Functional role of the N-terminal region of the Lon protease from Mycobacterium smegmatis.
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.
Roudiak
S G
SG
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Shrader
T E
TE
D049069:ATP-Dependent Proteases; D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002364:Caseins / Q000494:pharmacology; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D005822:Genetic Vectors / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D009161:Mycobacterium / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D049070:Protease La; D017510:Protein Folding; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D015843:Serpins / Q000494:pharmacology
D001426:Bacterial Proteins; D002364:Caseins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D015843:Serpins; C109893:cpsB protein, Streptomyces roseosporus; C067572:RcsA protein, E coli; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La; D000251:Adenosine Triphosphatases; D005998:Glycine