A fluorescent reporter and single-turnover kinetics reveal insight into BAM complex function

Area of Science:

• Bacterial outer membrane protein biogenesis
• Protein folding and assembly
• Molecular machines

Background:

• The β-barrel assembly machine (BAM) complex is crucial for folding and inserting outer membrane proteins (OMPs) into bacterial membranes.
• Previous studies relied on cell-based assays, limiting in vitro quantitative analysis of BAM component function.
• Structural data provided insights into the BamA β-barrel's role, but functional mechanisms remain incompletely understood.

Purpose of the Study:

• To develop an in vitro assay for quantitative analysis of BAM complex function.
• To determine kinetic parameters for OMP folding and insertion by the BAM complex.
• To investigate the roles of individual BAM components, including accessory lipoproteins and POTRA domains.

Main Methods:

• Development of a fluorescent reporter (bOmpA-A488) for OMP folding.
• Single-turnover kinetic analysis of wild-type BAM complex in vitro using liposomes.
• Assessment of subcomplexes (BamAB, BamAD) and POTRA domain deletion mutants.

Main Results:

• The wild-type BAM complex exhibits a folding rate (kfold) of 0.78 ± 0.15 min⁻¹ and substrate affinity of 3.1 ± 1.1 µM.
• BamA alone is inactive, but BamAB and BamAD subcomplexes show significant activity, comparable to the holo-BAM complex.
• POTRA domain deletions, including the essential POTRA3, did not significantly impair activity under single-turnover conditions.

Conclusions:

• Accessory lipoproteins (BamB/C/D) are essential for maintaining BamA in a catalytically active state for OMP folding and insertion.
• The first three POTRA domains of BamA may not be critical for substrate binding or folding under these specific in vitro conditions.
• The findings suggest a revised model for BAM complex function, highlighting the importance of accessory lipoproteins and questioning the primary role of early POTRA domains in catalysis.