ATP binding, but not its hydrolysis, is required for assembly and proteolytic activity of the HslVU protease in Escherichia coli

HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. To gain an insight into the role of ATP hydrolysis in protein breakdown, we determined the insulin B-chain-degrading activity and assembly of HslVU in the presence of ATP and its nonhydrolyzable analogs. While β,γ-methylene-ATP could not support the proteolytic activity, β,γ-imido-ATP supported it to an extent less than 10% of that seen with ATP. Surprisingly, however, HslVU degraded insulin B-chain even more rapidly in the presence of ATPγS than with ATP. Furthermore, the ability of ATP and its analogs in supporting the proteolytic activity was closely correlated with their ability in supporting the oligomerization of HslU and the formation of the HslVU complex. However, ADP, which is capable of supporting the HslU oligomerization, could not support the HslVU complex formation or the proteolytic activity, suggesting that the conformation of the ADP-bound HslU oligomer is different from that of ATP-bound form. Thus, it appears that ATP-binding, but not its hydrolysis, is essential for assembly and proteolytic activity of HslVU.