Membrane thinning and lateral gating are consistent features of BamA across multiple species

Abstract

In Gram-negative bacteria, the folding and insertion of β-barrel outer membrane proteins (OMPs) to the outer membrane are mediated by the β-barrel assembly machinery (BAM) complex. Two leading models of this process have been put forth: the hybrid barrel model, which claims that a lateral gate in BamA’s β-barrel can serve as a template for incoming OMPs, and the passive model, which claims that a thinned membrane near the lateral gate of BamA accelerates spontaneous OMP insertion. To examine the key elements of these two models, we have carried out 45.5 μs of equilibrium molecular dynamics simulations of BamA with and without POTRA domains from Escherichia coli, Salmonella enterica, Haemophilus ducreyi and Neisseria gonorrhoeae, together with BamA’s homolog, TamA from E. coli, in their native, species-specific outer membranes. In these equilibrium simulations, we consistently observe membrane thinning near the lateral gate for all proteins. We also see occasional spontaneous lateral gate opening and sliding of the β-strands at the gate interface for N. gonorrhoeae, indicating that the gate is dynamic. An additional 14 μs of free-energy calculations shows that the energy necessary to open the lateral gate in BamA/TamA varies by species, but is always lower than the Omp85 homolog, FhaC. Our combined results suggest OMP insertion utilizes aspects of both the hybrid barrel and passive models.

The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding

“…An alternative model proposes that BAM may disorder its lipid environment, lowering the kinetic barrier to OMP folding, potentially allowing OMPs to fold and insert into the outer membrane without direct interaction with the β1-β16 seam. This 'BamA-assisted' model 18,[30][31][32] is supported by molecular dynamics (MD) simulations which show lipid disordering and bilayer thinning by BamA 20,25,[30][31][32][33][34][35] , and by BAM-mediated distortion of a nanodisc 18 . Both protein dynamics and lipid disordering may act synergistically to maximise the efficiency of OMP folding, and different OMPs may depend on each effect to different degrees.…”

The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding.

“…Lipid destabilisation by BAM has been proposed previously as a potentially important facet of the catalysis of OMP folding and insertion into the OM 3,25,53 . This has been supported by MD simulations that reveal destabilisation of the membrane surrounding BamA 20,24,25,[30][31][32][33][34][35] , and a recent cryoEM structure of BAM in a nanodiscs containing E. coli polar lipids that shows distortion of the bilayer adjacent to the lateral gate 18 . Whilst these effects are localised to the BamA barrel, the laurdan fluorescence data provide direct biochemical evidence that BAM causes global destabilisation of a bilayer, as revealed by a reduction in the lipid phase transition temperature of DMPC liposomes (Fig.…”

“…An alternative model proposes that BAM may disorder its lipid environment, lowering the kinetic barrier to OMP folding, potentially allowing OMPs to fold and insert into the outer membrane without direct interaction with the β1-β16 seam. This 'BamA-assisted' model 18,[30][31][32] is supported by molecular dynamics (MD) simulations which show lipid disordering and bilayer thinning by BamA 20,25,[30][31][32][33][34][35] , and by BAM-mediated distortion of a nanodisc 18 . Both protein dynamics and lipid disordering may act synergistically to maximise the efficiency of OMP folding, and different OMPs may depend on each effect to different degrees.…”

“…In vitro studies have shown that spontaneous OMP folding rates and efficiencies are increased in membranes with decreased thickness, increased fluidity, or containing bilayer defects [42][43][44][45] . As well as directly interacting with its substrate OMPs 27,29 , BAM is also thought to reduce the stability of the lipid bilayer to facilitate folding, due to asymmetry in the hydrophobic thickness of the BamA β-barrel (which is narrowest in the vicinity of the lateral gate) 18,32 . Evidence for membrane destabilisation has been provided by molecular dynamics (MD) simulations of BamA in lipid bilayers 20,24,25,[30][31][32][33][34][35] and by cryoEM and MD simulations of BAM in nanodiscs formed from E. coli polar lipids 18 .…”

“…As well as directly interacting with its substrate OMPs 27,29 , BAM is also thought to reduce the stability of the lipid bilayer to facilitate folding, due to asymmetry in the hydrophobic thickness of the BamA β-barrel (which is narrowest in the vicinity of the lateral gate) 18,32 . Evidence for membrane destabilisation has been provided by molecular dynamics (MD) simulations of BamA in lipid bilayers 20,24,25,[30][31][32][33][34][35] and by cryoEM and MD simulations of BAM in nanodiscs formed from E. coli polar lipids 18 . To determine how the different conformational states of BAM affect bilayer stability more directly, we measured the effect of the different BAM complexes studied above on the lipid phase transition of liposomes formed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, diC 14:0 PC) using the fluorescent lipid probe laurdan (Supplementary Fig.…”