Dataset Information

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

ABSTRACT: Although the barrel assembly machinery (Bam) complex has been shown to facilitate the insertion of ? barrel proteins into the bacterial outer membrane (OM), the stage at which ? barrels fold is unknown. Here, we describe insights into ? 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 ? 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 ? barrel domain and the secretion of the passenger domain but that precedes proteolytic maturation. Our results demonstrate that ? 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 ? barrel domain is fully assembled, our results also provide evidence against the long-standing hypothesis that autotransporters are autonomous protein secretion systems.

SUBMITTER: Peterson JH

PROVIDER: S-EPMC6195845 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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

Peterson Janine H JH Hussain Sunyia S Bernstein Harris D HD

Molecular microbiology 20180928 1

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 ...[more]

PMID: 30107065

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Project description:The Escherichia coli outer membrane beta-barrel enzyme PagP and its homologues are unique in that the eight-stranded barrel is tilted by about 25 degrees with respect to the membrane normal and is preceded by a 19-residue amphipathic alpha-helix. To investigate the role of this helix in the folding and stability of PagP, mutants were generated in which the helix was deleted (Delta(1-19)), or in which residues predicted to be involved in helix-barrel interactions were altered (W17A or R59L). The ability of the variants to insert into detergent micelles or liposomes was studied in vitro using circular dichroism, fluorescence, Fourier transform infrared spectroscopy, electrophoretic mobility and gain of enzyme activity. The data show that PagP, initially unfolded in 5% (w/v) perfluoro-octanoic acid or 6 M guanidinium chloride, inserts spontaneously and folds quantitatively to an active conformation into detergent micelles of cyclofos-7 or into large vesicles of diC(12:0)-phosphatidylcholine (diC(12:0)PC), respectively, the latter in the presence of 7 M urea. Successful refolding of all variants into both micelles and liposomes ruled out an essential role for the helix or helix-barrel interactions in folding and membrane insertion. Measurements of thermal stability indicated that the variants R59L, W17A/R59L and Delta(1-19) were destabilised substantially compared with wild-type PagP. However, in contrast to the other variants, destabilisation of the W17A variant relative to wild-type PagP was much greater in liposomes than in micelles. Analysis of the kinetics of folding and unfolding of all variants in diC(12:0)PC liposomes suggested that this destabilisation arises predominantly from an increased dissociation of the refolded variant proteins from the lipid-inserted state. The data support the view that the helix of PagP is not required for folding and assembly, but instead acts as a clamp, stabilising membrane-inserted PagP after folding and docking with the membrane are complete.

Dataset Information

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

Publications

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

Similar Datasets

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