An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated RidA.

Area of Science:

• Biochemistry
• Molecular Biology
• Oxidative Stress Response

Background:

• Escherichia coli RidA functions as an enamine/imine deaminase under normal conditions.
• Hypochlorous acid (HOCl) modification transforms RidA into a chaperone-like holdase, protecting the proteome during oxidative stress.
• The specific residues targeted by HOCl for RidA activation remain unidentified.

Purpose of the Study:

• To identify the key residues in Escherichia coli RidA that are chlorinated by HOCl to gain chaperone-like activity.
• To elucidate the molecular mechanism underlying HOCl-induced chaperone activity in RidA.
• To understand how RidA's structural and functional changes contribute to oxidative stress protection.

Main Methods:

• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze HOCl-modified RidA.
• Chemoproteomic approach using 5-(dimethylamino)naphthalene-1-sulfinic acid (DANSO2H) to label N-chlorinated lysines.
• Site-directed mutagenesis to probe the function of specific arginine and lysine residues.
• Structural analysis and molecular modeling of RidA and its HOCl-modified forms.

Main Results:

• LC-MS/MS confirmed chlorination of one arginine residue in RidA.
• Chemoproteomics identified N-chlorination of six additional lysine residues.
• Mutagenesis revealed that R105 and R128 are critical for RidA's HOCl-induced chaperone activity.
• HOCl-mediated activation correlates with loss of positive charges and increased protein hydrophobicity.
• A RidA variant engineered to be active without HOCl was successfully designed.

Conclusions:

• HOCl activates RidA's chaperone function through specific chlorination of arginine and lysine residues, notably R105 and R128.
• The observed increase in hydrophobicity due to charge loss is key to RidA's holdase activity.
• This study provides a molecular mechanism for HOCl-activated chaperones, including RidA.