Structural basis for the interaction of BamB with the POTRA3–4 domains of BamA

2016 ◽  
Vol 72 (2) ◽  
pp. 236-244 ◽  
Author(s):  
Zhen Chen ◽  
Li-Hong Zhan ◽  
Hai-Feng Hou ◽  
Zeng-Qiang Gao ◽  
Jian-Hua Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Outer Membrane ◽  
Essential Role ◽  
Biochemical Analysis ◽  
Hydrophobic Interactions ◽  
Outer Membrane Proteins ◽  
Structural Basis ◽  
Conserved Residues ◽  
Bam Complex

InEscherichia coli, the Omp85 protein BamA and four lipoproteins (BamBCDE) constitute the BAM complex, which is essential for the assembly and insertion of outer membrane proteins into the outer membrane. Here, the crystal structure of BamB in complex with the POTRA3–4 domains of BamA is reported at 2.1 Å resolution. Based on this structure, the POTRA3 domain is associated with BamBviahydrogen-bonding and hydrophobic interactions. Structural and biochemical analysis revealed that the conserved residues Arg77, Glu127, Glu150, Ser167, Leu192, Leu194 and Arg195 of BamB play an essential role in interaction with the POTRA3 domain.

scholarly journals Classifying β-Barrel Assembly Substrates by Manipulating Essential Bam Complex Members

2016 ◽  
Vol 198 (14) ◽  
pp. 1984-1992 ◽  
Author(s):  
Tara F. Mahoney ◽  
Dante P. Ricci ◽  
Thomas J. Silhavy
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Mechanistic Model ◽  
Outer Membrane Proteins ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Bam Complex ◽  
Periplasmic Chaperones

ABSTRACTThe biogenesis of the outer membrane (OM) ofEscherichia coliis a conserved and vital process. The assembly of integral β-barrel outer membrane proteins (OMPs), which represent a major component of the OM, depends on periplasmic chaperones and the heteropentameric β-barrel assembly machine (Bam complex) in the OM. However, not all OMPs are affected by null mutations in the same chaperones or nonessential Bam complex members, suggesting there are categories of substrates with divergent requirements for efficient assembly. We have previously demonstrated two classes of substrates, one comprising large, low-abundance, and difficult-to-assemble substrates that are heavily dependent on SurA and also Skp and FkpA, and the other comprising relatively simple and abundant substrates that are not as dependent on SurA but are strongly dependent on BamB for assembly. Here, we describe novel mutations inbamDthat lower levels of BamD 10-fold and >25-fold without altering the sequence of the mature protein. We utilized these mutations, as well as a previously characterized mutation that lowers wild-type BamA levels, to reveal a third class of substrates. These mutations preferentially cause a marked decrease in the levels of multimeric proteins. This susceptibility of multimers to lowered quantities of Bam machines in the cell may indicate that multiple Bam complexes are needed to efficiently assemble multimeric proteins into the OM.IMPORTANCEThe outer membrane (OM) of Gram-negative bacteria, such asEscherichia coli, serves as a selective permeability barrier that prevents the uptake of toxic molecules and antibiotics. Integral β-barrel proteins (OMPs) are assembled by the β-barrel assembly machine (Bam), components of which are conserved in mitochondria, chloroplasts, and all Gram-negative bacteria, including many clinically relevant pathogenic species. Bam is essential for OM biogenesis and accommodates a diverse array of client proteins; however, a mechanistic model that accounts for the selectivity and broad substrate range of Bam is lacking. Here, we show that the assembly of multimeric OMPs is more strongly affected than that of monomeric OMPs when essential Bam complex components are limiting, suggesting that multiple Bam complexes are needed to assemble multimeric proteins.

Investigation of the role of the BAM complex and SurA chaperone in outer-membrane protein biogenesis and type III secretion system expression in Salmonella

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1613-1622 ◽  
Author(s):  
Yann Fardini ◽  
Jérôme Trotereau ◽  
Elisabeth Bottreau ◽  
Charlène Souchard ◽  
Philippe Velge ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Type Iii Secretion ◽  
Outer Membrane Proteins ◽  
Transcriptional Analysis ◽  
Secretion Systems ◽  
Type Iii ◽  
Bam Complex ◽  
Periplasmic Chaperones

In Escherichia coli, the assembly of outer-membrane proteins (OMP) requires the BAM complex and periplasmic chaperones, such as SurA or DegP. Previous work has suggested a potential link between OMP assembly and expression of the genes encoding type-III secretion systems. In order to test this hypothesis, we studied the role of the different lipoproteins of the BAM complex (i.e. BamB, BamC, BamD and BamE), and the periplasmic chaperones SurA and DegP, in these two phenotypes in Salmonella. Analysis of the corresponding deletion mutants showed that, as previously described with the ΔbamB mutant, BamD, SurA and, to a lesser extent, BamE play a role in outer-membrane biogenesis in Salmonella Enteritidis, while the membrane was not notably disturbed in ΔbamC and ΔdegP mutants. Interestingly, we found that BamD is not essential in Salmonella, unlike its homologues in Escherichia coli and Neisseria gonorrhoeae. In contrast, BamD was the only protein required for full expression of T3SS-1 and flagella, as demonstrated by transcriptional analysis of the genes involved in the biosynthesis of these T3SSs. In line with this finding, bamD mutants showed a reduced secretion of effector proteins by these T3SSs, and a reduced ability to invade HT-29 cells. As ΔsurA and ΔbamE mutants had lower levels of OMPs in their outer membrane, but showed no alteration in T3SS-1 and flagella expression, these results demonstrate the absence of a systematic link between an OMP assembly defect and the downregulation of T3SSs in Salmonella; therefore, this link appears to be related to a more specific mechanism that involves at least BamB and BamD.

scholarly journals The role of BamA in the biogenesis of beta-barrel membrane proteins

2014 ◽  
Vol 70 (a1) ◽  
pp. C578-C578
Author(s):  
Nicholas Noinaj ◽  
Adam Kuszak ◽  
Curtis Balusek ◽  
JC Gumbart ◽  
Petra Lukacik ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Crystal Structures ◽  
Outer Membrane Proteins ◽  
Hydrophobic Surface ◽  
Md Simulations ◽  
Structural Features ◽  
Bam Complex ◽  
Beta Barrel

Beta-barrel membrane proteins are essential for nutrient import, signaling, motility, and survival. In Gram-negative bacteria, the beta-barrel assembly machinery (BAM) complex is responsible for the biogenesis of beta-barrel outer membrane proteins (OMPs), with homologous complexes found in mitochondria and chloroplasts. Despite their essential roles, exactly how these OMPs are formed remains unknown. The BAM complex consists of a central and essential component called BamA (an OMP itself) and four lipoproteins called BamB-E. While the structure of the lipoproteins have been reported, the structure of full length BamA has been elusive. Recently though, we described the structure of BamA from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane beta-barrel domain. Together, our crystal structures and molecule dynamics (MD) simulations revealed several structural features which gave clues to the mechanism by which BamA catalyzes beta-barrel assembly. The first is that the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the beta-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. Third, the beta-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane. And fourth, a surface exposed exit pore positioned above the lateral opening site which may play a role in the biogenesis of extracellular loops. In this presentation, the crystal structures and MD simulations of BamA will be presented along with our work looking at the role of these four structural features in the role of BamA within the BAM complex.

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