RESUMO
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.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Mutação , Protease La/química , Protease La/genética , Cromatografia de Afinidade , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Homooligomeric ATP-dependent LonA proteases are bifunctional enzymes belonging to the superfamily of AAA+ proteins. Their subunits are formed by five successively connected domains: N-terminal (N), α-helical (HI(CC)), nucleotide binding (NB), the second α-helical (H) and proteolytic (P). The presence of the inserted HI(CC) domain defines the uniqueness of LonA proteases among AAA+ proteins. The role of α-helical domains in the LonA protease functioning is investigated on the example of E. coli Lon protease (Ec-Lon). A comparative study of properties of the intact Ec-Lon and its mutants of Lon-R164A and Lon-R542A with the substitutions of arginine residues located in similar positions in the HI(CC) and H domains is carried out. The H domain is shown to play a crucial role for the ATP hydrolysis and enzyme binding to the target protein. HI(CC) domain does not have a fundamental significance for the catalytic properties of the enzyme. However, it affects the functioning of Lon ATPase and peptidase sites and is involved in maintaining the enzyme stability. The participation of HI(CC) domain in formation of the spatial structures of LonA proteases and/or formation of their complexes with DNA is suggested.