Distinct Structural Elements Govern the Folding, Stability, and Catalysis in the Outer Membrane Enzyme PagP

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• The outer membrane enzyme PagP is crucial for lipid A palmitoylation in Gram-negative bacteria.
• PagP plays a role in bacterial resistance to host immune defenses.
• PagP has an unusual, tilted barrel domain and an N-terminal amphipathic helix.

Purpose of the Study:

• To investigate the role of the N-terminal helix in PagP folding and stability.
• To identify residues influencing PagP's catalytic rates.
• To understand the molecular factors governing PagP stability and function.

Main Methods:

• Constructing chimeric barrel sequences of PagP from E. coli and S. typhimurium.
• Measuring thermodynamic stability of PagP variants.
• Analyzing phospholipase activity of PagP mutants in vitro.
• Employing coevolution analysis to detect interaction networks.

Main Results:

• The N-terminal helix of E. coli PagP confers a 2-fold greater stability to the S. typhimurium barrel.
• A W78F substitution in S. typhimurium PagP increases specific activity nearly 20-fold compared to E. coli PagP.
• Coevolution analysis revealed an interaction network within the membrane protein.

Conclusions:

• The N-terminal helix is critical for the thermodynamic stability of the PagP barrel domain.
• Specific residues, like phenylalanine at position 78, act as key regulators of PagP catalysis.
• Structural and functional exchanges between homologous PagP enzymes illuminate mechanisms of membrane protein stability and function.