Objective: ATP-dependent Lon proteases are key players in the quality control system of cellular proteins. The Lon family includes three main subfamilies (A, B, and C), whose representatives are common in bacteria, eukaryotes, and archaea. Recently, enzymes that potentially form a new "hybrid" subfamily LonBA have been discovered in bacteria of the Bacilli and Clostridia classes. This study aims to characterize the structure of LonBA proteases and elucidate their features by comparison with classical LonA and LonB proteases. Methods: Bioinformatics analysis methods and approaches were used to comparatively characterize Lon proteases of different subfamilies. Results and Discussion: Analysis of sequences of the common pool of Firmiqutes' Lons showed that they contain both classical LonA and "hybrid" LonBA proteases. The ATPase component of the latters is similar to the ATPase fragment of LonB proteases, while the catalytic P domain is similar to the P domain of LonA proteases. Degrees of similarity of different structural fragments of LonBAs were estimated. Groups of short and long LonBA proteases were identified. Sources of short-LonBAs are both bacilli and clostridia, but long ones-only clostridia. Conclusions: The new subfamily of LonBA proteases was shown to consist of two communities of enzymes that differ in the structure of their N-terminal fragments and protease domains.

The Lon protease family belongs to the key peptide hydrolases of the protein quality control (PQC) system, which plays a leading role in maintaining the integrity of the cellular proteome in all natural kingdoms. Moreover, Lon proteases are the only family of ATP-dependent proteases of PQC which comprises a number of structurally distinct subfamilies. Recently, it has been suggested that the Lon family contains a previously unclassified LonBA subfamily, which includes enzymes from bacteria of the Bacilli and Clostridia classes. Using bioinformatics analysis, data were obtained on the structural features of enzymes of the putative new subfamily and on the existence of two different groups of Lon proteases in this subfamily.

Lon proteases, members of the AAA+ superfamily of enzymes, are key components of the protein quality control system in bacterial cells, as well as in the mitochondria and other specialized organelles of higher organisms. These enzymes have been subject of extensive biochemical and structural investigations, resulting in 72 crystal and solution structures, including structures of the individual domains, multi-domain constructs, and full-length proteins. However, interpretation of the latter structures still leaves some questions unanswered. Based on their amino acid sequence and details of their structure, Lon proteases can be divided into at least three subfamilies, designated as LonA, LonB, and LonC. Protomers of all Lons are single-chain polypeptides and contain two functional domains, ATPase and protease. The LonA enzymes additionally include a large N-terminal region, and different Lons may also include non-conserved inserts in the principal domains. These ATP-dependent proteases function as homohexamers, in which unfolded substrates are translocated to a large central chamber where they undergo proteolysis by a processive mechanism. X-ray crystal structures provided high-resolution models which verified that Lons are hydrolases with the rare Ser-Lys catalytic dyad. Full-length LonA enzymes have been investigated by cryo-electron microscopy (cryo-EM), providing description of the functional enzyme at different stages of the catalytic cycle, indicating extensive flexibility of their N-terminal domains, and revealing insights into the substrate translocation mechanism. Structural studies of Lon proteases provide an interesting case for symbiosis of X-ray crystallography and cryo-EM, currently the two principal techniques for determination of macromolecular structures.

The Protein Quality Control (PQC) system plays a leading role in maintaining the safety of the cellular proteome in all natural kingdoms. This review summarizes information about the structural and functional characteristics of molecular chaperones and energy-dependent proteases that form the PQC system, emphasizing the crucial role of proteins belonging to the AAA+ superfamily, with the focus on ATP-dependent Lon proteases as a special family in the PQC. Similarities and differences among the enzymes of individual Lon subfamilies are discussed in detail using up-to-date data, elucidating the structural features and unique mechanisms of functioning of these proteins.

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.

АТР-зависимая Lon-протеаза Escherichia coli ( Ес Lon), относящаяся к суперсемейству ААА + -белков, является ключевым участником системы контроля качества клеточного протеома, в которой она отвечает за расщепление потенциально опасных для клетки мутантных, поврежденных и короткоживущих регуляторных белков. Ес Lon функционирует как гомоолигомер, субъединица которого включает центральный характеристический ААА + -модуль, С-концевой протеазный домен, а также N-концевую некаталитическую область, образованную двумя доменами – собственно N-концевым и вставочным α-спирализованным. Анализ пространственной структуры N-домена позволил выявить остатки E34, K35 и R38 на поверхности молекулы фермента, предположительно вовлеченные в формирование стабильной, функционально активной Ec Lon-протеазы. Получен тройной мутант LonEKR, несущий замены остатков E34, K35 и R38 на аланин. Показано, что введение указанных замен влияет на конформационную стабильность и межцентровые аллостерические взаимодействия в ферменте, обусловленные действием нуклеотидов, а также на формирование корректного сайта связывания белкового субстрата.

LonA proteases and ClpB chaperones are key components of the protein quality control system in bacterial cells. LonA proteases form a unique family of ATPases associated with diverse cellular activities (AAA+) proteins due to the presence of an unusual N‐terminal region comprised of two domains: a β‐structured N domain and an α‐helical domain, including the coiled‐coil fragment, which is referred to as HI(CC). The arrangement of helices in the HI(CC) domain is reminiscent of the structure of the H1 domain of the first AAA+ module of ClpB chaperones. It has been hypothesized that LonA proteases with a single AAA+ module may also contain a part of another AAA+ module, the full version of which is present in ClpB. Here, we established and tested the structural basis of this hypothesis using the known crystal structures of various fragments of LonA proteases and ClpB chaperones, as well as the newly determined structure of the Escherichia coli LonA fragment (235–584). The similarities and differences in the corresponding domains of LonA proteases and ClpB chaperones were examined in structural terms. The results of our analysis, complemented by the finding of a singular match in the location of the most conserved axial pore‐1 loop between the LonA NB domain and the NB2 domain of ClpB, support our hypothesis that there is a structural and functional relationship between two coiled–coil fragments and implies a similar mechanism of engagement of the pore‐1 loops in the AAA+ modules of LonAs and ClpBs.

Energy-dependent Lon proteases play a key role in cellular regulation by degrading short-lived regulatory proteins and misfolded proteins in the cell. The structure of the catalytically inactive S679A mutant of Escherichia coli LonA protease (EcLon) has been determined by cryo-EM at the resolution of 3.5 Å. EcLonA without a bound substrate adopts a hexameric open-spiral quaternary structure that might represent the resting state of the enzyme. Upon interaction with substrate the open-spiral hexamer undergoes a major conformational change resulting in a compact, closed-circle hexamer as in the recent structure of a complex of Yersinia pestis LonA with a protein substrate. This major change is accomplished by the rigid-body rearrangement of the individual domains within the protomers of the complex around the hinge points in the interdomain linkers. Comparison of substrate-free and substrate-bound Lon structures allows to mark the location of putative pivotal points involved in such conformational changes.

The effect of the coiled-coil (CC) region of the α-helical inserted domain of Escherichia coli Lon protease (Ec-Lon) on the functional activity of the enzyme has been characterized. A recombinant form des-CC(G5)-Lon in which the deleted CC fragment is replaced by a pentaglycine peptide has been obtained and investigated. It has been shown that the CC region is involved in the recognition of the nucleotide nature by the enzyme and the interaction of the enzyme with the protein substrate. It has been also established that the CC region is necessary for the formation and functioning of the ATPase and peptidase active centers, the occurrence of allosteric interactions between them, and for the implementation of proteolysis by a unique processive mechanism.

The truncated form of E. coli LonA protease ( Ec Lon) lacking the N -terminal fragment 1–172 (Lon173) and the variant with deleted coiled-coil (CC) fragment 173–283 (dCC-Lon, a deletion form) are produced and characterized to study the role of the N -terminal region in the functioning of this protease. A comparative analysis of the properties of full-length Ec Lon protease, dCC-Lon, and Lon173 as well as an earlier produced form with retained C -terminal region (235–280) of CC fragment, Lon235, is performed. As is shown, fragment 1–280 plays an important role in both formation of the ATPase site and maintenance of a stable Ec Lon protease conformation. Fragment 107–172 is of a paramount importance for implementation of the processive mechanism of ATP-dependent proteolysis.

The binding to Lon protease through biotinylated aptamers whose structures contain G-quadruplex fragments with magnetic nanoparticles (MNPs) functionalized by streptavidin was investigated. The conditions of binding of target aptamers to MNPs are met. The resulting complexes are proposed for detection of Lon protease in different biological sources and for constructing a novel biomagnetic nanosensor immunoassay system.

Мультидоменная АТР-зависимая протеаза Lon из Escheriсhia coli (Ес-Lon) - один из ключевых ферментов системы контроля качества клеточного протеома. C целью изучения роли инсерционного HI(CC)-домена Ес-Lon получен и охарактеризован рекомбинантный фермент с делецией HI(CC)-домена (Lon-dHI(CC)). Проведено сравнительное исследование АТР-азной, протеолитической и пептидазной активности интактной Lon-протеазы и Lon-dHI(CC), изучена возможность автолиза обеих форм фермента и их способность к связыванию ДНК. Показано, что HI(CC)-домен необходим для формирования функционально активной структуры Ec-Lon-протеазы и реализации белок-белковых взаимодействий.

Bifunctional Escherichia coli LonA protease (Ec-Lon) belongs to the superfamily of AAA + proteins. It is a key member of the quality control system of the cell proteome. The enzyme degrades abnormal and defective polypeptides, as well as a number of regulatory proteins, by the processive mechanism. In addition to the ATPase module and the proteolytic domain, Ec-Lon subunit includes a two-domain N-terminal noncatalytic region. A comparative study of the enzyme properties and the DNA-binding ability of full-size Ec-Lon and its form with a deletion of 106 amino acid residues at the N-end has been carried out to reveal the role of the missing fragment in the Ec-Lon function. It has been shown that the fragment does not affect the enzyme peptidase site function or the hydrolysis of the protein substrate by the processive mechanism. However, it is essential for the manifestation of proper ATPase activity and for the implementation of the conformational rearrangements in the ATPase domain, stemming from the coordination of different nucleotides or their complexes by magnesium ions. The loss of the (1–106) fragment destabilizes the active Ec-Lon structure and results in intense Ec-Lon autolysis.

ATP-dependent Lon protease of E. coli (Ec-Lon) is a key enzyme of the quality control system of the cell proteome. The 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 of the complex Ec-Lon/DNA have not yet been characterized. Aptamers, small nucleic acids with high specificity to organic compounds of different natures, are known to be a promising tool for the study of molecular mechanisms of interaction between nucleic acids and protein ligands. 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 planned for studying the mechanism of the enzyme-DNA complexation.

АТР-зависимая LonА-протеаза из E. coli (Ес-Lon), относящаяся к суперсемейству ААА+-белков, является ключевым участником системы контроля качества клеточного протеома. Ес-Lon функционирует как гомогексамер и расщепляет аномальные и дефектные полипептиды, а также ряд регуляторных белков по процессивному механизму. Субъединица Ес-Lon включает АТР-азный и протеолитический компоненты (ААА+-модуль и Р-домен), а также уникальную для ААА+-белков некаталитическую область, образованную N-концевым (N) и инсерционным -спирализованным (HI(СС)) доменами. С целью выявления роли HI(СС)-домена в функционировании фермента получены мутанты Ес-Lon с заменами остатков R164, R192 и Y294, локализованных в этом домене, и изучены их свойства. Показано, что С-концевая часть НI(СС)-домена аллостерически влияет на эффективность функционирования АТР-азного и протеолитического центров фермента, а его coiled-coil(СС)-область вовлечена во взаимодействие с белковым субстратом.

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 α-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.