abolished ATPase activity. The effects of the mutations on th e protease activity of FtsH correlated strikingly with their effects on the ATPase activity. The ATPase-deficient SRH mutants underwent an ATP-induced conformational change similar to wild type FtsH, suggesting an important r ole for the SRH in ATP hydrolysis but not ATP binding. Analysis of the data in the light of the crystal structure of the hexamerization domain of N-et hylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes an intermolecular catalytic r ole for the SRH.