Abstract
The β-barrel assembly machinery (BAM) complex is an essential component of Escherichia coli that inserts and folds outer membrane proteins (OMPs). The natural antibiotic compound darobactin inhibits BamA, the central unit of BAM. Here, we employ dynamic single-molecule force spectroscopy (SMFS) to better understand the structure-function relationship of BamA and its inhibition by darobactin. The five N-terminal polypeptide transport (POTRA) domains show low mechanical, kinetic, and energetic stabilities. In contrast, the structural region linking the POTRA domains to the transmembrane β-barrel exposes the highest mechanical stiffness and lowest kinetic stability within BamA, thus indicating a mechano-functional role. Within the β-barrel, the four N-terminal β-hairpins H1-H4 expose the highest mechanical stabilities and stiffnesses, while the four C-terminal β-hairpins H5-H6 show lower stabilities and higher flexibilities. This asymmetry within the β-barrel suggests that substrates funneling into the lateral gate formed by β-hairpins H1 and H8 can force the flexible C-terminal β-hairpins to change conformations.
Highlights
Integral outer membrane proteins (OMPs) of Gram-negative bacteria participate in a variety of crucial cellular functions, including nutrient transport (Klebba and Newton, 1998), protein secretion (Lauber et al, 2018), and adhesion (Heras et al, 2014)
The b-barrel assembly machinery (BAM) complex is an essential component of Escherichia coli that inserts and folds outer membrane proteins (OMPs)
We pushed the tip of the atomic force microscopy (AFM) cantilever onto BamA-enriched regions with a force of 1 nN for 0.5 s to promote the non-specific attachment of the tip to the N-terminal region (Thoma et al, 2018a, 2018bbib_Thoma_et_al_2018a) of BamA (Figure 2A)
Summary
Integral outer membrane proteins (OMPs) of Gram-negative bacteria participate in a variety of crucial cellular functions, including nutrient transport (Klebba and Newton, 1998), protein secretion (Lauber et al, 2018), and adhesion (Heras et al, 2014). The BAM complex consists of five protein components These are BamA, an integral OMP and a member of the Omp superfamily (Gentle et al, 2004; Voulhoux et al, 2003), and four lipoproteins, BamB, BamC, BamD, and BamE, which are anchored to the inner leaflet of the outer membrane (Wu et al, 2005). BamA, the central component of the BAM complex, consists of a C-terminal 16-stranded transmembrane b-barrel domain and five periplasmic N-terminal polypeptide transport (POTRA) domains (Gentle et al, 2005; Sanchez-Pulido et al, 2003). All components of the BAM complex are necessary to efficiently insert proteins into the outer membrane (Hagan et al, 2010), only BamA and BamD are essential (Malinverni et al, 2006; Voulhoux et al, 2003)
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