THE ATP-DEPENDENT HSIVU PROTEASE FROM ESCHERICHIA-COLI IS A 4-RING STRUCTURE RESEMBLING THE PROTEASOME

HslVU is a new two-component protease in Escherichia coli composed of the proteasome-related peptidase HslV and the ATPase HslU. We have use d electron microscopy and image analysis to examine the structural org anization of HslV and HslU homo-oligomers and the active HslVU enzyme. Electron micrographs of HslV reveal ring-shaped particles, and averag ing of top Views reveal six-fold rotational symmetry, in contrast to o ther beta-type proteasome subunits, which form rings with seven-fold s ymmetry. Side views of HslV show two rings stacked together, thus, Hsl V behaves as dodecamer. The ATPase HslU forms ring-shaped particles in the presence of ATP, AMP-PNP or ADP, suggesting that nucleotide bindi ng, but not hydrolysis, is required for oligomerization. Subunit cross linking, 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 microsc opy shows that HslV and HslU form cylindrical four-ring structures in which the HslV dodecamer is flanked at each end by a HslU ring.