Mutational analysis demonstrates different functional roles for the two ATP-binding sites in ClpAP protease from Escherichia coli.

ClpA, the regulatory subunit of Clp protease from Escherichia coli, has two ATP-binding sites in non-homologous regions of the protein, referred to as domain I and domain II. We have mutated the invariant lysine in the ATP-binding sites of domain I and domain II and studied the enzymatic properties of the purified mutant ClpA proteins. The domain I mutant, ClpA-K220Q, was unable to form a hexamer in the presence of nucleotide, but the comparable domain II mutant, ClpA-K501Q, associated into a hexamer in the presence of ATP, indicating that nucleotide binding to domain I favors a conformation required to stabilize the quaternary structure of ClpA. ClpA-K220Q was defective in ATPase activity and in the ability to activate protein and peptide degradation by ClpP, but the defects could be partially overcome by formation of hybrid hexamers with wild-type ClpA. Another domain I mutant, ClpA-K220R, readily formed hexamers in the presence of ATP and retained > or = 60% of the wild-type ATPase activity and ability to activate ClpP. These results indicate that hexamer formation is a prerequisite for expression of enzymatic activity. Domain II mutants ClpA-K501Q and ClpA-K501R had very low ATPase activity (< 10% of wild-type) and a severe defect in activation of protein degradation, which requires ATP hydrolysis. Domain II mutants were able to activate ClpP to degrade a peptide whose degradation required nucleotide binding but not hydrolysis. Nucleotide binding to domain II of ClpA is important to form a productive complex with ClpP, and domain II appears to be primarily responsible for an energy-requiring step in the catalytic cycle unique to the degradation of large proteins.