Bam Lipoproteins Assemble BamA <i>in Vitro</i>

Hagan, Christine L.; Westwood, David B; Kahne, Daniel

doi:10.1021/bi400865z

Cited by 67 publications

(96 citation statements)

References 35 publications

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“…However, BamD is an essential protein in bacteria and must therefore play an important role (23,37). We previously demonstrated that BamD interacts with unfolded OMPs (21,38), and here we have shown that interfering with that interaction inhibits β-barrel assembly in vitro and compromises cell growth and OM integrity in vivo. Together, these observations suggest that BamD's essential function is to bind unfolded OMP substrates during the assembly process.…”

Section: Discussionsupporting

confidence: 47%

“…As a first step toward developing an inhibitor of the Bam complex, we assessed whether BamD interacts with substrates in a sequencespecific manner and whether that interaction is important in the folding mechanism. We previously demonstrated that unfolded BamA binds to and can be copurified with soluble BamD-His (i.e., a BamD construct lacking its N-terminal lipid acylation site) in vitro (21). We performed similar copurification experiments with unfolded fragments of BamA to determine which region of the BamA sequence BamD recognizes.…”

Section: Resultsmentioning

confidence: 99%

“…This study thus provides a starting point for the development of new antibiotics that directly target outer membrane biogenesis. Bam complex in proteoliposomes (21,26,27). In these assays, folding of the substrate protein, BamA, is detected by production of a band of lower apparent molecular weight on a seminative SDS/PAGE gel; folded β-barrels do not denature under the conditions of these gel assays and consequently migrate faster than their unfolded forms.…”

Section: Resultsmentioning

confidence: 99%

“…Do the β-barrel assembly complexes in all species use the same mechanism (perhaps with different accessory proteins), or have they evolved different essential components to meet their organism-specific requirements? We have previously demonstrated that BamD binds to two unfolded OMP substrates, OmpA and BamA, and that it directly facilitates the assembly of BamA (21). In this study, we have characterized this interaction to understand its role in the assembly process carried out by the complete Bam complex and to assess whether interfering with the binding of substrates to BamD might be a viable strategy for developing new antibiotics.…”

mentioning

confidence: 98%

“…The POTRA domains bind the other four components of the assembly complex, BamB-E, which are lipoproteins. Structural and biochemical studies have suggested that the POTRA domains, BamB, and BamD may interact with substrate proteins during their assembly (13,15,(19)(20)(21)(22). However, more detailed information is required about the nature of those interactions and whether they are critical in the assembly process to develop an integrated mechanistic model.…”

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confidence: 99%

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Inhibition of the β-barrel assembly machine by a peptide that binds BamD

Hagan

Wzorek

Kahne

2015

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

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107

145

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The protein complex that assembles integral membrane β-barrel proteins in the outer membranes of Gram-negative bacteria is an attractive target in the development of new antibiotics. This complex, the β-barrel assembly machine (Bam), contains two essential proteins, BamA and BamD. We have identified a peptide that inhibits the assembly of β-barrel proteins in vitro by characterizing the interaction of BamD with an unfolded substrate protein. This peptide is a fragment of the substrate protein and contains a conserved amino acid sequence. We have demonstrated that mutations of this sequence in the full-length substrate protein impair the protein's assembly, implying that BamD's interaction with this sequence is an important part of the assembly mechanism. Finally, we have found that in vivo expression of a peptide containing this sequence causes growth defects and sensitizes Escherichia coli to antibiotics to which they are normally resistant. Therefore, inhibiting the binding of substrates to BamD is a viable strategy for developing new antibiotics directed against Gram-negative bacteria.β-barrel | outer membrane | protein folding | Bam complex | inhibition

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Section: Discussionsupporting

confidence: 47%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 98%

mentioning

confidence: 99%

See 3 more Smart Citations

Inhibition of the β-barrel assembly machine by a peptide that binds BamD

Hagan

Wzorek

Kahne

2015

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

Self Cite

107

145

View full text Add to dashboard Cite

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ATP‐independent assembly machinery of bacterial outer membranes: BAM complex structure and function set the stage for next‐generation therapeutics

George,

Patil,

Mahalakshmi

2024

Protein Science

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Diderm bacteria employ β‐barrel outer membrane proteins (OMPs) as their first line of communication with their environment. These OMPs are assembled efficiently in the asymmetric outer membrane by the β‐Barrel Assembly Machinery (BAM). The multi‐subunit BAM complex comprises the transmembrane OMP BamA as its functional subunit, with associated lipoproteins (e.g., BamB/C/D/E/F, RmpM) varying across phyla and performing different regulatory roles. The ability of BAM complex to recognize and fold OM β‐barrels of diverse sizes, and reproducibly execute their membrane insertion, is independent of electrochemical energy. Recent atomic structures, which captured BAM–substrate complexes, show the assembly function of BamA can be tailored, with different substrate types exhibiting different folding mechanisms. Here, we highlight common and unique features of its interactome. We discuss how this conserved protein complex has evolved the ability to effectively achieve the directed assembly of diverse OMPs of wide‐ranging sizes (8–36 β‐stranded monomers). Additionally, we discuss how darobactin—the first natural membrane protein inhibitor of Gram‐negative bacteria identified in over five decades—selectively targets and specifically inhibits BamA. We conclude by deliberating how a detailed deduction of BAM complex—associated regulation of OMP biogenesis and OM remodeling will open avenues for the identification and development of effective next‐generation therapeutics against Gram‐negative pathogens.

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The β-Barrel Assembly Machinery Complex

Leyton

Belousoff

Lithgow

2015

Methods in Molecular Biology

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The outer membranes of gram-negative bacteria contain integral membrane proteins, most of which are of β-barrel structure, and critical for bacterial survival. These β-barrel proteins rely on the β-barrel assembly machinery (BAM) complex for their integration into the outer membrane as folded species. The central and essential subunit of the BAM complex, BamA, is a β-barrel protein conserved in all gram-negative bacteria and also found in eukaryotic organelles derived from bacterial endosymbionts. In Escherichia coli, BamA docks with four peripheral lipoproteins, BamB, BamC, BamD and BamE, partner subunits that add to the function of the BAM complex in outer membrane protein biogenesis. By way of introduction to this volume, we provide an overview of the work that has illuminated the mechanism by which the BAM complex drives β-barrel assembly. The protocols and methodologies associated with these studies as well as the challenges encountered and their elegant solutions are discussed in subsequent chapters.

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Bam Lipoproteins Assemble BamA in Vitro

Cited by 67 publications

References 35 publications

Inhibition of the β-barrel assembly machine by a peptide that binds BamD

Inhibition of the β-barrel assembly machine by a peptide that binds BamD

ATP‐independent assembly machinery of bacterial outer membranes: BAM complex structure and function set the stage for next‐generation therapeutics

The β-Barrel Assembly Machinery Complex

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