The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome.

HslVU is a new two-component protease in Escherichia coli composed of the proteasome-related peptidase HslIV and the ATPase HsIU. We have used electron microscopy and image analysis to examine the structural organization of HslV and HslU homo-oligomers and the active HslVU enzyme. Electron micrographs of HslV reveal ring-shaped particles, and averaging of top views reveal six-fold rotational symmetry, in contrast to other beta-type proteasome subunits, which form rings with seven-fold symmetry. Side views of HslV show two rings stacked together, thus, HslV behaves as dodecamer. The ATPase HslU forms ring-shaped particles in the presence of ATP, AMP-PNP or ADP, suggesting that nucleotide binding, but not hydrolysis, is required for oligomerization. Subunit crosslinking, STEM mass estimation, and analysis of HslU top views indicate that HslU exists both as hexameric and heptameric rings. With AMP-PNP present, maximal proteolytic activity is observed with a molar ratio of HslU to HslV subunits of 1:1, and negative staining electron microscopy shows that HslV and HsIU form cylindrical four-ring structures in which the HsIV dodecamer is flanked at each end by a HslU ring.