ABSTRACT In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum , the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC , tprD , and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 ( tp0548 ) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum . They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development. IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum , little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC , tprD , and bamA , in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.
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The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli
The outer membrane of Gram-negative bacteria acts as an additional diffusion barrier for solutes and nutrients. It is perforated by outer membrane proteins (OMPs) that function most often as diffusion pores, but sometimes also as parts of larger cellular transport complexes, structural components of the cell wall, or even as enzymes. These OMPs often have large loops that protrude into the extracellular environment, which have promise for biotechnological applications and as therapeutic targets. Thus, understanding how modifications to these loops affect OMP stability and folding is critical for their efficient application. In this work, the small outer membrane protein OmpX was used as a model system to quantify the effects of loop insertions on OMP folding and stability. The insertions were varied according to both hydrophobicity and size, and their effects were determined by assaying folding into detergent micelles in vitro by SDS-PAGE and in vivo by isolating the outer membrane of cells expressing the constructs. The different insertions were also examined in molecular dynamics simulations to resolve how they affect OmpX dynamics in its native outer membrane. The results indicate that folding of OMPs is affected by both the insert length and by its hydrophobic character. Small insertions sometimes even improved the folding efficiency of OmpX, while large hydrophilic inserts reduced it. All the constructs that were found to fold in vitro could also do so in their native environment. One construct that could not fold in vitro was transported to the OM in vivo , but remained unfolded. Our results will help to improve the design and efficiency of recombinant OMPs used for surface display.
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- Award ID(s):
- 1828187
- NSF-PAR ID:
- 10398106
- Date Published:
- Journal Name:
- Frontiers in Molecular Biosciences
- Volume:
- 9
- ISSN:
- 2296-889X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Outer membrane protein (OMP) biogenesis in gram‐negative bacteria is managed by a network of periplasmic chaperones that includes SurA, Skp, and FkpA. These chaperones bind unfolded OMPs (uOMPs) in dynamic conformational ensembles to suppress aggregation, facilitate diffusion across the periplasm, and enhance folding. FkpA primarily responds to heat‐shock stress, but its mechanism is comparatively understudied. To determine FkpA chaperone function in the context of OMP folding, we monitored the folding of three OMPs and found that FkpA, unlike other periplasmic chaperones, increases the folded yield but decreases the folding rate of OMPs. The results indicate that FkpA behaves as a chaperone and not as a folding catalyst to influence the OMP folding trajectory. Consistent with the folding assay results, FkpA binds all three uOMPs as determined by sedimentation velocity (SV) and photo‐crosslinking experiments. We determine the binding affinity between FkpA and uOmpA171by globally fitting SV titrations and find it to be intermediate between the known affinities of Skp and SurA for uOMP clients. Notably, complex formation steeply depends on the urea concentration, suggesting an extensive binding interface. Initial characterizations of the complex using photo‐crosslinking indicate that the binding interface spans the entire FkpA molecule. In contrast to prior findings, folding and binding experiments performed using subdomain constructs of FkpA demonstrate that the full‐length chaperone is required for full activity. Together these results support that FkpA has a distinct and direct effect on OMP folding that it achieves by utilizing an extensive chaperone‐client interface to tightly bind clients.
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Abstract SurA is thought to be the most important periplasmic chaperone for outer membrane protein (OMP) biogenesis. Its structure is composed of a core region and two peptidylprolyl isomerase domains, termed P1 and P2, connected by flexible linkers. As such these three independent folding units are able to adopt a number of distinct spatial positions with respect to each other. The conformational dynamics of these domains are thought to be functionally important yet are largely unresolved. Here we address this question of the conformational ensemble using sedimentation equilibrium, small‐angle neutron scattering, and folding titrations. This combination of orthogonal methods converges on a SurA population that is monomeric at physiological concentrations. The conformation that dominates this population has the P1 and core domains docked to one another, for example, “P1‐closed” and the P2 domain extended in solution. We discovered that the distribution of domain orientations is defined by modest and favorable interactions between the core domain and either the P1 or the P2 domains. These two peptidylprolyl domains compete with each other for core‐binding but are thermodynamically uncoupled. This arrangement implies two novel insights. Firstly, an open conformation must exist to facilitate P1 and P2 exchange on the core, indicating that the open client‐binding conformation is populated at low levels even in the absence of client unfolded OMPs. Secondly, competition between P1 and P2 binding paradoxically occludes the client binding site on the core, which may serve to preserve the reservoir of binding‐competent apo‐SurA in the periplasm.
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Abstract We examine the influence of cellular interactions in all‐atom models of a section of the
Homo sapiens cytoplasm on the early folding events of the three‐helix bundle protein B (PB). While genetically engineered PB is known to fold in dilute water box simulations in three microseconds, the three initially unfolded PB copies in our two cytoplasm models using a similar force field did not reach the native state during 30‐microsecond simulations. We did however capture the formation of all three helices in a compact native‐like topology. Folding in vivo is delayed because intramolecular contact formation within PB is in direct competition with intermolecular contacts between PB and surrounding macromolecules. In extreme cases, intermolecular beta‐sheets are formed. Interactions with other macromolecules are also observed to promote structure formation, for example when a PB helix in our simulations is shielded from solvent by macromolecular crowding. Sticking and crowding in our models initiate sampling of helix/sheet structural plasticity of PB. Relatedly, in past in vitro experiments, similar GA domains were shown to switch between two different folds. Finally, we also observed that stickiness between PB and the cellular environment can be modulated in our simulations through the reduction in protein hydrophobicity when we reversed PB back to the wild‐type sequence. This study demonstrates that even fast‐folding proteins can get stuck in non‐native states in the cell, making them useful models for protein–chaperone interactions and early stages of aggregate formation relevant to cellular disease. -
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Abstract The outer membrane (OM) of Gram-negative bacteria such as
Escherichia coli is an asymmetric bilayer with the glycolipid lipopolysaccharide (LPS) in the outer leaflet and glycerophospholipids in the inner. Nearly all integral OM proteins (OMPs) have a characteristic β-barrel fold and are assembled in the OM by the BAM complex, which contains one essential β-barrel protein (BamA), one essential lipoprotein (BamD), and three non-essential lipoproteins (BamBCE). A gain-of-function mutation inbamA enables survival in the absence of BamD, showing that the essential function of this protein is regulatory. Here, we demonstrate that the global reduction in OMPs caused by BamD loss weakens the OM, altering cell shape and causing OM rupture in spent medium. To fill the void created by OMP loss, phospholipids (PLs) flip into the outer leaflet. Under these conditions, mechanisms that remove PLs from the outer leaflet create tension between the OM leaflets, which contributes to membrane rupture. Rupture is prevented by suppressor mutations that release the tension by halting PL removal from the outer leaflet. However, these suppressors do not restore OM stiffness or normal cell shape, revealing a possible connection between OM stiffness and cell shape.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmolb.2022.918480
