Lipid-regulated assembly mechanisms and functional energetics of the essential bacterial chaperone BamA

Abstract

BamA is the highly conserved essential outer membrane chaperone of all Gram-negative bacteria. Our understanding of the BamA machinery remains incomplete, delaying knowledge-based antibacterials design. Here, we report the first detailed identification of molecular elements indispensable for BamA folding, stability, and function. BamA displays two unique transition state structures and folding pathways in phosphatidylethanolamine (PE)– and phosphatidylglycerol (PG)– containing membranes. PE retards BamA folding; once folded, PE lowers stability of the N-terminal β-strands. In interesting contrast, PG promotes directional folding of BamA, and rigidifies the protein structure by lowering its conformational sampling space. We demonstrate that BamA β5–L4–β8 is an obligatory late-assembly zone in both PE and PG. Thermodynamic free energy measurements show BamA as membrane-anchored at β11–β15, destabilized at β2–β7 for its N-terminal gating function, with a C-terminal structural kink at β16. We show how BamA function links directly with (i) structures of PE-specific transition states, and (ii) zonal (de)stabilization hotspots at β5–L4–β8, β9–L5–β10, and β16-K808. We propose that these sites can now serve as novel hotspots for structure-based design of peptidomimetics to target multi-drug resistant Gram-negative pathogens.