Mutational studies on HslU and its docking mode with HslV

HslVU is an ATP-dependent prokaryotic: protease complex. Despite detailed c rystal and molecular structure determinations of free HslV and HslU, the me chanism of ATP-dependent peptide and protein hydrolysis remained unclear, m ainly because the productive complex of HslV and HslU could not be unambigu ously identified from the crystal data. In the crystalline complex, the I d omains of HslU interact with HslV. Observations based on electron microscop y data were interpreted in the light of the crystal structure to indicate a n alternative mode of association with the intermediate domains away from H slV. By generation and analysis of two dozen HslU mutants, we find that the amidolytic: and caseinolytic activities of HslVU are quite robust to mutat ions on both alternative docking surfaces on HslU. In contrast, HslVU activ ity against the maltose-binding protein-SulA fusion protein depends on the presence of the I domain and is also sensitive to mutations in the N-termin al and C-terminal domains of HslU, Mutational studies around the hexameric pore of HslU seem to show that it is involved in the recognition/translocat ion of maltose-binding protein-SulA but not of chromogenic small substrates and casein. ATP-binding site mutations, among other things, confirm the es sential role of the "sensor arginine" (R393) and the "arginine finger" (R32 5) in the ATPase action of HslU and demonstrate an important role for E321. Additionally, we report a better refined structure of the HslVU complex cr ystallized along with resorufin-labeled casein.