Aug 2026· PLoS Biology· Vol 24 8, pp.
e3003961
· 0 citations· 67 references
Medicine
TL;DR
The newly developed "Gradient Enrichment of Native Targets from Lipid Environments" (GENTLE) methodology is used to elucidate the structures of Campylobacter jejuni OMPs directly from native, detergent-solubilized crude membranes and provides novel structural information for functionally important OMPs.
Abstract
Bacterial outer membrane proteins (OMPs) are critical players in host-pathogen interactions and environmental adaptation. Here we describe the newly developed "Gradient Enrichment of Native Targets from Lipid Environments" (GENTLE) methodology and use this approach to elucidate the structures of Campylobacter jejuni OMPs directly from native, detergent-solubilized crude membranes. We identify and solve high-resolution cryo-EM structures of PorA, OMP50, and Cj0034c from C. jejuni membranes, all of which are required for Campylobacter invasion, adhesion, and initiation of host infection. Notably, our results provide the first structural information of OMP50, revealing a two-domain architecture constructed with an all β-stranded transmembrane domain and an all α-helical periplasmic domain. This structure depicts that all tyrosine residues, many of which are expected to be critical for phosphorylation and host-pathogen interaction, are localized to the outer membrane of C. jejuni. Our studies also led to the first structure of the full-length Cj0034c protein, which assembles as a nonamer with each protomer containing a single-spanning transmembrane helix and a large periplasmic domain. The nine protomers stack side-by-side to form a channel that spans the entire lipid bilayer. However, whether Cj0034c spans the outer membrane (OM) or inner membrane (IM) of C. jejuni must await further experimental studies. In addition, we observed that the surface-exposed extracellular loop L4 of PorA is very flexible, which may be critical for the virulence of this porin. Collectively, this work provides novel structural information for functionally important OMPs and sheds light on how they assemble in native bacterial membranes. These findings further demonstrate that it is possible to obtain high-resolution structural information for targeted membrane proteins from crude native membranes without their overexpression and purification.
Campylobacter jejuni is the leading bacterial cause of foodborne enteric disease worldwide. The increasing emergence of antibiotic-resistant C. jejuni strains underscores the need to identify new protein targets for antibacterial drug development through the functional characterization of previously unstudied C. jejuni proteins. One such protein whose function has not been experimentally investigated is CJ1041C. To gain insight into the role of CJ1041C, we determined its crystal structures in both the apo form and in complex with Ca2+ ions. CJ1041C adopts a six-bladed β-propeller architecture, in which six four-stranded β-sheets are radially arranged around a central channel. This channel is occluded in the middle and harbors two oppositely oriented, negatively charged cavities. Notably, the upper cavity coordinates a Ca2+ ion through highly conserved residues and additionally accommodates a glycerol molecule presumably as a substrate–water mimic. The Ca2+-binding configuration of CJ1041C closely resembles that observed in β-propeller lactonases, suggesting that CJ1041C functions as a lactonase or lactonase-like enzyme. However, the unique glycerol-binding mode of CJ1041C, combined with the results of phylogenetic and sequence analyses, indicates that CJ1041C represents a distinct member of the β-propeller lactonase family that likely exerts catalytic activity toward noncanonical substrates.
Minwoo Park, Sung-il Yoon· Scientific Reports· 0 citations
Lipoprotein and OmpW are two major components of the outer membrane (OM) in Gram-negative bacteria and play essential roles in various physiological processes, e.g., protein secretion, folding, and localization. They typically assist in maintaining client proteins in a folded state or form pore channels during secretion. However, how OM proteins facilitate the secretion of proteins that lack classical signal peptides remains elusive. Here, we demonstrate that they contribute to the secretion of unconventional B-type dye-decolorizing peroxidase (DypB2985) in Pseudomonas putida. The lipoprotein, Lpp1528, which contains a Sec signal peptide, is translocated to the periplasm through the Sec pathway and anchored in the inner leaflet of the OM. Lpp1528 recognizes the C-terminal hydrophobic region of DypB2985 in the cytoplasm and facilitates its coupling to the Sec machinery for inner membrane translocation, despite DypB2985 lacking a canonical N-terminal Sec signal peptide. Following translocation, the two proteins appear to dissociate in the periplasm. Subsequently, another OM protein, OmpW4836, recognizes the N-terminal hydrophobic region of periplasmic DypB2985 and mediates its incorporation into outer membrane vesicles (OMVs) for extracellular delivery. Our study reveals the crosstalk between the Sec pathway and OMVs in the secretion of a non-canonical peroxidase, in which OM proteins, acting as the molecular tethers, mediate the stepwise secretion process. It expands our understanding of non-classical protein secretion mechanisms and bacterial survival strategies. Moreover, the identified OMV sorting mechanism offers potential for the further functionalization of OMVs as versatile biotechnological platforms.
Cong-Ying Liang, Wenping Zhu, Lu Lin· PLoS Genetics· 0 citations
Members of the Mammalian Cell Entry (MCE) superfamily interact with other proteins to form diverse architectures for the transport of hydrophobic molecules across the cell envelope in Gram-negative bacteria. Some of these trans-envelope MCE protein complexes include a PqiC-like outer membrane (OM) lipoprotein component. The best-studied member of this group of OM lipoproteins is E. coli PqiC, from the PqiABC system, which can form an octameric ring. How PqiC-like lipoproteins interact with their MCE protein binding partners to facilitate transport is not well understood. Here we report the cryo-electron microscopy structures of Pseudomonas aeruginosa PA3214, a homolog of PqiC, in the context of the full MCE transport PA3211-PA3214 system. Our structure provides insight into the biological assembly of the lipoprotein and interactions with its binding partner, MCE protein PA3213. We utilize deep mutational scanning to identify functionally important sites in E. coli PqiC in an unbiased manner. Through phenotypic and biochemical experiments, we characterize the interactions of the lipoproteins PqiC and PA3214 with their associated MCE proteins PqiB and PA3213, thus providing a model for how some MCE proteins employ a C-terminal peptide to mediate key interactions with their cognate lipoproteins at the OM.
Sabrina I. Giacometti, Nicolas Coudray, Rachel L. Redler et al.· Journal of Biological Chemis...· 0 citations
The outer membrane (OM) of Gram-negative bacteria acts as a permeability barrier against toxic compounds. Its integrity is maintained by various outer membrane proteins (OMPs), which are inserted into the OM by the β-barrel assembly machinery (BAM) complex. The periplasmic chaperone SurA delivers unfolded OMPs to BAM; however, the mechanism of substrate transfer remains unclear. Here, we show that the flexible P1 and P2 domains of SurA regulate the function of its Core domain and interact with BAM components, including BamE, whose interaction with the P2 domain is crucial for efficient OMP assembly. Moreover, cryo-electron microscopy reveals four distinct Escherichia coli SurA–BAM structures, suggesting dynamic domain rearrangements of SurA. Based on these findings, we propose a dynamic model in which SurA transfers substrates to BAM through multiple conformational changes, providing a unified framework for chaperone-assisted OMP biogenesis. Outer membrane proteins are delivered to the BAM complex by the chaperone SurA for proper assembly. Here, the authors report multiple structures of the SurA–BAM complex and show that SurA undergoes conformational changes to pass substrates to BAM.
ABSTRACT Chlamydia species secrete a unique class of effector proteins termed inclusion membrane proteins (Incs) that are translocated into the inclusion membrane and reprogram host cell processes to support intracellular growth. Using affinity purification–mass spectrometry, we previously identified an interaction between the early expressed Inc CT226 and a human multiprotein complex comprising leucine-rich repeat Flightless-I-interacting protein 1 (LRRFIP1), LRRFIP2, Flightless-I (FLII), and tropomodulin 3 (TMOD3). Here, we define the molecular basis and functional consequences of this interaction during Chlamydia trachomatis infection. We show that CT226 is both necessary and sufficient to recruit the LRRFIP1/2:FLII:TMOD3 (LFT) complex to the inclusion membrane. In vitro reconstitution with purified proteins demonstrates that the predicted coiled-coil region of CT226 directly binds the N-terminal helix of LRRFIP1. Using in silico structural modeling with AlphaFold-Multimer coupled with mutational validation, we identify key residues that mediate an electrostatic CT226:LRRFIP1 interaction required for LFT recruitment to the inclusion. Unexpectedly, genetic disruption of CT226 or its interaction with the LFT complex results in modest increases in inclusion size and recovery of infectious progeny late in infection, demonstrating that the CT226:LFT interaction modulates C. trachomatis developmental progression. Together, these findings establish the molecular basis of CT226-mediated recruitment of the LFT complex to the inclusion membrane and uncover a previously unrecognized role for this effector–host interaction at late timepoints of the C. trachomatis intracellular developmental cycle. IMPORTANCE Chlamydia trachomatis is a major cause of sexually transmitted infections and blindness worldwide. This bacterium survives by replicating inside host cells within a membrane-bound compartment, where it deploys specialized proteins to manipulate host processes. These proteins, known as inclusion membrane proteins (Incs), help organize the host–pathogen interface, but the molecular mechanisms by which many Incs engage host factors remain poorly understood. Here, we show that the Inc protein CT226 recruits a host multiprotein complex to the bacterial compartment by directly binding one of its components. Using structural modeling and targeted mutations, we define the molecular interface required for this interaction. Disruption of CT226 or its ability to engage this host complex results in modest increases in inclusion size and production of infectious progeny, suggesting a role for the CT226:host interaction in regulating Chlamydia intracellular development. These findings reveal how a bacterial effector can engage a host protein complex to influence the outcome of infection. Chlamydia trachomatis is a major cause of sexually transmitted infections and blindness worldwide. This bacterium survives by replicating inside host cells within a membrane-bound compartment, where it deploys specialized proteins to manipulate host processes. These proteins, known as inclusion membrane proteins (Incs), help organize the host–pathogen interface, but the molecular mechanisms by which many Incs engage host factors remain poorly understood. Here, we show that the Inc protein CT226 recruits a host multiprotein complex to the bacterial compartment by directly binding one of its components. Using structural modeling and targeted mutations, we define the molecular interface required for this interaction. Disruption of CT226 or its ability to engage this host complex results in modest increases in inclusion size and production of infectious progeny, suggesting a role for the CT226:host interaction in regulating Chlamydia intracellular development. These findings reveal how a bacterial effector can engage a host protein complex to influence the outcome of infection.
C. Elwell, Amy Diallo, B. S. Sixt et al.· mBio· 0 citations
Sortase A (SrtA) enzymes covalently anchor surface proteins to Gram-positive bacterial cell walls, promoting colonization and virulence. In Streptococcus pneumoniae, previous studies identified both a domain-swapped dimer and an active refolded monomer, but the active enzyme has not been characterized at the structural and residue-specific level. Here, we performed quantitative proteomic comparisons of wild-type and SrtA knockout strains that confirmed the loss of multiple LPxTG-containing virulence factors, including ZmpB, NanA, and IgA1 protease, consistent with an essential role for SrtA in surface protein anchoring. To enable mechanistic studies, we established a biochemical framework to produce monomeric Streptococcus pneumoniae SrtA by refolding and developed a gel-based assay using recombinant substrates to monitor catalytic activity. The refolded monomer, but not the swapped dimer, catalyzed cleavage and transpeptidation of a canonical LPxTG substrate in a metal-independent manner under the conditions examined. We further report high-resolution NMR backbone assignments for the active monomer and identify substrate-induced chemical shift perturbations that localize to the active site. Together, these findings provide an integrated proteomic, biochemical, and NMR characterization of monomeric, catalytically active Streptococcus pneumoniae SrtA and reveal residue-specific interactions with a canonical LPNTG recognition peptide.
Eunjeong Lee, Blaine H. Gordon, J. Redzic et al.· Biomolecules· 0 citations
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