Role of the Inserted α-Helical Domain in E. coli ATP-Dependent Lon Protease Function.

Multidomain ATP-dependent Lon protease of E. coli (Ec-Lon) is one of the key enzymes of the quality control system of the cellular proteome. A recombinant form of Ec-Lon with deletion of the inserted characteristic α-helical HI(CC) domain (Lon-dHI(CC)) has been prepared and investigated to understand the role of this domain. A comparative study of the ATPase, proteolytic, and peptidase activities of the intact Lon protease and Lon-dHI(CC) has been carried out. The ability of the enzymes to undergo autolysis and their ability to bind DNA have been studied as well. It has been shown that the HI(CC) domain of Ec-Lon protease is required for the formation of a functionally active enzyme structure and for the implementation of protein-protein interactions.

[The Effect of Mutations in the Inserted Domain of ATP-Dependent Lon Protease from E. coli on the Enzyme Function].

ATP-Dependent protease LonA from E. coli (Ec-Lon), belonging to the superfamily of AAA+ proteins, is a key member of the protein quality control system in bacterial cells. Ec-Lon functions as homohexamer and degrades abnormal and defective polypeptides as well as a number of regulatory proteins by the processive mechanism. Ec-Lon subunit includes--the both ATPase and proteolytic components (AAA+ module and P domain) in addition to the unique non-catalytic region formed by the N-terminal (N) and the inserted c-helical (HI(CC)) domains. The mutant forms Lon-R164A, Lon-R192A and Lon-Y294A have been obtained and characterized in order to reveal the role of the HI (CC) domain for the enzyme functioning. C-Terminal part of the HI (CC) domain is shown to display an allosteric effect on the efficiency of the enzyme ATPase and proteolytic sites while its coiled-coil (CC) region is involved in the interaction with the protein substrate.