The effect of mutations in the inserted domain of ATP-dependent Lon protease from E-coli on the enzyme function

The ATP-dependent protease LonA from E. coli (Ec-Lon) belongs to the superfamily of AAA(+) proteins and plays a key role in the quality control system of the cell proteome. Ec-Lon functions as a homohexamer and destroys abnormal and defective polypeptides, as well as a number of regulatory proteins, according to a "processive degradation" mechanism. A Ec-Lon subunit includes an ATPase component and a proteolytic component (AAA(+) module and P-domain, respectively), as well as a noncatalytic region formed by the N-terminal (N) domain and an inserted alpha-helical (HI(CC)) domain; this region is unique for AAA(+) proteins. Mutant forms of Ec-Lon were obtained by replacing R164, R192, or Y294 residues localized in the HI(CC) domain, and the properties of these proteins were investigated in order to elucidate the role of the HI(CC) domain in enzyme functioning. The C-terminal part of the HI(CC) domain was shown to have an allosteric effect on the efficiency of the functioning of both ATPase and proteolytic sites of the enzyme, while the coiled-coil (CC) fragment of this domain was shown to interact with the protein substrate.