How does a β-barrel integral membrane protein insert into the membrane?

Integral membrane proteins are categorized into two major groups: transmembrane β-helical bundles and β-barrels. β-Barrel membrane proteins are usually located in the outer membranes of Gram-negative bacteria, as well as mitochondria and chloroplasts of eukaryotic cells. The mechanism of membrane insertion of β-barrel membrane proteins is not well understood compared with that of β-helical membrane proteins. Recently reported crystal structures of the β-barrel assembly machinery (Bam) complex from Escherichia coli shed new light on the mechanism of folding and membrane insertion of β-barrel membrane proteins. Expanding upon existing models, Zhang and his colleagues propose and discuss a general mechanism in which the Bam complex provides a chaperone that is specific for the unfolded nascent peptide, a template for the initiation of barrel building, and a channel for the hydrophilic, extracellular loops of the β-barrel to move across the membrane. —Chih-chen Wang* Integral membrane proteins (MPs) are categorized into two major groups: transmembrane (TM) α-helical bundles and β-barrels (Cymer et al., 2014). Both types of structures permit the backbones of their peptides to fulfill their hydrogen-bonding potential in a lipid-bilayer environment. The thermal stability of a typical β-barrel MP is usually much higher than that of a helical MP. Thus, β-barrel MPs are able to withstand near boiling-temperatures during purification (Han et al., 2016), compared with the typical melting temperatures of 50°C or lower for α-helical TM proteins. In fact, unfolding a β-barrel MP in a single-molecule assay typically requires multiple steps, each of which requires a force of 100–300 pN (Thoma et al., 2015). Considering the thickness of the membrane as well as the multiple steps, unfolding one mole of β-barrel MP molecules would require ~103 kJ of energy (equivalent to 400 RT or energy from hydrolyzing 20–30 moles of ATP). Such high thermal stability is considered to be the sole source of the energy that drives the folding of β-barrel MPs (Fleming, 2015). Nearly all α-helical MPs are located in the plasma membrane or its equivalents (e.g., the inner membrane of Gram-negative bacteria and the endoplasmic reticulum membrane of eukaryotic cells). The mechanism of membrane insertion of α-helical MPs is much better understood (Cymer et al., 2014), at least conceptually, than that of β-barrel MPs. Hydrophobic TM helices, usually one pair at a time, insert co-translationally into the membrane, with assistance from translocon machineries (e.g., the Sec, YidC, and Tat complexes) (Gogala et al., 2014; Kumazaki et al., 2014; Widdick et al., 2006). In this case, the translocon provides a TM hydrophilic slot/channel to overcome the kinetic energy barrier of the hydrophobic lipid bilayer, thereby facilitating the movement of the hydrophilic, exo-membrane loops of the helix-hairpins across the membrane. The driving force of the membrane insertion comes from the favorable hydrophobic interaction between the TM helices and the lipid bilayer. The orientations of the TM helices are guided by the positive-inside rule (von Heijne, 1992), which dictates that the more positively-charged ends of TM helices remain on the intracellular side of the membrane, which carries a negative-inside electrostatic potential. For Gram-negative bacteria, only the inner membrane carries a potential, and the co-translational folding system ensures that most (if not all) α-helical MPs reside in the inner membrane. Therefore, to perform its biological functions, the outer membrane (OM) has to exploit a fundamentally different type of integral MP, namely β-barrel MPs (referred to hereafter as OMPs), as well as a different folding mechanism, for OMPs to avoid being stuck in the inner membrane. In addition to their different membrane location compared with α-helical MPs, OMPs must overcome additional energy barriers during folding (Fleming, 2015). After translocation into the periplasmic space, nascent OMPs must remain unfolded and pass through the aqueous periplasm. Like the exo-membrane loop in a helix-hairpin, the extracellular loops (ECLs) of a β-barrel must overcome the energy barrier of the hydrophobic OM during membrane insertion. It has been shown that a thinned membrane allows faster folding and assembly of OMPs (Burgess et al., 2008; Gessmann et al., 2014). In addition, the β-strands of a nascent OMP need to insert sequentially into the OM to avoid misfolding. For most known structures of OMPs, the β-barrels usually possess an even number of β-strands (Fairman et al., 2011), with both the amino (N)- and carboxyl (C)-termini residing in the periplasmic space (Rollauer et al., 2015). The number of β-strands of known β-barrel structures varies from 8 to 36. For example, OmpA (Pautsch and Schulz, 1998), FhaC (Clantin et al., 2007), FhuA (Ferguson et al., 1998), LptD (Qiao et al., 2014), and CsgG (Cao et al., 2014) contain eight, 16, 22, 26, and 36 (i.e., 4 × 9) strands, respectively (Note that the voltage-dependent anion channel of the mitochondrial OM is known to have 19 β-strands (PDB ID: 3EMN) (Ujwal et al., 2008).). In addition, extracellular loops are usually longer than periplasmic loops. Furthermore, the outside surface of a β-barrel is hydrophobic, while the hydrophobicity and charge distribution inside the cavities of β-barrels vary from protein to protein. Thus, each strand of a β-barrel often shows a pattern of alternating hydrophobic and hydrophilic residues. The hydrophobic residues will eventually form the surface of the β-barrel that faces the lipid bilayer. Assembly of β-barrel integral MPs into the target membrane is catalyzed by insertases of the Omp85 superfamily. In Escherichia coli, the β-barrel assembly machinery (Bam) complex is an Omp85 superfamily member and contains five subunits, BamA–E (Gu et al., 2016; Han et al., 2016; Noinaj et al., 2015). While BamA itself is a 16-strand β-barrel protein (Noinaj et al., 2013), all other accessary proteins, BamB–E, are lipoproteins, each attaching to the inner leaflet of the OM via an N-terminal, triple-acylated moiety. BamA and BamD are essential parts of this complex, but other components are required for maximum OMP folding activity (Malinverni et al., 2006; Sklar et al., 2007a; Wu et al., 2005). The BamA β-barrel is partially closed from the extracellular side by a large capping dome consisting of extended inter-strand loops (mainly from the β11-β12 connection loop, ECL6), presumably to prevent membrane leakage. The cavity of the BamA β-barrel appears to be too small to house a fully folded OMP substrate, yet large enough to accommodate a couple of substrate β-hairpins (Noinaj et al., 2015). While most of the β-strands of BamA resemble those of a typical β-barrel, its C-terminal strand, β16, is short and kinks at its C-terminus. These structural features are evolutionarily conserved in the Omp85 superfamily. The β16 strand interacts only loosely with the β1 strand in the middle of the lipid bilayer. The corresponding region between the β1 and β16 strands is termed a portal or lateral opening (Fig. 1). In fact, this portal is the only transmembrane gap in the wall of the BamA β-barrel that allows the substrate peptide to exit from the cavity and enter the OM while avoiding topologic crossing-over between peptides of the substrate and the BamA β-barrel. Thus, the portal is proposed to be the exit through which the substrate β-strands are released. Meanwhile, an opening on the extracellular cap of the cavity (termed the exit pore) likely serves as the exit for ECLs of the substrate OMP (Noinaj et al., 2013). Schematic of the BamA structure. The BamA β-barrel is represented by a green cylinder. The edges of the β-sheets of the five POTRA domains (P1–P5) are represented by blue arrows. The nascent OMP is orange, and its β-strands are shown as arrows. The N-terminal strand is labeled “1”, and so on BamA contains five periplasmic domains that are N-terminal to its β1 strand, and they are referred to as polypeptide transport-associated (POTRA) domains 1–5 (numbered sequentially from the N-terminus). Each of these POTRA domains contains a three-stranded β-sheet and two α-helices (with an order of β1-α1-α2-β2-β3) (Noinaj et al., 2013). Truncation experiments showed that only the last POTRA domain, POTRA-5, is essential for BamA function (Bos et al., 2007; Gessmann et al., 2014). POTRA1–4 are consistently missing in the mitochondrial Omp85 homolog SAM50 (Bohnert et al., 2015), while another Omp85 protein in E. coli, TamA, contains three POTRA domains (PDB ID: 4C00) (Gruss et al., 2013). In the recently published crystal structure of the BamA–E complex, POTRA1–5 form a right-handed spiral structure that is stabilized by the accessory proteins BamB–E (PDB ID: 5AYW) (Han et al., 2016). Together, POTRA2–5 and BamB–D form a ring near the periplasm-OM interface, with POTRA-1 located below the ring (Fig. 1). The periplasmic ring is ~40 Å thick and contains a chamber that is ~40 Å in diameter. Furthermore, near the periplasm-OM interface and the β1-β16 portal, there is an ~16 × 42 Å surface hole that connects the chamber of the periplasmic ring to the outside of the complex. This surface hole was proposed by the authors to be the exit for the OMP substrate (Han et al., 2016). Consistent with their essential roles in Bam functions, POTRA-5 and BamD have been observed to directly interact with each other and to participate in the formation of the surface hole. Interestingly, in the spiral structure of the five POTRA domains, all β-sheets are exposed to the solvent or the interior surface the chamber (Fig. 1). Furthermore, the β-sheet edges (i.e., the β2 strands) from the five POTRA domains form a nearly connected spiral track. This track runs from the bottom of the periplasmic ring all the way to the β1 strand of the BamA β-barrel. The distances between consecutive β-sheet edges (midpoint-to-midpoint) range from 22 Å to 36 Å in E. coli BamA. None of these edge-strands contains proline residues, which might potentially block β-sheet extension. The accessory proteins BamB–E probably function to stabilize this POTRA track. For instance, BamD stabilizes the connection between POTRA-5 and the β-barrel, as well as the POTRA1–2 connection. In addition, BamB stabilizes the POTRA2–3 connection, and BamE, together with BamD, stabilizes the POTRA4–5 connection. A similar, yet more flexible, arrangement of POTRA domains relative to the β-barrel has also been observed in TamA (PDB ID: 4C00), which has three POTRA domains (Gruss et al., 2013), and FhaC (PDB ID: 2QDZ), which has two POTRA domains (Clantin et al., 2007). In contrast to the complete complex, the spatial arrangement of the POTRA domains relative to the β-barrel is drastically different in the absence of the accessory subunits BamB–E (Noinaj et al., 2013). For instance, POTRA-5 blocks the periplasmic entry of the β-barrel in isolated BamA. Therefore, we believe that the newly published complete BamA–E complex is more informative in terms of understanding the β-barrel assembly mechanism. Intriguingly, in the crystal structure of E. coli BamA POTRA1–5 (PDB ID: 2QCZ), the β2 strand of POTRA-3 of one molecule interacts with a short peptide from POTRA-5 of a symmetry-related neighboring molecule through parallel β-strand hydrogen bonding (Kim et al., 2007). Similarly, in the crystal structure of TamA (PDB ID: 4BZA), POTRA-3 (corresponding to POTRA-5 of BamA) was found to interact in a parallel fashion with the β-sheet of POTRA-2 from another molecule via their β2 strands (Gruss et al., 2013). These structural observations suggest that the POTRA domains may bind the unfolded peptide substrate through β-sheet expansion (also called β-augmentation). It is probable that the POTRA track in the BamA–E complex provides a loading path for the unfolded peptide substrate (Bergal et al., 2016; Knowles et al., 2008), and the chamber of the periplasmic ring provides a path for ECLs to move from the periplasmic space into the cavity of the BamA β-barrel. Interestingly, the proposed binding modes between the nascent peptide and the β2 strands from the POTRA domains in the periplasmic ring are also in a parallel fashion (Fig. 1). This hypothesis can be tested by introducing proline residues at the β-sheet edges (i.e., the β2 stands) of POTRA domains. Each point mutation of proline-substitution would eliminate two potential hydrogen bonds for β-augmentation. A triple proline-mutation in the β2 strand of POTRA-5 of E. coli BamA consistently impaired cell growth (Gu et al., 2016). However, the mutation positions in this variant are in a registration that disrupts the three-strand β-sheet rather than directly blocks β-augmentation. In addition, for a BamA-like protein with multiple POTRA domains, a mutation in one or two β2 strands may not always be sufficient to have a noticeable effect on β-barrel assembly. In short, we propose that POTRA domains function essentially as a chaperone for the unfolded peptides of β-barrel OMPs. To move across the aqueous environment of the periplasmic space that separates the inner and outer membranes, unfolded peptides of OMPs are escorted by chaperones to ensure that they remain assembly-competent (Thoma et al., 2015). Among the major chaperones in the periplasm, the 45-kDa SurA (survival protein A) has been shown to be directly involved in Bam-mediated OMP assembly (Behrens et al., 2001; Rollauer et al., 2015). A physical interaction between SurA and the N-terminal POTRA-1 of BamA has been reported (Bennion et al., 2010). In particular, Arg64 of POTRA-1, which faces the chamber entrance of the periplasmic ring of BamA, interacts with SurA. In fact, SurA is the only periplasmic chaperone that can be chemically cross-linked with BamA in vivo (Sklar et al., 2007b); in contrast, evidence of direct binding between SurA and other accessory Bam proteins (e.g., BamD) is lacking. SurA was found to bind specifically to peptides containing an Ar-X-Ar sequence motif (where Ar stands for an aromatic residue and X stands for any residue) (Goemans et al., 2014), which is common in β-barrel OMPs. The crystal structure of SurA contains four domains: a substantial N-terminal domain, two peptidylprolyl isomerase (PPIase) domains, and a C-terminal tail. Among them, the PPIase-1 domain was shown to bind the Ar-X-Ar motif of an unfolded peptide of OMP (Bitto and McKay, 2002; Xu et al., 2007). However, both PPIase domains have been shown to be dispensable for in vivo chaperone activity (Behrens et al., 2001). Intriguingly, a β-hairpin from the N-terminal domain forms a three-stranded β-sheet with the very C-terminal end of SurA, and formation of this β-sheet is essential for the chaperone activity of SurA (Chai et al., 2014). In particular, the length and β-strand propensity of the C-terminal peptide, but not its detailed amino acid sequence, are important for the chaperone activity. Furthermore, SurA has been shown to bind to β-hairpins, which are considered to be building blocks of β-barrels (Thoma et al., 2015). These observations suggest that the chaperone function of SurA requires β-augmentation with its substrate, rather than recognition of a specific sequence. Multiple SurA proteins may bind to potential β-strands from the unfolded peptide of a nascent OMP in a pearl-necklace fashion. SurA proteins are probably released sequentially from the substrate peptide when the latter is transferred to the downstream chaperone, the POTRA track. In this scenario, multiple spatially organized POTRAs may compete effectively with individual SurA for substrate binding. Hypothetically, the peptide of a β-barrel contains two types of β-hairpins: those consisting of odd-even strands and those consisting of even-odd strands. The difference between the two types lies in their hairpin loops. For an odd-even hairpin, the two β-strands connect to an ECL, which is usually longer than the periplasmic loop. Thus, it is possible that with a β-augmentation mechanism, SurA binds to only one type (e.g., odd-even) of hairpin in a specific orientation, based on the properties of the substrate, such as its connection loop and hydrophobic surface (e.g., the Ar-X-Ar motif), as well as the peptide direction. Moreover, the assembly of a few OMPs, e.g., TolC and BamA, has been shown to be SurA-independent (Bennion et al., 2010). These OMPs usually contain large, auto-folded, periplasmic domains, which are located at their N-termini, as shown in BamA (Han et al., 2016), or attached to β-hairpins that are to be inserted into the OM, as exemplified by TolC (Koronakis et al., 2000). These soluble periplasmic domains may help to prevent aggregation of the unfolded peptide, in a manner that is similar to those of well-folded soluble proteins (e.g., maltose-binding protein), which help fused peptides to stay soluble during protein et al., 2013). The soluble periplasmic domains may also contain to a β-barrel directly to the complex help from SurA. two of Bam-mediated OMP assembly have been proposed based on structures of Bam the and (Rollauer et al., 2015). one the that BamA a thinned lipid region near the portal, which the peptide of the OMP substrate to overcome the kinetic energy barrier during membrane insertion. the β1-β16 has hydrogen than other inter-strand in the BamA β-barrel. A that BamA disrupts the lipid near the β1-β16 portal region (Noinaj et al., 2013). the other the (Gruss et al., that the BamA β-barrel its exposed in the β1 strand, as a template for the substrate peptide to barrel The OMP peptide would to into the β-barrel the barrel eventually from BamA and is released into the this there is a the is also called a β-augmentation mechanism (Noinaj et al., 2015). Consistent with this mechanism, it has been that a lateral opening of the β-barrel at the portal is essential for BamA function (Noinaj et al., 2014). the and are not and both are likely to roles in OMP assembly. Interestingly, all known structures of β-barrel OMPs have a when from the extracellular space, the pattern as that of the BamA β-barrel (Fig. on the the observed of β-barrels is as a general of OMPs, the of the OMP substrate barrel building by binding to the β16 of the BamA β-barrel, or the of the OMP substrate binds to the other the gap in the of the complex must be with the β1 or β16 strand of the BamA β-barrel. that while the complex its as the the exposed of the barrel is likely to remain in a so that it is for substrate Such a mechanism may be important for the assembly of a β-barrel from multiple of the For example, CsgG is an channel that subunits for crystal structure shows of the which forms a β-barrel, the one known to (Cao et al., 2014). In this case, there is force between the peptides of neighboring Therefore, the complex appears to have to when loading each new of the and it the Schematic of the mechanism of β-barrel assembly. Each β-barrel is by a of and is from the extracellular Each β-strand is represented by a and those β-strands point to the extracellular space (i.e., strands) are with an additional The strands of BamA are light to green from the to POTRA-5 is N-terminal to the β1 strand, and it serves as the for β-hairpin The strands of the newly substrate β-barrel are to from the to The an extracellular loop of the β-barrel The of the β-barrel peptide, as from the extracellular space, is It is to that the function of the β1 strand, as well as POTRA-5, is to an unfolded peptide to the This may also to the essential of the BamD which stabilizes the relative between the BamA β-barrel and In contrast, on the β16 side of the portal, there is for substrate In addition, of a of interaction with the periplasmic ring (Han et al., 2016), the C-terminal β16 strand is more likely to be than the β1 Therefore, we that the opening of the located near the β1 strand, while strand β16 provides a template from which the and this would require the folding of an OMP β-barrel to from its by attaching to strand β16 of BamA. of the of such a is that the folding may be peptide and translocation are This is by an from a in which the OM a large of nascent proteins et al., However, based on that the of an OMP may contain a that is important for assembly et al., 2006; et al., it is that the of the substrate peptide the binding with BamA. there is structural evidence for such a C-terminal In contrast, it was observed that of the β-strand or in the last β-strand in substrate of the Bam the assembly et al., et al., In addition, the C-terminal not prevent membrane insertion of the substrate, but to the folding of In contrast to the common N-terminal initiation that building of the β-barrel of an OMP substrate at its while the C-terminal may be for the of barrel A to the of β-barrel expansion in a to be the substrate β-strands from SurA to the POTRA track of BamA, the nascent peptide the chamber of the periplasmic ring and the β1 by both hydrophobic with the lipid and with the of the the peptide at the β1 strand and the POTRA-5 region form a which from the β1 strand and into the cavity of the BamA β-barrel, bonds with its N-terminal neighboring (i.e., that the β16 strand serves as a template during the very of the assembly The is one hairpin at a time, with the hydrophobic of the hairpin the lipid and the hydrophilic through the partially hydrophilic cavity of the the extracellular side of the Meanwhile, more of the unfolded peptide is into the periplasmic chamber the POTRA track. In we propose that the Bam complex a spiral POTRA track that as a chaperone for binding the unfolded peptide of the OMP a template (i.e., the β16 for initiation of β-barrel and a TM hydrophilic channel for the of each β-hairpin building Such a mechanism would be similar to that of the insertion of α-helical TM proteins by the YidC, in which TM helix-hairpins function as the of membrane insertion (Cymer et al., 2014). for and for the of the BamA–E complex it to the This was by the the of and the of Zhang and Han that they have of This not contain any with by any of the