Involvement of the N Domain Residues E34, K35, and R38 in the Functionally Active Structure of Escherichia coli Lon Protease.

ATP-dependent Lon protease of Escherichia coli (EcLon), which belongs to the superfamily of AAA+ proteins, is a key component of the cellular proteome quality control system. It is responsible for the cleavage of mutant, damaged, and short-lived regulatory proteins that are potentially dangerous for the cell. EcLon functions as a homooligomer whose subunits contain a central characteristic AAA+ module, a C-terminal protease domain, and an N-terminal non-catalytic region composed of the actual N-terminal domain and the inserted α-helical domain. An analysis of the N domain crystal structure suggested a potential involvement of residues E34, K35, and R38 in the formation of stable and active EcLon. We prepared and studied a triple mutant LonEKR in which these residues were replaced with alanine. The introduced substitutions were shown to affect the conformational stability and nucleotide-induced intercenter allosteric interactions, as well as the formation of the proper protein binding site.

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.

[Interaction of DNA Aptamers with the ATP-Dependent Lon Protease from Escherichia coli].

ATP-dependent Lon protease of E. coli (Ec-Lon) is a key enzyme of the quality control system of the cell proteome. Ec-Lon subunit comprises N-terminal non-catalytic region, ATPase module and proteolytic domain (serine-lysine endopeptidase). A distinctive feature of the Ec-Lon is its ability to interact with DNA, however either DNA binding site(s) or the role ofthe complex Ec-Lon · DNA have not yet been characterized. A promising tool for the study of molecular mechanisms of interaction between nucleic acids and protein ligands are known to be aptamers (small nucleic acids with high specificity to organic compounds of different nature). Ec-Lon-protease was found to form complexes with the previously obtained thrombin aptamers whose molecules comprise the duplex domains and G-quadruplex region. The aptamer affinities to the enzyme have been characterized. The synthesis of novel aptamers specific to Ec-Lon protease is planed for studying the mechanism of the enzyme-DNA complexation.

[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.

[Role of the α-helical domains in the functioning of ATP-dependent Lon protease of Escherichia coli].

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.

[Forms of LonB protease from Archaeoglobus fulgidus devoid of the transmembrane domain: the contribution of the quaternary structure to the regulation of enzyme proteolytic activity].

Deletion of the transmembrane domain (TM-domain) of Archaeoglobus flggidus LonB protease (AfLon) was shown to result in uncontrollable activation of the enzyme proteolytic site and in vivo autolysis yielding a stable and functionally inactive fragment consisting of both alpha-helical and proteolytic domains (alphaP). The deltaTM-AfLonTM-S590A enzyme form, obtained by site-directed mutagenesis of the catalytic Ser residue, is capable of recombination with the alphaP fragment. The mixed oligomers were shown to be proteolytically active, which indicates a crucial role of subunit interactions in the activation of the AfLon proteolytic site. The thermophilic nature of AfLon protease was found to be due to the special features of the enzyme activity regulation, the structure of ATPase domain, and the quaternary structure.