Polypeptide stimulators of the Ms-Lon protease

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Lon proteases exhibit peptidase and ATPase activities, often stimulated by unstructured proteins like alpha-casein.
• This stimulation suggests simultaneous interaction with multiple substrates, highlighting the complexity of Lon protease regulation.
• Understanding the properties of Lon stimulators is crucial for elucidating its cellular functions.

Purpose of the Study:

• To investigate the ability of random copolymers of amino acids (rcAAs) to stimulate the peptidase and ATPase activities of Mycobacterium smegmatis Lon protease (Ms-Lon).
• To determine the physical properties of rcAAs that influence Ms-Lon stimulation.
• To explore the complex interactions between Ms-Lon, rcAAs, and alpha-casein.

Main Methods:

• Enzyme activity assays measuring peptidase and ATPase activities of Ms-Lon and a truncated mutant (N-E226) in the presence of various rcAAs and alpha-casein.
• Active site labeling experiments to probe enzyme-substrate interactions.
• Comparative analysis of stimulation effects by different types of rcAAs and alpha-casein.

Main Results:

• Cationic, but not anionic, rcAAs significantly stimulated Ms-Lon's peptidase activity, with stimulation correlating with hydrophobicity.
• rcAAs stimulated peptidase activity without affecting ATPase activity, unlike alpha-casein which stimulates both.
• Interactions were complex: low alpha-casein concentrations further stimulated rcAA-induced peptidase activity, while higher concentrations inhibited it; alpha-casein inhibited the truncated mutant's activity.

Conclusions:

• Ms-Lon can simultaneously interact with an rcAA, alpha-casein, and a peptide substrate, requiring its N-terminal domain.
• These findings support models of Lon protease with distinct allosteric polypeptide binding sites.
• The N-terminal domain plays a critical role in mediating complex interactions with multiple ligands.