The Catalytic Domain of Escherichia coli Lon Protease Has a Unique Fold and a Ser-Lys Dyad in the Active Site

Botos, Istvan; Melnikov, Edward E.; Cherry, Scott; Tropea, Joseph E.; Khalatova, Anna G.; Rasulova, Fatima; Dauter, Zbigniew; Maurizi, Michael R.; Rotanova, T. V.; Wlodawer, Alexander; Gustchina, Alla

doi:10.1074/jbc.m312243200

Cited by 175 publications

(191 citation statements)

References 61 publications

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“…Of the soluble ATP-dependent proteases within the AAA + superfamily, Lon more closely resembles proteases within the proteasome family (26,27,29,42). As such, we began our search for a Lon inhibitor by screening a series of commercially available peptide-based proteasome inhibitors.…”

Section: Discussionmentioning

confidence: 99%

Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

2006

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Lon is a homo-oligomeric ATP-dependent serine protease which functions in the degradation of damaged and certain regulatory proteins. The importance of Lon activity in bacterial pathogenicity has led to its emergence as a target in the development of novel antibiotics. As no potent inhibitors of Lon activity have been reported to date, we sought to identify an inhibitor which could serve as a lead compound in the development of a potent Lon-specific inhibitor. To determine whether a nucleotide-or peptide-based inhibitor would be more effective, we evaluated the steady-state kinetic parameters associated with both ATP and peptide hydrolysis by human and Salmonella enterica serovar Typhimurium Lon. Although the ATP hydrolysis activities of both homologs are kinetically indistinguishable, they display marked differences in peptide substrate specificity. This suggests that a peptide-based inhibitor could be developed which would target bacterial Lon, thereby decreasing side-effects due to cross-reactivity with human Lon. Using Salmonella enterica serovar Typhimurium Lon as a model, we evaluated the IC 50 values of a series of commercially available peptide-based inhibitors. Those inhibitors which behave as transition state analogs were the most useful in inhibiting Lon activity. The peptidyl boronate, MG262, was the most potent inhibitor tested (IC 50 = 122 ± 9 nM) and required binding, but not hydrolysis of ATP to initiate inhibition. We hope to use MG262 as a lead compound in the development of future Lon specific inhibitors.The number of pathogenic, antibiotic resistant bacteria increases each year, however the development of new antibiotics to treat them lags behind (1). Recent studies aimed at identifying proteins necessary for virulence have implicated the importance of Lon protease (2,3). Pathogenic Salmonella enterica are responsible for causing a range of human diseases from mild gastroenteritis (serovar Typhimurium and serovar Enteritidis) to typhoid fever (serovar Typhi). It has been demonstrated that Salmonella enteria serovar Typhimurium (S. Typhimurium) Lon protease activity is required for systemic infection in mice, a common study model for S. Typhi infection in humans (3). In fact, Lon-deficient S. Typhimurium, when administered as an oral vaccine to mice, conferred subsequent protection against infection by virulent S. Typhimurium (4). Taken together, these studies highlight Lon as an important target in the development of novel therapeutic agents.Lon, also known as the protease La, is a homo-oligomeric ATP-dependent serine protease, which functions in the degradation of damaged and certain short-lived regulatory proteins (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Homologs exist ubiquitously in nature, however they localize to the cytosol in prokaryotes and to the mitochondrial matrix in eukaryotes (8,15,16 (11,(18)(19)(20)(21)(22)(23).Lon protease is a member of the AAA + superfamily (ATPases Associated with different cellular Activities) along with other ATP-dependent proteases such as ClpXP, H...

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Section: Discussionmentioning

confidence: 99%

Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

2006

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“…The CapR9 mutant was later proved to carry a single amino acid mutation (E614K) on its C-terminal domain, which has a higher isoelectric point than that of the wild type (55). Judging from the three-dimensional structure of the proteolytic domain of E. coli Lon, the point mutation (E614K) is located at ␤-sheet 1 that plays an important role in forming the subunit interface (56). The crystal structure data also showed that the subunit interface is characterized by mostly hydrophilic interactions (56), implying that the hydrophilic interactions among subunit interfaces stabilize the oligomerization.…”

Section: The N-terminal Domain Of Lon Proteases Is Essential Formentioning

confidence: 99%

“…Judging from the three-dimensional structure of the proteolytic domain of E. coli Lon, the point mutation (E614K) is located at ␤-sheet 1 that plays an important role in forming the subunit interface (56). The crystal structure data also showed that the subunit interface is characterized by mostly hydrophilic interactions (56), implying that the hydrophilic interactions among subunit interfaces stabilize the oligomerization. In our experiments, Bt-LonN316 mutant lacking the C-terminal domain forms an octamer as the major species and a dimer as the minor one by analytical gel filtration chromatography.…”

Section: The N-terminal Domain Of Lon Proteases Is Essential Formentioning

confidence: 99%

Functional Domains of Brevibacillus thermoruber Lon Protease for Oligomerization and DNA Binding

Lee

Hsu

2004

Journal of Biological Chemistry

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Lon protease is a multifunctional enzyme, and its functions include the degradation of damaged proteins and naturally short lived proteins, ATPase and chaperonelike activities, as well as DNA binding. A thermostable Lon protease from Brevibacillus thermoruber WR-249 (BtLon) has been cloned and characterized with an N-terminal domain, a central ATPase domain that includes a sensor and substrate discrimination (SSD) domain, and a C-terminal protease domain. Here we present a detailed structure-function characterization of Bt-Lon, not only dissecting the individual roles of Bt-Lon domains in oligomerization, catalytic activities, chaperone-like activity, and DNA binding activity but also describing the nature of oligomerization. Seven truncated mutants of Bt-Lon were designed, expressed, and purified. Our results show that the N-terminal domain is essential for oligomerization. The truncation of the N-terminal domain resulted in the failure of oligomerization and led to the inactivation of proteolytic, ATPase, and chaperone-like activities but retained the DNA binding activity, suggesting that oligomerization of Bt-Lon is a prerequisite for its catalytic and chaperone-like activities. We further found that the SSD is involved in DNA binding based on gel mobility shift assays. On the other hand, the oligomerization of Bt-Lon proceeds through a dimer 7 tetramer 7 hexamer assembly model revealed by chemical cross-linking experiments. The results also showed that hydrophobic interactions may play important roles in the dimerization of Bt-Lon, and ionic interactions are mainly responsible for the assembly of hexamers.Lon protease (La), the first ATP-dependent protease purified from Escherichia coli (1, 2), is a member of the ATPases associated with diverse cellular activities (AAA ϩ ) 1 superfamily (3).Lon consists of a variable N-terminal domain, a central ATPase domain, and a C-terminal protease domain on a single polypeptide (4, 5). The N-terminal domain with its still obscure function contains a potential coiled coil region built from ␣-helices and is proposed to be involved in binding and recognition of substrate proteins (5, 6). Lon is active as a homo-oligomer (7-10), which is distinct from other ATP-dependent proteases, Clp/HSP100 proteins, forming a hetero-oligomer. Nevertheless, the oligomeric state of Lon is still vague. Lon behaves as a tetramer or an octamer in E. coli (7), as a tetramer to a hexamer in Mycobacterium (5, 10), and as a hexamer or a heptamer in yeast mitochondria (8, 9). More recently, we have shown that Brevibacillus thermoruber Lon (Bt-Lon) is a hexameric structure (11). On the other hand, Clp/HSP100 proteins form a hexameric structure in the presence of ATP (12-15). Furthermore, the N-terminal domain of many Clp/HSP100 proteins, which is involved in binding of protein substrates (16 -18), is not necessary for oligomerization (17)(18)(19). By analogy with Clp/HSP100 proteins, it is quite reasonable to predict that the regulation of oligomerization of Lon may occur through an N-terminal doma...

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“…Purified sensor and substrate discrimination domain binds to known Lon substrates (10), suggesting that it plays a role in substrate recognition. Although E. coli Lon is thought to be a homo-tetramer or -octamer, recent structural analysis of the proteolytic domain of Lon revealed that it assembles into hexameric rings (11).…”

mentioning

confidence: 99%

Effects of Inorganic Polyphosphate on the Proteolytic and DNA-binding Activities of Lon in Escherichia coli

Nomura

Kato

Takiguchi

et al. 2004

Journal of Biological Chemistry

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Lon belongs to a unique group of proteases that bind to DNA and is involved in the regulation of several important cellular functions, including adaptation to nutritional downshift. Previously, we revealed that inorganic polyphosphate (polyP) increases in Escherichia coli in response to amino acid starvation and that it stimulates the degradation of free ribosomal proteins by Lon. In this work, we examined the effects of polyP on the proteolytic and DNA-binding activities of Lon. An order-of-addition experiment suggested that polyP first binds to Lon, which stimulates Lon-mediated degradation of ribosomal proteins. A polyP-binding assay using Lon deletion mutants showed that the polyP-binding site of Lon is localized in the ATPase domain. Because the same ATPase domain also contains the DNA-binding site, polyP can compete with DNA for binding to Lon. In fact, an equimolar amount of polyP almost completely inhibited DNA-Lon complex formation, suggesting that Lon binds to polyP with a higher affinity than it binds to DNA. Collectively, our results showed that polyP may control the cellular activity of Lon not only as a protease but also as a DNA-binding protein.

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The Catalytic Domain of Escherichia coli Lon Protease Has a Unique Fold and a Ser-Lys Dyad in the Active Site

Abstract: ATP-dependent

Cited by 175 publications

References 61 publications

Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

Functional Domains of Brevibacillus thermoruber Lon Protease for Oligomerization and DNA Binding

Effects of Inorganic Polyphosphate on the Proteolytic and DNA-binding Activities of Lon in Escherichia coli

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