Structure and Function of an Essential Component of the Outer Membrane Protein Assembly Machine

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Science (Impact Factor: 33.61). 09/2007; 317(5840):961-4. DOI: 10.1126/science.1143993
Source: PubMed


Integral beta-barrel proteins are found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. The machine that assembles these proteins contains an integral membrane protein, called YaeT in Escherichia coli, which has one or more polypeptide transport-associated (POTRA) domains. The crystal structure of a periplasmic fragment of YaeT reveals the POTRA domain fold and suggests a model for how POTRA domains can bind different peptide sequences, as required for a machine that handles numerous beta-barrel protein precursors. Analysis of POTRA domain deletions shows which are essential and provides a view of the spatial organization of this assembly machine.

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    • "Again the functional relevance is supported by conservation analysis (Figure 5B) that shows high conservation on the interfaces of not only P4 and P5 but of all neighboring POTRA domains. The BamA P5 domain, subsequently, is the critical hub for b-barrel assembly because it has to interact with substrate OMPs as well as the essential lipoprotein BamD (Kim et al., 2007; Malinverni et al., 2006). Our data on the E373 mutants that weaken binding of BamD in vivo (Ricci et al., 2012; Rigel et al., 2013) provide evidence for a role of P5 plasticity in this interaction. "
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    ABSTRACT: BamA is the main component of the β-barrel assembly machinery (BAM) that folds and inserts outer membrane proteins in Gram-negative bacteria. Crystal structures have suggested that this process involves conformational changes in the transmembrane β-barrel of BamA that allow for lateral opening, as well as large overall rearrangements of its periplasmic POTRA domains. Here, we identify local dynamics of the BamA POTRA 5 domain by solution and solid-state nuclear magnetic resonance. The protein region undergoing conformational exchange is highly conserved and contains residues critical for interaction with BamD and correct β-barrel assembly in vivo. We show that mutations known to affect the latter processes influence the conformational equilibrium, suggesting that the plasticity of POTRA 5 is related to its interaction with BamD and possibly to substrate binding. Taken together, a view emerges in which local protein plasticity may be critically involved in the different stages of outer membrane protein folding and insertion. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Structure 05/2015; 23(7). DOI:10.1016/j.str.2015.04.014 · 5.62 Impact Factor
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    • "To answer this, we built two partially diploid strains expressing either BamA WT or BamA- F474L from the chromosome and a His-tagged copy of wild-type BamA (BamA WT-His ) from a low-copynumber plasmid, pZS21 [24] [27]. A null recA allele was introduced to prevent recombination between the wild-type and mutant alleles of bamA. "
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    ABSTRACT: Assembly of the β-barrel outer membrane proteins (OMPs) is an essential cellular process in Gram negative bacteria and in the mitochondria and chloroplasts of eukaryotes-two organelles of bacterial origin. Central to this process is the conserved β-barrel OMP that belongs to the Omp85 superfamily. In Escherichia coli, BamA is the core β-barrel OMP, and together with four outer membrane lipoproteins, BamBCDE, constitute the β-barrel assembly machine (BAM). In this paper, we investigated the roles of BamD, an essential lipoprotein, and BamB in BamA biogenesis. Depletion of BamD caused impairment in BamA biogenesis and cessation of cell growth. These defects of BamD depletion were partly reversed by single amino acid substitutions mapping within the β-barrel domain of BamA. However, in the absence of BamB, the positive effects of the β-barrel substitutions on BamA biogenesis under BamD depletion conditions were nullified. By employing a BamA protein bearing one such substitution, F494L, it was demonstrated that the mutant BamA protein could not only assemble without BamD, but it could also facilitate the assembly of wild-type BamA expressed in trans. Based on these data, we propose a model in which the Bam lipoproteins, which are localized to the outer membrane by the BAM-independent Lol pathway, aid in the creation of new BAM complexes by serving as outer membrane receptors and folding factors for nascent BamA molecules. The newly assembled BAM holocomplex then catalyzes the assembly of substrate OMPs and BamA. These in vivo findings are corroborated by recently published in vitro data.
    Journal of Molecular Biology 05/2014; 427(5). DOI:10.1016/j.jmb.2014.04.021 · 4.33 Impact Factor
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    • "Our results suggest that both domains are rigid with respect to each other. The angle between POTRA 2 and 3 has been under more debate as the two crystal structures of POTRA 1–4 strikingly differ in this aspect, resulting in either a " fish hook " [8] or extended conformation [11]. In the structure of FL N. gonorrhoeae BamA, the POTRA domains however adopt a conformation that is in between these two folds [6]. "
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    ABSTRACT: The outer membrane protein BamA is the key player in β-barrel assembly in Gram-negative bacteria. Despite the availability of high-resolution crystal structures, the dynamic behavior of the transmembrane domain and the large periplasmic extension consisting of five POTRA domains remains unclear. We demonstrate reconstitution of full-length BamA in proteoliposomes at low lipid-to-protein ratio, leading to high sensitivity and resolution in solid-state NMR (ssNMR) experiments. We detect POTRA domains in ssNMR experiments probing rigid protein segments in our preparations. These results suggest that the periplasmic region of BamA is firmly attached to the β-barrel and does not experience fast global motion around the angle between POTRA 2 and 3. We show that this behavior holds at lower protein concentrations and elevated temperatures. Chemical-shift variations observed after reconstitution in lipids with different chain lengths and saturation levels are compatible with conformational plasticity of BamA's transmembrane domain. Electron microscopy of the ssNMR samples shows that BamA can cause local disruptions of the lipid bilayer in proteoliposomes. The observed interplay between protein-protein and protein-lipid interactions may be critical for BamA-mediated insertion of substrates into the outer membrane.
    Journal of Molecular Biology 02/2014; 426(9). DOI:10.1016/j.jmb.2014.02.007 · 4.33 Impact Factor
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