Calcium-induced folding and stabilization of the Pseudomonas aeruginosa alkaline protease

Area of Science:

• Microbiology and Molecular Biology
• Protein Structure and Function

Background:

• Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections, particularly in immunocompromised individuals and cystic fibrosis patients.
• The bacterium's virulence is enhanced by biofilm formation and secreted factors like calcium-regulated alkaline protease (AP).
• AP, an RTX family protein, plays a significant role in P. aeruginosa pathogenesis.

Purpose of the Study:

• To investigate the structural and functional role of calcium ions (Ca(2+)) in the folding and activation of Pseudomonas aeruginosa alkaline protease (AP).
• To elucidate the mechanism by which Ca(2+) regulates AP and other RTX family virulence factors.

Main Methods:

• Purification of full-length and truncation mutants of AP.
• Structural and functional studies to assess the impact of Ca(2+) on AP folding and stability.

Main Results:

• Ca(2+) binding was found to induce the folding of the RTX domain of AP.
• The folded RTX domain acts as a molecular chaperone, promoting the correct folding of the protease domain.
• Association of the RTX domain with an N-terminal α-helix further stabilizes the activated AP.

Conclusions:

• Ca(2+) is a critical regulator of AP folding and activation, essential for its function as a virulence factor.
• The findings provide insights into the Ca(2+)-mediated regulatory mechanisms governing the RTX family of secreted bacterial toxins.