Functional Role of the N-Terminal Region of the Lon Protease from Mycobacterium smegmatis

Abstract

Lon protease homologues contain a poorly conserved N-terminal region of variable length. To better understand the role of the N-terminal region of Lon in the complicated reaction cycle of ATP-dependent protein degradation, we expressed and characterized mutants of the Lon protease from Mycobacterium smegmatis (Ms-Lon) lacking 90, 225, and 277 N-terminal residues (N-G91, N-E226, and N-I278, respectively). N-I278 displayed neither peptidase nor ATPase activity despite the fact that it was stable and soluble in vivo, had a near-wild-type CD spectrum, and the deleted residues included neither the catalytic nucleophile for peptide bond hydrolysis (S675) nor the ATP binding regions. N-G91 and N-E226 retained peptidase activities against small unstructured peptides that were stimulated, to near-wild-type levels, by the Ms-Lon substrate protein alpha-casein. By contrast, N-G91 and N-E226 retained basal ATPase activities, but these activities were only stimulated weakly by alpha-casein. Ms-Lon, N-E226, and N-G91 all exhibited low-level peptidase activity in assays containing nonhydrolyzed nucleotide analogues. However, these peptidase activities were stimulated strongly by alpha-casein in the case of Ms-Lon but weakly by alpha-casein in the cases of N-G91 and N-E226. Strikingly, despite the near-wild-type peptidase activities of N-G91 and N-E226, both were severely impaired in their degradation of the Ms-Lon protein substrates alpha-casein in vitro and RcsA in vivo. Overall, N-G91 and N-E226 displayed catalytic properties similar to Escherichia coli Lon (Ec-Lon) in the presence of the PinA inhibitor, suggesting that PinA inhibits Ec-Lon protease by inhibiting the function of Ec-Lon's N-terminal region. In vivo protease assays further revealed that, in contrast to the inactive Ms-Lon point mutant S675A, N-G91 and N-E226 did not reduce the cellular activity of RcsA. This same defect was observed previously for Ms-Lons with multiple mutations in their peptidase active sites. We conclude that proteolytically inactive mutants of Ms-Lon retain the ability to reduce the cellular activity of RcsA but that both the N-terminal region and the peptidase active site region of Ms-Lon are required for this activity of wild-type Ms-Lon. The inabilities of N-G91 and N-E226 to degrade larger protein substrates and to reduce the cellular activity of RcsA were not the result of drastic alterations in their quaternary structures. Gel filtration profiles of N-G91 and N-E226 revealed that each was primarily tetrameric, with an increased percentage of dimeric species and a decreased percentage of trimeric species relative to Ms-Lon. The observed shifts in the dimer/trimer ratios of the N-terminal truncation mutants suggest that the Ms-Lon tetramer contains two types of subunit-subunit interactions.

Degrons in protein substrates program the speed and operating efficiency of the AAA+ Lon proteolytic machine

“…At sufficiently high concentrations, such inhibitors fill most sites and prevent substrate binding. Previous studies also showed that protein substrates activate ATP-independent Lon cleavage of very small peptide mimics (14,7). Evidence for two conformations of Lon, whose populations change as a function of substrate binding, was obtained by assaying degradation and ATP hydrolysis under identical conditions as a function of titin-I27 CM -␤20 concentration (Fig.…”

“…This model also accounts for the stimulation of Lon degradation of one substrate by another substrate and for previous reports that proteins substrates stimulate Lon peptidase activity (7,14).…”

“…Functional Lon is a homohexamer. Each subunit consists of an N domain, an AAAϩ ATPase domain that provides mechanical power, and a peptidase domain (7)(8). Lon's ability to distinguish misfolded variants from correctly folded proteins depends on recognition of degradation tags rich in aromatic and hydrophobic residues, which tend to be hidden in the core of native proteins but become accessible upon denaturation (9).…”

“…Prior studies have shown that substrates and other ligands interact with Lon allosterically (7,(14)(15). However, little is known about the importance of these mechanisms in controlling protein degradation.…”

DNA and RNA Binding by the Mitochondrial Lon Protease Is Regulated by Nucleotide and Protein Substrate

“…To demonstrate the protease activity of purified Lon, casein covalently labeled with fluorescein isothiocyanate (FITC-casein) was used as a substrate (46,47,49). The energy-dependent degradation of casein has been routinely employed to characterize bacterial, yeast, and mammalian Lon (1,8,46,50).…”