HslU is the ATPase component of the ATP-dependent HslVU protease in Escherichia coli. To gain an insight into the structure and function of HslU, site-directed mutagenesis was performed to generate a mutation in the ATP-binding site of the ATPase (i.e., to replace the Lys 63 with Thr). Unlike the wild-type HslU, the mutant form (referred to as HslU/K63T) could not hydrolyze ATP or support the ATP-dependent hydrolysis of N-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methyl coumarin by HslV. The wild-type HslU (a mixture of monomer and dimer) formed a multimer containing 6-8 subunits in the presence of either ATP or ADP, indicating that ATP-binding, but not its hydrolysis, is required for oligomerization of HslU. However, HslU/K63T remained as a monomer whether or not the adenine nucleotides were present. Furthermore, ATP or ADP could protect HslU, but not HslU/K63T, from degradation by trypsin. These results suggest that the mutation in the ATP-binding site results in prevention of the binding of the adenine nucleotides to HslU and hence in impairment of both oligomerization and ATPase function of HslU.
Shin, D. H., Yoo, S. J., Shim, Y. K., Seol, J. H., Kang, M. S., & Chung, C. H. (1996). Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli. FEBS Letters, 398(2–3), 151–154. https://doi.org/10.1016/S0014-5793(96)01223-9