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.

[Unique structural organization of ATP-dependent LonA proteases].

Homooligomeric LonA proteases are the key components of the protein quality control system in bacteria and eukaryotes. Domain organization of the common pool of LonA proteases is determined by comparative analysis of primary and secondary structures of a number of bacterial and eukaryotic enzymes. The similarity of individual enzyme domains was estimated, domain-domain linker areas were revealed, regions that are capable to include intercalated peptide fragments were identified. LonA proteases were shown to be unique AAA+ proteins, because in addition to the classic AAA+ module they contain a part of another AAA+ module, namely the alpha-helical domain including a coiled-coil region, which is similar to the alpha-helical domain of the AAA(+)-1 module of the chaperone-disagregases ClpB/Hsp104.

[Novel view of the structure of the non-catalytic N-terminal region of ATP-dependent LonA proteases].

ATP-Dependent Lon proteases are components of the protein quality control system, which maintains a keeping of cellular proteome. Lon family consists of two subfamilies A and B, differing in subunit architecture and intracellular location. The reinterpretation of the domain organization of the non-catalytic N-terminal region of ATP-dependent LonA proteases is proposed. Using Escherichia coli LonA protease (EcLon) as an example it has been shown that a fragment (alphaN-domain), which is located between the N-terminal domain and the AAA+ module of that protein, is similar to the alpha1-domain of the first AAA+ module of chaperone-disaggregase C1pB. A coiled-coil (CC) region included in the alphaN-domain of LonA is similar to the M domain of C1pB chaperones, which is inserted into the alpha1-domain. This region is suggested to adopt the structure similar to the propeller-like (PL) domain. The typical architecture of the N-terminal region of LonA proteases is postulated to be characterized by the obligatory presence of a PL domain, included in the alphaN-domain, but may vary in the length and topology of the preceding N-terminal domain.

[Molecular chaperones].

Chaperones are unique remodeling proteins that participate in a great number of intracellular processes and are involved in the correction of protein structure, the prevention of the aggregation of misfolded proteins, the destruction of protein aggregates, and also the unfolding of native protein targets for their translocation across a membrane. In addition to this, Chaperones assist in the dismantling of active oligomers into inactive unfolded monomers for their subsequent photolytic degradation and the assembly of folded subunits into protein assemblies and specific complexes. Data on the structure and functioning of molecular chaperones from five basic families are summarized in the review.

[ATP-dependent proteases and proteolytic complexes involved into intracellular protein degradation].

The review characterized the main enzymatic systems of selective proteolysis responsible for maintenance of intracellular proteome in prokaryotes, eukaryotes and archea. The features of proteolytic components of the ATP-dependent proteases as well as similarity and diversity of their regulatory AAA(+)-ATPases are discussed.

[VI Symposium on "Chemistry of Proteolytic Enzymes"].

The VI Symposium on the Chemistry of Proteolytic Enzymes took place in Moscow on April 23-25, 2007. It was dedicated to the memory of Corresponding Member of the Russian Academy of Sciences Vladimir Konstantinovich Antonov. At the symposium, 40 reports were delivered and 103 posters were presented in the following sections: (1) expression of genes, isolation and general characterization of proteases; (2) structure-function studies of proteases; (3) regulation of the activity of proteolytic enzymes; (4) regulatory functions of proteolytic enzymes; (5) proteases in biotechnology, protein engineering, and peptide synthesis; and (6) proteolysis and medicine. In addition to Russian scientists, researchers from the United States, the Netherlands, France, Ukraine, Belarus, Azerbaijan and Uzbekistan took part in the work of the symposium. Note that, in the five years since the V Symposium, the geography of Russian scientific centers working in the area of proteolysis has been considerably extended. Participating in the forum were researchers from, Novosibirsk, Tomsk, Penza, and Stavropol in addition to scientists from Moscow, Saint Petersburg, Petrozavodsk, Kazan, Nizhni Novgorod, and Krasnodar.

[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.

[Proteolysis coupled with ATP. Regulation of activity of proteolytic centers of Escherichia coli lon protease]. / Proteoliz, sopriazhennyi s gidrolizom ATP, reguliatsiia aktivnosti proteoliticheskikh tsentrov Lon-proteinazy Escherichia coli.

Regulation of activity of the proteolytic sites of Lon protease was studied. It was found that ATP-Mg has the properties of a noncompetitive activator of peptidase sites. The processive mechanism of the hydrolysis of protein substrates by Lon protease was experimentally confirmed under the conditions of ATP hydrolysis. It was shown that the oligomeric state of the enzyme is the necessary prerequisite for the processive proteolysis by the native Lon protease. The study of the properties of the mixed mutant Lon-K362Q/S679A confirmed the existence of the intra- and intersubunit pathways of signal transduction from the ATPase to proteolytic sites. The mutual influence of substrates of Lon protease was studied, and the existence of cooperative interactions between the peptidase sites in the oligomeric enzyme was suggested.

[Catalytic dyad Ser-Lys at the active site of Escherichia coli ATP-dependent Lon-proteinase]. / Katalitcheskaia diada Ser-Lys v aktivnom tsentre ATP-zavisimoi Lon-proteinazy Escherichia coli.

Two subfamilies of Lon proteases that differ in the structure of the fragments containing the catalytically active Ser residue were revealed by the comparison of more than sixty sequences of Lon proteases from various sources. The absence of the classic catalytic triad in the active site of Lon proteases was confirmed. The catalytic site of Lon proteases was shown to be represented by the Ser-Lys dyad.

[Peptide hydrolases with catalytic dyad Ser-Lys. Similarity and distinctions of the active centers of ATP-dependent Lon proteases, LexA repressors, signal peptidases and C-terminal processing proteases]. / Peptidgidrolazy s kataliticheskoi diadoi Ser-Lys. Skhodstvo i razlichiia aktivnykh tsentrov ATP-zavisimykh Lon-proteinaz, repressorov LexA, signal'nykh peptidaz i proteinaz C-kontsevogo protsessinga.

It is established that ATP-dependent protease Lon family belongs to the serine-lysine peptide hydrolases clan. Significant similarity of amino acid sequences of proteases Lon and repressors LexA in the regions including the catalytic serine and lysine residues is revealed by comparing primary structures of different families of the enzymes with Ser-Lys catalytic dyad. The both Lon and LexA families are shown to be divided into two subfamilies in accordance with the nature of amino acids in the catalytically active serine environment. Putative DNA binding sites are revealed in proteolytic domains of Lon A subfamily. Similarities and distinctions of the all families peptide hydrolases of the clan in the regions of their active centers are discussed.

[Energy-dependent selective intracellular proteolysis. Structure, active sites and specificity of ATP-dependent proteinases]. / Energozavisimyi selektivnyi vnutrikletochnyi proteoliz. Stroenie, aktivnye tsentry i spetsifichnost' ATP-zavisimykh proteinaz.

The enzymatic systems of selective proteolysis serving for the maintenance of cell homeostasis and functioning in prokaryotic and eukaryotic cells are characterized. The data on structure, active sites and specificity towards protein targets are given.

[Coupling of proteolysis and hydrolysis of ATP upon functioning of Lon proteinase of Escherichia coli. II. Hydrolysis of ATP and activity of peptide hydrolase sites of the enzyme]. / Sopriazhenie proteoliza i gidroliza ATP pri funktsionirovanii Lon-proteinazy Escherichia coli. II. Gidroliz ATP i aktivnost' peptidgidrolaznykh tsentrov fermenta.

The absence of direct correlation between the efficiency of functioning of ATPase and peptidehydrolase sites of Lon protease was revealed. It was shown that Lon protease is an allosteric enzyme, in which the catalytic activity of peptidehydrolase sites is determined by the binding of nucleotides, their magnesium complexes, and free magnesium ions in the enzyme's ATPase sites. It was revealed that complex ADP-Mg, an inhibitor of the native enzyme, is an activator of the Lon-K362Q form of the Lon protease mutant in the ATPase site. Considered are variants of intersite functional contacts realizing in the enzyme. The existence of two ways of signal transduction was established from the ATPase sites to peptidehydrolase ones in the Lon protease oligomer--intra- and intersubunit ways. Location of the enzyme ATPase sites is suggested in the areas of the complementary surfaces of subunits. It is hypothesized that ATP hydrolysis upon degradation of protein substrates by the E. coli Lon protease in vivo acts as a factor of restriction of the enzyme's degrading activity.

[Coupling of proteolysis with ATP hydrolysis by Escherichia coli Lon proteinase. I. Kinetic aspects of ATP hydrolysis]. / Sopriazhenie proteoliza i gidroliza ATP pri funktsionirovanii Lon-proteinazy Escherichia coli. I. Kineticheskie aspekty gidroliza ATP.

Some aspects of the ATPase function of the Escherichia coli Lon protease were studied around the optimum pH value. It was revealed that, in the absence of the protein substrate, the maximum ATPase activity of the enzyme is observed at an equimolar ratio of ATP and Mg2+ ions in the area of their millimolar concentrations. Free components of the substrate complex (ATP-Mg)2- inhibit the enzyme ATPase activity. It is hypothesized that the effector activity of free Mg2+ ions is caused by the formation of the "ADP-Mg-form" of the ATPase centers. It was shown that the activation of ATP hydrolysis in the presence of the protein substrate is accompanied by an increase in the affinity of the (ATP-Mg)2- complex to the enzyme, by the elimination of the inhibiting action of free Mg2+ ions without altering the efficiency of catalysis of ATP hydrolysis (based on the kcat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg)- complex (without changing the Ki value). Interaction of the Lon protease protein substrate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment.

[Intracellular proteolysis: signals of selective protein degradation]. / Vnutrikletochnyi proteoluz. Signaly selektivnoi degradatsii belkov.

Selective proteolysis is one of the mechanisms for the maintenance of cell homeostasis via rapid degradation of defective polypeptides and certain short-lived regulatory proteins. In prokaryotic cells, high-molecular-mass oligomeric ATP-dependent proteases are responsible for selective protein degradation. In eukaryotes, most polypeptides are attacked by the multicatalytic 26S proteasome, and the degradation of the majority of substrates involves their preliminary modification with the protein ubiquitin. The proteins undergoing the selective proteolysis often contain specific degradation signals necessary for their recognition by the corresponding proteases.

[A test system based on the lux-regulon from Vibrio fischeri for studying the functional activity of mutant forms of Escherichia coli lon-proteinase]. / Test-sistema na osnove lux-regulona Vibrio fischeri pri issledovanii funktsional'noi aktivnosti mutantnykh form lon-proteinazy Escherichia coli.

The possibility of application of the bioluminescence method (Lux-test) for studying in vivo functional activity of Escherichia coli protease Lon and its mutants was demonstrated. This assay is based on the capacity of protease Lon and its mutant forms for specific degradation of the LuxR protein, a positive transcriptional activator of the right operon luxICDABE from the marine bacterium Vibrio fischeri, and thus to affect the level of AB luciferase in the cells. A correlation between in vitro activity of the protease Lon mutants and the intensity of bioluminescence measured by the Lux-test was revealed.