Fold and function of polypeptide transport‐associated domains responsible for delivering unfolded proteins to membranes

Knowles, Timothy J.; Jeeves, Mark; Bobat, Saeeda; Dancea, Felician; McClelland, Darren; Palmer, Tracy; Overduin, Michael; Henderson, Ian R.

doi:10.1111/j.1365-2958.2008.06225.x

Cited by 143 publications

(188 citation statements)

References 42 publications

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“…However, this conclusion does not imply that each of the >50 different OMPs present in the OM specifically requires BamD for its assembly (39). Other components of the Bam complex also interact with substrates (13,15,(19)(20)(21)(22)38) and may have functions that overlap with that of BamD such that certain OMPs can be assembled in its absence. Rather, we suggest that BamD's essentiality in substrate binding reflects a kinetic effect on In the in vitro experiments, full-length BamA substrates containing mutations in the β-signal sequence were denatured and then diluted into solutions containing 0.5% lauryldimethylamine-N-oxide (LDAO) or BamABCDE proteoliposomes to assess, respectively, the effects of the mutations on the stability of the folded β-barrel and on the folding mechanism carried out by the Bam complex.…”

Section: Discussionmentioning

confidence: 94%

“…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.

112

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: Discussionmentioning

confidence: 94%

mentioning

confidence: 99%

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

Hagan

Wzorek

Kahne

2015

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

112

145

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“…YaeT (also known as Omp85) is an essential OMP with a large periplasmic domain containing 5 polypeptide-transport-associated (POTRA) domains (32). Recent data suggest that these POTRA domains recognize unfolded OMPs and may serve to nucleate the formation of ␤-strands in OMPs before their membrane insertion (33)(34)(35). Importantly, there is no ATP in the periplasm that could be used to power the unfolding of proteins before their insertion in the membrane.…”

Section: Skp Prevents the Aggregation Of Ompa By Forming A Stable Commentioning

confidence: 99%

The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains

Walton

Sandoval

Fowler

et al. 2009

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

114

116

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Outer membrane proteins (OMPs) of Gram-negative bacteria are synthesized in the cytosol and must cross the periplasm before insertion into the outer membrane. The 17-kDa protein (Skp) is a periplasmic chaperone that assists the folding and insertion of many OMPs, including OmpA, a model OMP with a membrane embedded ␤-barrel domain and a periplasmic ␣␤ domain. Structurally, Skp belongs to a family of cavity-containing chaperones that bind their substrates in the cavity, protecting them from aggregation. However, some substrates, such as OmpA, exceed the capacity of the chaperone cavity, posing a mechanistic challenge. Here, we provide direct NMR evidence that, while bound to Skp, the ␤-barrel domain of OmpA is maintained in an unfolded state, whereas the periplasmic domain is folded in its native conformation. Complementary cross-linking and NMR relaxation experiments show that the OmpA ␤-barrel is bound deep within the Skp cavity, whereas the folded periplasmic domain protrudes outside of the cavity where it tumbles independently from the rest of the complex. This domain-based chaperoning mechanism allows the transport of ␤-barrels across the periplasm in an unfolded state, which may be important for efficient insertion into the outer membrane.cavity-based ͉ outer membrane M any molecular chaperones have evolved cavity-like structures to bind their non-native substrates. Classic examples, such as the chaperonins, bind the substrate in the cavity formed by their open rings and protect them from aggregation. Cycles of ATP binding and hydrolysis, coupled with substrate binding and release, then help the substrate achieve its native conformation (1). Because the cavities of these chaperones have limited capacities, binding substrates larger than the cavity requires a portion of the substrate to be bound inside the cavity while the rest of the substrate remains outside. In these cases, however, the entire substrate remains unfolded while bound to the open ring of the chaperone (2-4).The 17-kDa protein (Skp) is a periplasmic chaperone present in many Gram-negative bacteria involved in the folding and insertion of proteins in the outer membrane (5-7). The crystal structure of Skp revealed a trimer with a ''jellyfish'' architecture where a central cavity is formed by long tentacle-like helical protrusions emanating from a body domain (Fig. 1A) (8, 9). The Skp structure was unexpectedly similar to that of prefoldin, a cytosolic molecular chaperone present in archea and eukarya (8-10). Although Skp is a trimer (8, 9, 11) and prefoldin is a hexamer (12, 13), both proteins share jellyfish architectures with a central cavity ( Fig. 1 A and B). In prefoldin, this cavity has been shown to bind substrate proteins (10, 13). Prefoldin thus belongs to a family of chaperones sometimes referred to as ''holdases.'' These chaperones are ATP independent and, in contrast to chaperonins, do not directly facilitate folding of their substrates. Instead they protect the substrates from aggregation by holding them in their cavities, and subs...

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“…A conformational change similar to that in BamA x-ray structures was proposed for full-length FhaC by electron paramagnetic resonance (EPR) spectroscopy (32). Analysis of the orientation and conformational flexibility of the POTRA domains of BamA by solution and solid-state NMR, EPR, or small-angle x-ray scattering measurements revealed a rigid connection between ecP1 and ecP2 with a reorientation at timescales not detectable by these experimental methods, especially in lipid bilayers (13,31,33,34). Similarly, ecP4-ecP5 has a stable/rigid conformation in lipid bilayers as well as in detergent (31,35,36), which is similar to the x-ray structure of those domains.…”

Section: Introductionmentioning

confidence: 76%

“…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). In line with this, NMR titration experiments revealed that ecP1 (for POTRA 1 of the E. coli protein BamA) and ecP2 interact with peptides comprising transmembrane b-strands of PhoE, which itself is a substrate of the BAM complex (13).…”

Section: Introductionmentioning

confidence: 82%

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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Fold and function of polypeptide transport‐associated domains responsible for delivering unfolded proteins to membranes

Cited by 143 publications

References 42 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

The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains

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

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