Extreme Dynamics in the BamA β-Barrel Seam

Doerner, Pamela A.; Sousa, Marcelo C.

doi:10.1021/acs.biochem.7b00281

Cited by 53 publications

(75 citation statements)

References 43 publications

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“…There, it was found through disulfide cross-linking that β-barrel precursors translocate into the Sam50 channel and then enter the lateral gate as β-hairpins, interacting with both N-and C-terminal strands of Sam50 (51). Nonetheless, in another recent study, it was found that a disulfide-locked lateral gate still accelerates the insertion of an eight-strand β-barrel OmpX, which runs contrary to the hybrid barrel model (34). Regardless of the exact mechanism, membrane thinning, as consistently observed in our simulations, plays a vital role in OMP insertion by significantly lowering the insertion free-energy barrier.…”

Section: Discussionmentioning

confidence: 98%

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C-terminal kink formation is required for lateral gating in BamA

Lundquist

Bakelar

Noinaj

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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In Gram-negative bacteria, the outer membrane contains primarily β-barrel transmembrane proteins and lipoproteins. The insertion and assembly of β-barrel outer-membrane proteins (OMPs) is mediated by the β-barrel assembly machinery (BAM) complex, the core component of which is the 16-stranded transmembrane β-barrel BamA. Recent studies have indicated a possible role played by the seam between the first and last β-barrel strands of BamA in the OMP insertion process through lateral gating and a destabilized membrane region. In this study, we have determined the stability and dynamics of the lateral gate through over 12.5 μs of equilibrium simulations and 4 μs of free-energy calculations. From the equilibrium simulations, we have identified a persistent kink in the C-terminal strand and observed spontaneous lateral-gate separation in a mimic of the native bacterial outer membrane. Free-energy calculations of lateral gate opening revealed a significantly lower barrier to opening in the C-terminal kinked conformation; mutagenesis experiments confirm the relevance of C-terminal kinking to BamA structure and function.

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Section: Discussionmentioning

confidence: 98%

“…The BamA β1 − β16 seam has become the centerpiece for several models for OMP assembly including the so-called "hybrid barrel model" (32,33) and the "passive model" (34). In the passive model, the principal role played by the seam is to prime the membrane for OMP insertion.…”

mentioning

confidence: 99%

C-terminal kink formation is required for lateral gating in BamA

Lundquist

Bakelar

Noinaj

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…Consistent with this model, engineered disulfide bonds that prevent the opening of the putative lateral gate are lethal in E. coli (17). Disulfide locking of the BamA b-barrel, however, does not effectively block OMP assembly in vitro (16,18,19). Molecular dynamics simulations (20, 21) and other studies (22) that suggest that the BamA β-barrel causes thinning of the adjacent membrane have led to an alternative model in which the Bam complex catalyzes OMP assembly by creating a local deformation of the lipid bilayer.…”

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confidence: 81%

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

Hussain

Bernstein

2018

Journal of Biological Chemistry

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Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "b-barrel" architecture, but the assembly of these proteins is poorly understood. The spontaneous assembly of OM proteins (OMPs) into pure lipid vesicles has been studied extensively, but often requires nonphysiological conditions and time scales and is strongly influenced by properties of the lipid bilayer including surface charge, thickness, and fluidity. Furthermore, the membrane insertion of OMPs in vivo is catalyzed by a heterooligomer called the b-barrel assembly machinery (Bam) complex. To determine the role of lipids in the assembly of OMPs under more physiological conditions, we exploited an assay in which the Bam complex mediates their insertion into membrane vesicles. After reconstituting the Bam complex into vesicles that contain a variety of different synthetic lipids, we found that two model OMPs, EspP and OmpA, folded efficiently regardless of the lipid composition. Most notably, both proteins folded into membranes composed of a gel phase lipid that mimics the rigid bacterial OM. Interestingly, we found that EspP, OmpA and another model protein (OmpG) folded at significantly different rates and that an a-helix embedded inside the EspP b-barrel accelerates folding. Our results show that the Bam complex largely overcomes effects that lipids exert on OMP assembly and suggest that specific interactions between the Bam complex and an OMP influence its rate of folding. __________________________ Gram-negative bacteria, a class that includes many pathogenic and emerging antibiotic-resistant organisms, are bound by a double cell membrane. Most of the proteins that reside in the outer membrane (OM) 2 , which serves as a robust barrier, have a distinctive "b-barrel" architecture. A bbarrel is essentially a b-sheet rolled into a closed cylinder that has a hydrophobic exterior and a hydrophilic interior and that is held together by hydrogen bonds between the first and last bstrands. The b-barrel scaffold is found exclusively in the OM of bacteria and organelles of bacterial origin. OM proteins (OMPs) mediate many different functions vital for bacterial survival and range considerably in size from 8-26 b-strands (1, 2). Some OMPs form oligomers (3), while others contain a segment that is embedded inside the bbarrel or a separately folded domain that is displayed on either the periplasmic or extracellular side of the OM (1).OMPs must first traverse the inner membrane (IM) and the periplasmic space, which is devoid of ATP (4), before they attain their final structure in the OM. After OMPs are transported across the IM in an unfolded conformation through the Sec translocon, a variety of periplasmic chaperones including SurA, Skp, and DegP prevent their aggregation in the periplasm (5-7). Integration into the OM is then catalyzed by the heterooligomeric β-barrel assembly machinery (Bam) complex (8,9). In E. coli, the Bam complex is composed of BamA, a b-barrel protein that contains five http://www.jbc.org/cgi

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“…In an alternative model, it has been proposed that the Bam complex facilitates the membrane integration of folded or partially folded β barrels by perturbing the lipid bilayer (Noinaj et al ., 2013 ). This model is supported by evidence that at least some OMPs begin to fold prior to membrane integration (Ieva et al ., 2008 ;Lee et al ., 2016 ;Sikdar et al ., 2017 ), that BamA lowers the kinetic barrier for OMP insertion imposed by lipid head groups (Gessmann et al ., 2014 ), that BamA exerts a greater catalytic effect on OMP folding in thicker bilayers (Schiffrin et al ., 2017 ), and that BamA is highly dynamic in liposomes (Doerner and Sousa, 2017 ). The crystal structure of the Bam complex shows that the POTRA domains and the four lipoproteins form a ring below the BamA β barrel.…”

Section: Introductionmentioning

confidence: 99%

Identification of a novel post‐insertion step in the assembly of a bacterial outer membrane protein

Peterson

Hussain

Bernstein

2018

Molecular Microbiology

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Although the barrel assembly machinery (Bam) complex has been shown to facilitate the insertion of β barrel proteins into the bacterial outer membrane (OM), the stage at which β barrels fold is unknown. Here, we describe insights into β barrel assembly that emerged from an analysis of a member of the autotransporter family of OM proteins (EspP) in Escherichia coli. EspP contains an extracellular 'passenger' domain that is translocated across the OM and then released from the covalently linked β barrel domain in an intra-barrel cleavage reaction. We found that the mutation of an unusual lipid-exposed lysine residue impairs a previously unidentified late folding step that follows both the membrane insertion of the β barrel domain and the secretion of the passenger domain but that precedes proteolytic maturation. Our results demonstrate that β barrel assembly can be completed at a post-insertion stage and raise the possibility that interactions with membrane lipids can promote folding in vivo. Furthermore, by showing that the passenger domain is secreted before the β barrel domain is fully assembled, our results also provide evidence against the long-standing hypothesis that autotransporters are autonomous protein secretion systems.

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Extreme Dynamics in the BamA β-Barrel Seam

Cited by 53 publications

References 43 publications

C-terminal kink formation is required for lateral gating in BamA

C-terminal kink formation is required for lateral gating in BamA

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

Identification of a novel post‐insertion step in the assembly of a bacterial outer membrane protein

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