Probing the mechanism of ATP hydrolysis and substrate translocation in theAAA protease FtsH by modelling and mutagenesis

We have built a homology model of the AAA domain of the ATP-dependent prote ase FtsH of Escherichia coil based on the crystal structure of the hexameri zation domain of N-ethylmaleimide-sensitive fusion protein. The resulting m odel of the hexameric ring of the ATP-bound form of the AAA ATPase suggests a plausible mechanism of ATP binding and hydrolysis, in which invariant re sidues of Walker motifs A and B and the second region of homology, characte ristic of the AAA ATPases, play key roles. The importance of these invarian t residues was confirmed by site-directed mutagenesis. Further modelling su ggested a mechanism by which ATP hydrolysis alters the conformation of the loop forming the central hole of the hexameric ring. It is proposed that un folded polypeptides are translocated through the central hole into the prot ease chamber upon cycles of ATP hydrolysis. Degradation of polypeptides by FtsH is tightly coupled to ATP hydrolysis, whereas ATP binding alone is suf ficient to support the degradation of short peptides. Furthermore, comparat ive structural analysis of FtsH and a related ATPase, HsIU, reveals interes ting similarities and differences in mechanism.