Crystal Structure of BamB Bound to a Periplasmic Domain Fragment of BamA, the Central Component of the β-Barrel Assembly Machine

Jansen, Katarina Bartoš; Baker, Susan L.; Sousa, Marcelo C.

doi:10.1074/jbc.m114.584524

Cited by 49 publications

(54 citation statements)

References 34 publications

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“…BamB is known to bind to BamA (37,71), and a crystal structure of a BamB-POTRA3-5 fragment complex (71) in addition to recent crystal structures of the BamA/B/C/ D/E complex (39,40) show that the BamB b-propeller contacts the face of the POTRA3 b-sheet. For BamD, the only essential BAM lipoprotein (29), a crystal structure of a BamD-POTRA4-5 fragment identified the interface structural elements between subunits (33) and this interface has been confirmed in crystal structures of the BAM complex (38)(39)(40).…”

Section: Binding Of Bamb and Bamd Is Compatible With Selected Conformmentioning

confidence: 94%

BamA POTRA Domain Interacts with a Native Lipid Membrane Surface

Fleming

Patel

et al. 2016

Biophysical Journal

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The outer membrane of Gram-negative bacteria is an asymmetric membrane with lipopolysaccharides on the external leaflet and phospholipids on the periplasmic leaflet. This outer membrane contains mainly β-barrel transmembrane proteins and lipidated periplasmic proteins (lipoproteins). The multisubunit protein β-barrel assembly machine (BAM) catalyzes the insertion and folding of the β-barrel proteins into this membrane. In Escherichia coli, the BAM complex consists of five subunits, a core transmembrane β-barrel with a long periplasmic domain (BamA) and four lipoproteins (BamB/C/D/E). The BamA periplasmic domain is composed of five globular subdomains in tandem called POTRA motifs that are key to BAM complex formation and interaction with the substrate β-barrel proteins. The BAM complex is believed to undergo conformational cycling while facilitating insertion of client proteins into the outer membrane. Reports describing variable conformations and dynamics of the periplasmic POTRA domain have been published. Therefore, elucidation of the conformational dynamics of the POTRA domain in full-length BamA is important to understand the function of this molecular complex. Using molecular dynamics simulations, we present evidence that the conformational flexibility of the POTRA domain is modulated by binding to the periplasmic surface of a native lipid membrane. Furthermore, membrane binding of the POTRA domain is compatible with both BamB and BamD binding, suggesting that conformational selection of different POTRA domain conformations may be involved in the mechanism of BAM-facilitated insertion of outer membrane β-barrel proteins.

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Section: Binding Of Bamb and Bamd Is Compatible With Selected Conformmentioning

confidence: 94%

BamA POTRA Domain Interacts with a Native Lipid Membrane Surface

Fleming

Patel

et al. 2016

Biophysical Journal

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“…In addition to BamA, the BAM complex is composed of several lipoproteins (BamB/C/D/E (7-10)). It is described that the third POTRA domain of BamA binds BamB (11) and thus is involved in complex formation. In turn, the first, and thus most N-terminal, POTRA domain is an essential interaction partner of SurA, which has been proposed to deliver b-barrel proteins from the plasma membrane to the BAM complex (12).…”

Section: Introductionmentioning

confidence: 99%

Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy

Dastvan

Brouwer

Schuetz

et al. 2016

Biophysical Journal

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Many proteins of the outer membrane of Gram-negative bacteria and of the outer envelope of the endosymbiotically derived organelles mitochondria and plastids have a b-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal b-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. Based on structural studies of Omp85 proteins, including the five POTRAdomain-containing BamA protein of Escherichia coli, it is predicted that anaP2 and anaP3 bear a fixed orientation, whereas anaP1 and anaP2 are connected via a flexible hinge. We challenged this proposal by investigating the conformational space of the N-terminal POTRA domains of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 using pulsed electron-electron double resonance (PELDOR, or DEER) spectroscopy. The pronounced dipolar oscillations observed for most of the double spinlabeled positions indicate a rather rigid orientation of the POTRA domains in frozen liquid solution. Based on the PELDOR distance data, structure refinement of the POTRA domains was performed taking two different approaches: 1) treating the individual POTRA domains as rigid bodies; and 2) using an all-atom refinement of the structure. Both refinement approaches yielded ensembles of model structures that are more restricted compared to the conformational ensemble obtained by molecular dynamics simulations, with only a slightly different orientation of N-terminal POTRA domains anaP1 and anaP2 compared with the x-ray structure. The results are discussed in the context of the native environment of the POTRA domains in the periplasm.

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“…Indeed, the lateral gate , Noinaj et al, 2013a conformational change simulated for BamA was also suggested for LptD (Dong et al, 2014). The BAM exists as a BamAB and BamACDE protein complex (Sklar et al, 2007a, Malinverni et al, 2006, Hagan et al, 2010, Jansen et al, 2015a. However, LptE differs from BamB in structure, size (BamB: 40KDa, LptE: 20KDa), and location in relation to the central protein component.…”

Section: Growth Characteristics Of Mg1655 Periplasmic Chaperone Mutanmentioning

confidence: 86%

“…BamA is the major protein that catalyses folding of OMPs and can act alone or in complex with either BamB or with BamC, BamD and BamE (Malinverni et al, 2006, Kim et al, 2007, Hagan et al, 2010, Plummer and Fleming, 2015. The Cterminal domain of BamA forms a transmembrane 16-stranded β-barrel core, while the Nterminal region of BamA forms five soluble periplasmic polypeptide-transport-associated domains (POTRA) (Jansen et al, 2015b, Kim et al, 2007, Gatzeva-Topalova et al, 2010, Gatzeva-Topalova et al, 2008. These POTRA domains contribute to BamA stability and directly interact with BamBD within the periplasm, nascent polypeptides and the periplasmic chaperone SurA (Bennion et al, 2010, Kim et al, 2007, Workman et al, 2012, Dong et al, 2012a.…”

Section: The β-Barrel Assembly Machine (Bam) Complexmentioning

confidence: 99%

“…BamE and BamC are thought to stabilise the interaction between BamA and BamD, and to modulate the functional activity of BamA and BamD, respectively (Kim et al, 2011a, Rigel et al, 2012. BamB is known to form a propeller like structure within the periplasm and is linked together with BamA via the third POTRA domain (Jansen et al, 2015a, Dong et al, 2012b. The junction between the second and third POTRA domains of BamA is flexible, creating a hinge like structure (Noinaj et al, 2013a, Gatzeva-Topalova et al, 2010, Gatzeva-Topalova et al, 2008.…”

Section: Growth Characteristics Of Mg1655 Periplasmic Chaperone Mutanmentioning

confidence: 99%

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Autotransporter proteins of Uropathogenic Escherichia coli: regulation, characterisation and the role of periplasmic proteins in their expression

Nichols¹

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Uropathogenic Escherichia coli (UPEC) are the primary cause for all urinary tract infections.Autotransporter (AT) proteins are virulence factors secreted by the type V secretion pathway. This project examined the prevalence, regulation and expression of the vacuolating AT toxin (Vat), and the role of periplasmic chaperone proteins in AT translocation.The vacuolating autotransporter (AT) toxin (Vat) contributes to Uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Vat is a serine protease AT of Enterobacteriaceae (SPATE) with cytotoxic activity. Here we characterised Vat and investigated its regulation in UPEC.We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed it that was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, 73 and 95) and these strains secreted the Vat protein.Further analysis of the vat genetic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator, which we termed vatX. The vatvatX genes were present in the UPEC reference strain CFT073 and RT-PCR revealed both genes are co-transcribed. Over-expression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator H-NS; thus the hns gene was mutated in CFT073 (to generate CFT073hns).Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073hns compared to wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients iv Declaration by authorThis thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

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Crystal Structure of BamB Bound to a Periplasmic Domain Fragment of BamA, the Central Component of the β-Barrel Assembly Machine

Cited by 49 publications

References 34 publications

BamA POTRA Domain Interacts with a Native Lipid Membrane Surface

BamA POTRA Domain Interacts with a Native Lipid Membrane Surface

Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy

Autotransporter proteins of Uropathogenic Escherichia coli: regulation, characterisation and the role of periplasmic proteins in their expression

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