Fig. 5: The MlaA hydrophilic channel exhibits solvent-accessibility changes due to interactions with MlaC and trimeric porins.
From: Molecular mechanism of phospholipid transport at the bacterial outer membrane interface
a Extracellular (left) and periplasmic (right) views of cartoon representations of the reported crystal structure of OmpK36 (green)-KpMlaA (cyan) (PDB: 5NUP)32, showing channel residues substituted with cysteine (for SCAM) in sticks. b, c Representative immunoblots showing Mal-PEG5k alkylation of MlaAK184C-His cysteine variant expressed from the pCDF plasmid, either in (b) ΔmlaA, ΔmlaA ΔmlaC, and ΔmlaA ΔmlaD backgrounds, or (c) ΔmlaA ΔmlaC background also producing wildtype or charge-reversed K84D/R90D (MlaC1) MlaC variants from the pET23/43-mlaCB plasmid. d Representative immunoblots showing Mal-PEG5k alkylation of MlaAK184C-His variant also harboring non-functional 3G3P or gain-of-function mlaA* mutations expressed from pET23/42 plasmids in ΔmlaA, ΔmlaA ΔmlaC, and ΔmlaA ΔmlaD background strains. e, f Representative immunoblots showing Mal-PEG5k alkylation of MlaAK184C-His cysteine variant expressed from the pCDF plasmid in various porin mutant backgrounds. In (e), OmpC or OmpF were expressed from pDSW206 plasmids where indicated. In SCAM, cells were labeled with membrane-permeable (NEM) or impermeable (MTSES) reagents, followed by alkylation with Mal-PEG5k, which introduces a ~5-kDa mass shift to MlaAK184C-His. The levels of solvent accessibility of K184C in MlaA in the various strains, i.e. fully, partially, or not blocked by MTSES, are highlighted in blue, orange, or red, respectively. These experiments had been performed at least three times with similar results. Source data are provided as a Source Data file.
