Engineering a Hyperstable Yersinia pestis Outer Membrane Protein Ail Using Thermodynamic Design

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Yersinia pestis, a tier I pneumopathogen, requires understanding its virulence factors for therapeutic development.
• The outer membrane protein Ail is crucial for Yersinia pathogenesis but difficult to study due to its instability and aggregation.

Purpose of the Study:

• To characterize the molecular determinants of Ail's metastability and identify strategies for enhancing its stability.
• To engineer a hyperstable variant of Ail for potential therapeutic applications.

Main Methods:

• Analysis of Ail's structure-thermodynamics relationship.
• Site-directed mutagenesis to identify key residues affecting stability (Q50, L88, L92, A94).
• Characterization of the engineered variant Out14, including its folding pathways and stability mechanisms.

Main Results:

• Four specific residues (Q50, L88, L92, A94) were found to significantly reduce Ail's stability.
• Engineered variant Out14 exhibited hyperthermostability and reduced aggregation.
• Out14 possesses a functional scaffold and distinct folding pathways compared to wild-type Ail.
• Enhanced stability in Out14 is attributed to favorable nonpolar surface changes and a unique folding intermediate.

Conclusions:

• Specific amino acid substitutions can re-engineer intrinsically unstable outer membrane proteins into hyperstable variants.
• Engineered hyperstable Ail (Out14) offers a promising scaffold for drug screening and developing countermeasures against Yersiniae.
• This strategy can be applied to design therapeutics for other biopathogens.