BamA is required for autotransporter secretion
Introduction
Many, if not all, pathogenic Gram-negative and Gram-positive bacteria use surface-localized virulence factors to infect and/or undermine the host immune system. One of the ways that Gram-negative bacteria export these virulence factors is via the type V secretion system, also known as autotransporters [1]. Two sub-types, type Va and Vc, are composed of an N-terminal passenger domain (which usually contains the virulence factor [2]) and a C-terminal β-barrel translocon [[3], [4], [5], [6], [7], [8]]. Some autotransporters contain an α-helical linker which connects the β-barrel to its passenger domain [3,5,6,9]. The name autotransporter originally comes from the understanding that they require no accessory factors nor external energy to secrete across the outer membrane [10,11], although more recently it has been recognized that insertion of the β-barrel translocon is mediated by the β-barrel assembly machinery (BAM) complex [12,13].
After secretion across the inner membrane in the unfolded form [14], autotransporters are guided by chaperone proteins [15,16] in the periplasm to the BAM complex, which assists their insertion into the outer membrane [[17], [18], [19], [20], [21]]. How the passenger domain is exported, however, is still not well understood, mainly due to the lack of traditional energy sources, i.e. ATP and ion gradients, in the periplasm [22]. One hypothesis is that passenger domain folding on the extracellular side provides the energy for secretion in a manner akin to a “Brownian ratchet” [23,24]. Based on this hypothesis, there are two leading models for passenger domain secretion: the hairpin model and the BamA-assisted model [25]. The hairpin model states that the passenger domain is exported through its own β-barrel domain via a hairpin intermediate without assistance from other proteins. In contrast, the BamA-assisted model states that the β-barrel domain stays in conjunction with the BAM complex while the passenger domain is secreted through a hybrid pore composed of BamA and its own β-barrel domain [26,27]. Experimental evidence supporting the latter model includes the observed association of BamA with stalled secretion intermediates [20,28].
Here, we test the validity of the two models using molecular dynamics simulations for two types of autotransporters, type Va (classical autotransporters) and type Vc (trimeric autotransporters). We use EspP as an example of a type Va classical autotransporter [12,29], and YadA as an example of a type Vc trimeric autotransporter adhesin [30]. EspP is a member of the serine protease autotransporter of Enterobacteriaceae (SPATE) family [31]. As a classical autotransporter, it is composed of a monomeric N-terminal passenger domain, a 12-stranded C-terminal β-barrel translocon, and an α-helical linker that gets cleaved after secretion, releasing the passenger domain from the cell surface via an autoproteolytic reaction [32]. Its cleavable α-linker region connecting the passenger domain and translocon has been found to be necessary for folding and stability of the β-barrel translocon [3,33,34]. EspP's known virulence functions include biofilm formation and cell adhesion [35]. It has been implicated in several diseases and symptoms, including diarrhea and inflammation [29,36]. Its possible use as a vaccine against Shiga toxins has also been explored [37,38].
YadA is a trimeric autotransporter adhesin (TAA) from the family of type Vc autotransporters [30]. It is expressed by both Yersinia enterocolitica and Yersinia pseudotuberculosis [39] and is present in Yersinia pestis with a frameshift mutation [40]. It mainly functions as an adhesin, although it also has been reported to act as an invasin in Y. pseudotuberculosis [41]. The passenger domain is an N-terminal trimeric β-roll, connected to the transmembrane domain via a trimeric coiled-coil stalk [42,43] and its transmembrane β-barrel domain is 12-stranded, with each YadA monomer contributing four β-strands. The structure of a shortened construct of YadA containing the β-barrel and part of the stalk, called YadA-M, has been resolved using solid-state NMR [[44], [45], [46]]. In this structure, a region in the coiled-coiled domain consisting of small residues, termed the ASSA region, was identified that exhibited low structural propensity and predicted increased dynamics. Later functional studies showed that mutating the first alanine of this linker region to a proline stalls passenger domain secretion [47]. This finding is similar to earlier studies, which showed that secretion can be stalled or inhibited by mutation of a conserved glycine residue [48], and the corresponding residue in Hia forms a stabilizing interaction between the α-helix and β-sheet [49]. Additionally, an arginine residue in the C-terminal β-barrel plays a role in preventing the misfolding of the passenger domain [50,51].
We used previous experimental evidence for the formation of a passenger domain hairpin intermediate to first address whether this intermediate could be accommodated by the fully-formed β-barrel domains of these two autotransporters. Our simulation results indicate that while this is possible, major structural distortions of the β-barrel domain would need to occur for passenger domain secretion. We next tested passenger domain transport through the BamA β-barrel via steered molecular dynamics, where we found the forces required for transport are lower than those required for transport through the autotransporter β-barrel domains alone. We therefore propose that passenger domain secretion likely occurs before the autotransporter β-barrel is fully folded and passes through an asymmetric hybrid barrel as recently proposed [52].
Section snippets
Molecular dynamics (MD)
All-atom MD simulations were performed using NAMD 2.11 [53] and the CHARMM36m [54,55] force field parameters for proteins and lipids with the TIP3P-CHARMM water model [56]. All simulations were performed under periodic boundary conditions with a cutoff at 12 Å for short-range electrostatic and Lennard-Jones interactions and a force-based switching function starting at 10 Å. The particle-mesh Ewald method [57] with a grid spacing of 1 Å was used for long-range electrostatic interaction
The YadA β-barrel domain is unable to accommodate the passenger domain as a hairpin intermediate
To test if the YadA β-barrel is large enough to accommodate the trimeric passenger domain in a hairpin conformation, we constructed a total of five YadA-M structures based on its NMR structure [44,45], which is a truncated construct composed of the β-barrel domain and a partial segment of the α-helical passenger domain (PDB ID:2LME). These five structures are (1) the wildtype structure (WT), (2) the linker region proline mutant from Chauhan et al. [47] (A354P), (3–4) the hairpin structures
Discussion
In this study, we explored the plausibility of the hairpin model of passenger domain secretion in autotransporters. MD simulations of two model autotransporters from the type Va (EspP) and type Vc (YadA) families were performed. For each autotransporter, a hairpin model was constructed and simulated at equilibrium to judge their stability and with steered MD to mimic secretion. Results for YadA (Fig. 1, Fig. 2) show that to accommodate the passenger domain in the hairpin conformation, the
Acknowledgements
This work was supported by the National Institutes of Health (R01-GM123169) and by the National Science Foundation Physics of Living Systems Student Research Network (PHY-1806833). Support from the Norwegian Center for Molecular Medicine (NCMM - to DL) is gratefully acknowledged. MOR acknowledges the Research Council of Norway for funding. Computational resources were provided through the Extreme Science and Engineering Discovery Environment (XSEDE; TG-MCB130173), which is supported by NSF
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2022, Advances in Protein Chemistry and Structural BiologyCitation Excerpt :Due to passenger and translocator being part of the same polypeptide, it was thought that these proteins could “secrete themselves,” and no other factors were needed; hence the name “autotransporter” (Klauser, Pohlner, & Meyer, 1993). This turned out to be a misconception, and now it is clear that the BAM plays a crucial role in autotransporter biogenesis (Doyle & Bernstein, 2021; Ryoo et al., 2020). However, the term “autotransporter” is still applicable, as the information for passenger secretion as well as the energy for secretion is provided by the autotransporter protein itself (Leo, Grin, & Linke, 2012).
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2021, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :The pentameric β-barrel assembly machinery (BAM) complex mediates the biogenesis of OMPs, and consists of the integral membrane protein BamA and four lipoproteins: BamB, BamC, BamD and BamE, each of which are attached to the membrane via a lipid anchor [1,8–11]. The substrates of the BAM complex range from simple monomeric β-barrels like OmpT [12], to more complicated β-barrels such as LptD [13] and FimD [5], and auto-transporters such as the homologs EspP and the plasmid-encoded toxin (Pet) [14–17]. Despite the structural insights into the BAM complex obtained through cryo-electron microscopy (cryo-EM) [12] and X-Ray diffraction [18,19], and the extensive mapping of subunit-subunit interfaces by cross linking in vivo and in vitro in the presence of substrate [20], the dynamics of the BAM complex in solution, embedded in a membrane environment and how this assists OMP folding and insertion remains to be defined.
Protein import and export across the bacterial outer membrane
2021, Current Opinion in Structural BiologyCitation Excerpt :These intermediate states have been detected in both classical and trimeric ATs, suggesting a common assembly mechanism [16–20]. The AT β-barrel targets BAM to promote membrane integration and facilitate secretion of the passenger domain [20,21]. The mechanism of BAM-mediated insertion of ATs into the OM is still a matter of debate [22∗].
Editorial: Advances in computational molecular biophysics
2021, Biochimica et Biophysica Acta - General SubjectsBamA forms a translocation channel for polypeptide export across the bacterial outer membrane
2021, Molecular CellCitation Excerpt :We also found that, in addition to catalyzing the assembly and integration of OM β-barrel proteins, BamA can function as a remarkable polypeptide translocase. The traversal of the BamA β-barrel lumen is consistent with recent molecular dynamics experiments that show that much less force is required to pull a polypeptide through BamA than EspP (Ryoo et al., 2020). Furthermore, we recently found that crosslinking between cysteines positioned in BamAβ16 and EspPβ1 is unexpectedly low (Doyle and Bernstein, 2019) (e.g., ∼5% for BamAT809C-MBP-76EspPG1040C), but we show here that crosslinking between BamAT809C and cysteines positioned in the passenger is exceptionally high (e.g., ∼50% for BamAT809C-MBP-76EspPT984C).