Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli

FEBS Letters
D H ShinC H Chung

Abstract

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 Lys63 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.

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Citations

Aug 16, 2011·Biochimica Et Biophysica Acta·Petra WendlerSebastian Kube
Aug 22, 1997·The Journal of Biological Chemistry·H Huang, A L Goldberg
Jun 23, 1998·The Journal of Biological Chemistry·E C SchirmerS Lindquist
Dec 14, 2001·The Journal of Biological Chemistry·Yo-hei WatanabeMasasuke Yoshida
Aug 14, 2020·Genome Biology and Evolution·Tiffany N BatarsehBrandon S Gaut
Jan 22, 1998·Biochemical and Biophysical Research Communications·C H ChungM S Kang

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