Resolving the native conformation of Escherichia coli OmpA

Area of Science:

• Structural biology
• Protein folding
• Membrane proteins

Background:

• The native structure of Escherichia coli outer membrane protein A (OmpA) is debated, with models proposing either narrow-pore or large-pore conformations.
• Previous work identified cytoplasmic modifications of N-terminal residues (Ser163, Ser167) with oligo-(R)-3-hydroxybutyrates (cOHBs), essential for N-terminal domain pore formation.

Purpose of the Study:

• To investigate the impact of periplasmic modifications on OmpA pore structure.
• To compare OmpA from outer membranes (M-OmpA) with OmpA from cytoplasmic inclusion bodies (I-OmpA).

Main Methods:

• Comparative analysis of M-OmpA and I-OmpA using chemical analysis, Western blotting, and 1H-NMR.
• Planar lipid bilayer experiments to assess pore characteristics and temperature-induced transitions.
• Investigation of disulfide bond formation by DsbA.

Main Results:

• Periplasmic OmpA (M-OmpA) exhibits cOHB modification in segment 264-325, unlike cytoplasmic OmpA (I-OmpA).
• A disulfide bond forms between Cys290 and Cys302 in M-OmpA, mediated by DsbA.
• M-OmpA narrow pores transition to large pores upon heating, a process dependent on cOHB modification and disulfide bond integrity.

Conclusions:

• The narrow pore of OmpA represents a folding intermediate.
• cOHB modification, disulfide bond formation, and temperature are critical factors in OmpA's native large-pore configuration.