A Mutation in the N Domain of Escherichia coli Lon Stabilizes Dodecamers and Selectively Alters Degradation of Model Substrates

Abstract

Escherichia coli Lon, an ATP-dependent AAA ؉ protease, recognizes and degrades many different substrates, including the RcsA and SulA regulatory proteins. More than a decade ago, the E240K mutation in the N domain of Lon was shown to prevent degradation of RcsA but not SulA in vivo. Here, we characterize the biochemical properties of the E240K mutant in vitro and present evidence that the effects of this mutation are complex. For example, Lon E240K exists almost exclusively as a dodecamer, whereas wild-type Lon equilibrates between hexamers and dodecamers. Moreover, Lon E240K displays degradation defects in vitro that do not correlate in any simple fashion with degron identity, substrate stability, or dodecamer formation. The Lon sequence segment near residue 240 is known to undergo nucleotide-dependent conformational changes, and our results suggest that this region may be important for coupling substrate binding with allosteric activation of Lon protease and ATPase activity. A AAϩ proteases play important biological roles in all organisms (1). Inhibition of the AAA ϩ Lon protease reduces or eliminates virulence in several pathogenic bacteria and is toxic to human lymphoma cells, whereas Lon overexpression improves the life span of some fungi but kills Escherichia coli (2-7). Like most AAA ϩ proteases, bacterial Lon recognizes substrates by binding to specific amino acid sequences called degrons or degradation tags (8, 9). In E. coli, Lon degrades native regulatory proteins, including the RcsA transcription factor and the SulA inhibitor of cell division, but also appears to be responsible for degrading the majority of misfolded proteins, including ␤-galactosidase (10-12). The degron that targets RcsA for Lon degradation is unknown, whereas degrons for SulA (called sul20) and ␤-galactosidase (called ␤20) have been indentified (8, 13). The binding of different degrons stabilizes enzyme conformations with high or low protease activities, and the latter state may allow Lon to function as a chaperone (14).E. coli Lon subunits assemble into a homohexamer, which appears to be the minimal unit of proteolytic function (15). Each subunit contains an N-terminal domain, a central AAA ϩ module, and a C-terminal peptidase domain (16,17). The active sites for peptide bond cleavage are sequestered within a bowl-like chamber formed by the peptidase domains of the hexamer (17, 18). The hexameric AAA ϩ ring of Lon appears to regulate access to this chamber by coupling ATP hydrolysis to conformational changes that unfold and translocate substrates through an axial pore and into the chamber (1). The N domain of Lon is required for stable hexamer formation (19,20), and recent studies suggest that it also stabilizes a dodecamer, with hexamers and dodecamers being populated at physiological Lon concentrations (21). The dodecamer degrades certain substrates as well as the hexamer but degrades other substrates, including casein, at substantially reduced rates (21). Although crystal structures are known for most individual domains of Lon...

Toxin-Antitoxin Modules Are Pliable Switches Activated by Multiple Protease Pathways

“…The oligomeric state of the proteases affects its ability to degrade small or partially folded protein domains. Lon has been identified to form a concentration-dependent dodecamer (a dimer of hexamers) with a large central chamber that could accommodate small clients with residual secondary structure [ 129 , 130 ], such as perhaps an antitoxin molecule. Similarly, the central pore of ClpP is opened significantly when it associates with an ATPase oligomer [ 131 ].…”

Stress-Induced Remodeling of the Bacterial Proteome

“…This may gate this chamber so that large substrates (>12–25 kDa) can no longer enter and be proteolyzed [180]. Importantly, the cellular concentration of Lon is high enough to support dodecamer formation, and constitutively dodecameric Lon mutants can complement many lon deletion phenotypes in vivo [180, 181]. As dodecamers cannot recognize large protein aggregates, dodecamer formation can realign the powerful degradation capacity of Lon to focus on important small regulatory proteins during times of high protein unfolding and aggregation [180].…”

Multiple domains of bacterial and human Lon proteases define substrate selectivity

“…While the precise contribution of the three Lon domains to substrate selection is unknown, the N domain of the Escherichia coli Lon (eLon) is required for substrate recognition and binding as exemplified by the direct binding between Lon and sul20 peptide 23 . Consistently, a E240K mutation in the N domain selectively alters degradation of substrates 25 . Furthermore, deletion of 124–304 amino acids in the N domain led to a complete loss of the proteolytic activity of eLon toward its substrate β-casein 26 .…”

“…The Francisella and E. coli proteins for in vivo degradation were prepared by cloning amplicons of the target coding sequences from the genomic DNA preparation of LVS and MG1655, respectively, and expressed as recombinant polypeptides with a C-terminal His tag in pACYCDuet-1 as described in our previous study 11 . The C-terminally peptide-tagged HUβ variants were generated as described previously 25 . The coding sequences of HUβ and each peptide were separately amplified from E. coli genomic DNA, linked by fusion PCR, and cloned into the Nco I/ Sal I site of pACYCDuet-1.…”