ATP-DEPENDENT DEGRADATION OF CCDA BY LON PROTEASE - EFFECTS OF SECONDARY STRUCTURE AND HETEROLOGOUS SUBUNIT INTERACTIONS

CcdA, the antidote protein of the ccd post-segregational killing syste m carried by the F plasmid, was degraded in vitro by purified Lon prot ease from Escherichia coli. CcdA had a low affinity for Lon (K-m great er than or equal to 200 mu M), and the peptide bond turnover number wa s similar to 10 min(-1). CcdA formed tight complexes with purified Ccd B, the killer protein encoded in the ccd operon, and fluorescence and hydrodynamic measurements suggested that interaction with CcdB convert ed CcdA to a more compact conformation. CcdB prevented CcdA degradatio n by Lon and blocked the ability of CcdA to activate the ATPase activi ty of Lon, suggesting that Lon may recognize bonding domains of protei ns exposed when their partners are absent. Degradation of CcdA require d ATP hydrolysis; however, CcdA41, consisting of the carboxyl-terminal 41 amino acids of CcdA and lacking the alpha-helical secondary struct ure present in CcdA, was degraded without ATP hydrolysis. Lon cleaved CcdA primarily between aliphatic and hydrophilic residues, and CcdA41 was cleaved at the same peptide bonds, indicating that ATP hydrolysis does not affect cleavage specificity, CcdA lost alpha-helical structur e at elevated temperatures (T-m similar to 50 degrees C), and its degr adation became independent of ATP hydrolysis at this temperature, ATP hydrolysis may be needed to disrupt interactions that stabilize the se condary structure of proteins allowing the disordered protein greater access to the proteolytic active sites.