The Lon Protease from <i>Mycobacterium smegmatis</i>:  Molecular Cloning, Sequence Analysis, Functional Expression, and Enzymatic Characterization

Roudiak, Stanislav G.; Seth, Anjali; Knipfer, Nancianne; Shrader, Thomas E.

doi:10.1021/bi971732f

Cited by 23 publications

(41 citation statements)

References 55 publications

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“…This result indicated that the ATPase and proteolytic domains function independently but interact structurally. These findings parallel the facts that substitution in the active site Ser nucleophile abolishes the peptidase activities but not the whole ATPase activities of Lon proteases (45)(46)(47). By contrast, Bt-Lon⌬N retained less than 1% of ATPase activity and only 1 Ϯ 0.5 and 2 Ϯ 0.3% of protease and peptidase activities, respectively, compared with those of the wild type.…”

Section: Design Of Bt-lon Mutants For Structure-function Studies-supporting

confidence: 77%

“…However, Smith et al (35) demonstrated that a small ␣-domain, present in most AAA ϩ modules, acts as a ''sensor and substrate discrimination'' domain. On the other hand, both peptidase and ATPase activities of Lon proteases are stimulated by unfolded proteins such as ␣-casein (47,60). The existing working model of Lon proteases indicates that the ␣-casein interaction site also resides in the N-terminal domain (5,61), which has been termed previously the allosteric site (or initiator site) (57,59).…”

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

confidence: 99%

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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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Section: Design Of Bt-lon Mutants For Structure-function Studies-supporting

confidence: 77%

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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“…HspR controls expression of the hsp70 and clpB genes in Mycobacterium tuberculosis and Mycobacterium leprae, but these bacteria do not contain any gene orthologous to lon. However, lon orthologues have been found in other mycobacteria, such as Mycobacterium smegmatis (Roudiak et al, 1998), and genome analysis revealed HAIR motifs upstream of lon in M. smegmatis, suggesting that the HAIR\HspR regulation of lon may be widespread among actinomycetes. Although Streptomyces spp.…”

Section: Discussionmentioning

confidence: 99%

The lon gene, encoding an ATP-dependent protease, is a novel member of the HAIR/HspR stress-response regulon in actinomycetes

et al. 2002

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Members of a family of ATP-dependent proteases related to Lon fromEscherichia coli are present in most prokaryotes and eukaryotes. These proteases are generally reported to be heat induced, and various regulatory systems have been described. The authors cloned and disrupted the lon gene and studied the regulation of its expression in Streptomyces lividans. lon is negatively regulated by the HspR/HAIR repressor/operator system, suggesting that Lon is produced concomitantly with the other members of this regulon, DnaK and ClpB. The lon mutant grew more slowly than the wild-type and spore germination was impaired at high temperature. Nevertheless its cell cycle was not greatly affected and it sporulated normally.

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“…The first discovered of these proteases and probably the most intensively studied biochemically is Lon (also called La) (4,5). Genes encoding Lon protease have been found in a large variety of prokaryotes, where they have been implicated in developmental pathways, as well as in the degradation of specific regulatory proteins (see below) (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). In eukaryotes, Lon is found in mitochondria and has been shown to be essential for mitochondrial function in Saccharomyces cerevisiae (18)(19)(20)(21).…”

mentioning

confidence: 99%

“…In Lon, a consensus site for ATP binding and hydrolysis has been shown by mutation to be necessary for proteolysis and ATPase activity; mutations in the active site serine ( Fig. 1) block proteolysis but do not fully abolish ATPase activity (14,(26)(27)(28)(29)(30). How and where substrate binding takes place and how substrates are presented to the active site, however, have not been clarified, and very few mutations other than those in the serine or ATPase sites have been described.…”

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confidence: 99%

Substrate sequestration by a proteolytically inactive Lon mutant

Melderen

Gottesman

1999

Proc. Natl. Acad. Sci. U.S.A.

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Lon protein of Escherichia coli is an ATPdependent protease responsible for the rapid turnover of both abnormal and naturally unstable proteins, including SulA, a cell division inhibitor made after DNA damage, and RcsA, a positive regulator of transcription. Lon is a multimer of identical 94-kDa subunits, each containing a consensus ATPase motif and a serine active site. We found that overexpressing Lon, which is mutated for the serine active site (LonS679A) and is therefore devoid of proteolytic activity, unexpectedly led to complementation of the UV sensitivity and capsule overproduction of a lon deletion mutant. SulA was not degraded by LonS679A, but rather was completely protected by the Lon mutant from degradation by other cellular proteases. We interpret these results to mean that the mutant LonS679A binds but does not degrade Lon substrates, resulting in sequestration of the substrate proteins and interference with their activities, resulting in apparent complementation. Lon that carried a mutation in the consensus ATPase site, either with or without the active site serine, was no longer able to complement a ⌬lon mutant. These in vivo results suggest that the pathway of degradation by Lon couples ATPdependent unfolding with movement of the substrate into protected chambers within Lon, where it is held until degradation proceeds. In the absence of degradation the substrate remains sequestered. Comparison of our results with those from a number of other systems suggest that proteins related to the regulatory portions of energy-dependent proteases act as energy-dependent sequestration proteins. Intracellular protein degradation is responsible for the rapid turnover of specific, unstable regulatory proteins and the clearing of abnormal proteins from the cytoplasm. The high molecular weight, ATP-dependent proteases that are primarily responsible for this degradation must recognize and select their protein substrates from among an enormous pool of other proteins that should not be degraded. Recent studies suggest that binding of substrate is followed by unfolding of the substrate or, at least of a portion of it, sufficient to gain access to and be cleaved by the proteolytic sites (1, 2). Processive degradation of the substrate then proceeds, leading to the release of short peptides of 6-15 aa.

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The Lon Protease from Mycobacterium smegmatis: Molecular Cloning, Sequence Analysis, Functional Expression, and Enzymatic Characterization

Cited by 23 publications

References 55 publications

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

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

The lon gene, encoding an ATP-dependent protease, is a novel member of the HAIR/HspR stress-response regulon in actinomycetes

Substrate sequestration by a proteolytically inactive Lon mutant

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