Structural basis for bacterial lipoprotein relocation by the transporter LolCDE.

Lipoproteins in the outer membrane of Gram-negative bacteria are involved in various vital physiological activities, including multidrug resistance. Synthesized in the cytoplasm and matured in the inner membrane, lipoproteins must be transported to the outer membrane through the Lol pathway mediated by the ATP-binding cassette transporter LolCDE in the inner membrane via an unknown mechanism. Here, we report cryo-EM structures of Escherichia coli LolCDE in apo, lipoprotein-bound, LolA-bound, ADP-bound and AMP-PNP-bound states at a resolution of 3.2-3.8 Å, covering the complete lipoprotein transport cycle. Mutagenesis and in vivo viability assays verify features of the structures and reveal functional residues and structural characteristics of LolCDE. The results provide insights into the mechanisms of sorting and transport of outer-membrane lipoproteins and may guide the development of novel therapies against multidrug-resistant Gram-negative bacteria.

Characterization of BamA reconstituted into a solid-supported lipid bilayer as a platform for measuring dynamics during substrate protein assembly into the membrane.

In Gram-negative bacteria, the multi-protein ß-barrel assembly machine (BAM) complex is a nanomachine playing a vital role in the process of assembling ß-barrel proteins into the outer membrane (OM). The core component of this multiprotein complex, BamA, is an evolutionarily conserved protein that carries five polypeptide-transport-associated (POTRA) domains that project from the outer membrane. BamA is essential for chaperoning the insertion of proteins into the OM surface of bacterial cells. In this work, we have reconstituted a membrane containing BamA on a gold substrate and characterized structure of each component and movement in different situation at the nanoscale level using quartz-crystal microbalance with dissipation and neutron reflectometry (NR). The purified BamA in n-dodecyl ß-D-maltoside (DDM) was first engineered onto a nickel-NTA (Nα, Nα-bis-(carboxymethyl)-l-lysine) modified gold surface followed by DDM removal and bilayer assembly. The system was then used to monitor the binding and insertion of a substrate membrane protein. The data shows the total reach of BamA was 120 Å and the embedding of membrane had no effect on the BamA morphology. However, the addition of the substrate enabled the periplasmic POTRA domain of BamA to extend further away from the membrane surface. This dynamic behaviour of BamA POTRA domains is consistent with models invoking the gathering of transported substrates from the periplasmic space between the inner and outer membranes in bacterial cells. This study provides evidence that NR is a reliable tool for diverse investigations in the future, especially for applications in the field of membrane protein biogenesis.

BBE31 from the Lyme disease agent Borrelia burgdorferi, known to play an important role in successful colonization of the mammalian host, shows the ability to bind glutathione.

Lyme disease is a tick-borne infection caused by Borrelia burgdorferi sensu lato complex spirochetes. The spirochete is located in the gut of the tick; as the infected tick starts the blood meal, the spirochete must travel through the hemolymph to the salivary glands, where it can spread to and infect the new host organism. In this study, we determined the crystal structures of the key outer surface protein BBE31 from B. burgdorferi and its orthologous protein BSE31 (BSPA14S_RS05060 gene product) from B. spielmanii. BBE31 is known to be important for the transfer of B. burgdorferi from the gut to the hemolymph in the tick after a tick bite. While BBE31 exerts its function by interacting with the Ixodes scapularis tick gut protein TRE31, structural and mass spectrometry data revealed that BBE31 has a glutathione (GSH) covalently attached to Cys142 suggesting that the protein may have acquired some additional functions in contrast to its orthologous protein BSE31, which lacks any interactions with GSH. In the current study, in addition to analyzing the potential reasons for GSH binding, the three-dimensional structure of BBE31 provides new insights into the molecular details of the transmission process as the protein plays an important role in the initial phase before the spirochete is physically transferred to the new host. This knowledge will be potentially used for the development of new strategies to fight against Lyme disease.

In situ observation of conformational dynamics and protein ligand-substrate interactions in outer-membrane proteins with DEER/PELDOR spectroscopy.

Observation of structure and conformational dynamics of membrane proteins at high resolution in their native environments is challenging because of the lack of suitable techniques. We have developed an approach for high-precision distance measurements in the nanometer range for outer-membrane proteins (OMPs) in intact Escherichia coli and native membranes. OMPs in Gram-negative bacteria rarely have reactive cysteines. This enables in situ labeling of engineered cysteines with a methanethiosulfonate spin label (MTSL) with minimal background signals. Following overexpression of the target protein, spin labeling is performed with E. coli or isolated outer membranes (OMs) under selective conditions. The interspin distances are measured in situ, using pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. The residual background signals, which are problematic for in situ structural biology, contribute specifically to the intermolecular part of the signal and can be selectively removed to extract the desired interspin distance distribution. The initial cloning stage can take 5-7 d, and the subsequent protein expression, OM isolation, spin labeling, PELDOR experiment, and data analysis typically take 4-5 d. The described protocol provides a general strategy for observing protein ligand-substrate interactions, oligomerization, and conformational dynamics of OMPs in their native OM and intact E. coli.

Structure of the Helicobacter pylori Cag type IV secretion system.

Bacterial type IV secretion systems (T4SSs) are molecular machines that can mediate interbacterial DNA transfer through conjugation and delivery of effector molecules into host cells. The Helicobacter pylori Cag T4SS translocates CagA, a bacterial oncoprotein, into gastric cells, contributing to gastric cancer pathogenesis. We report the structure of a membrane-spanning Cag T4SS assembly, which we describe as three sub-assemblies: a 14-fold symmetric outer membrane core complex (OMCC), 17-fold symmetric periplasmic ring complex (PRC), and central stalk. Features that differ markedly from those of prototypical T4SSs include an expanded OMCC and unexpected symmetry mismatch between the OMCC and PRC. This structure is one of the largest bacterial secretion system assemblies ever reported and illustrates the remarkable structural diversity that exists among bacterial T4SSs.

Single-Molecule Force Spectroscopy of Transmembrane ß-Barrel Proteins.

Single-molecule force spectroscopy (SMFS) has been widely applied to study the mechanical unfolding and folding of transmembrane proteins. Here, we review the recent progress in characterizing bacterial and human transmembrane ß-barrel proteins by SMFS. First, we describe the mechanical unfolding of transmembrane ß-barrels, which follows a general mechanism dictated by the sequential unfolding and extraction of individual ß-strands and ß-hairpins from membranes. Upon force relaxation, the unfolded polypeptide can insert stepwise into the membrane as single ß-strands or ß-hairpins to fold as the native ß-barrel. The refolding can be followed at a high spatial and temporal resolution, showing that small ß-barrels are able to fold without assistance, whereas large and complex ß-barrels require chaperone cofactors. Applied in the dynamic mode, SMFS can quantify the kinetic and mechanical properties of single ß-hairpins and reveal complementary insight into the membrane protein structure and function relationship. We further outline the challenges that SMFS experiments must overcome for a comprehensive understanding of the folding and function of transmembrane ß-barrel proteins.

Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump.

The MacA-MacB-TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism.

Role of OmpA2 surface regions of Porphyromonas gingivalis in host-pathogen interactions with oral epithelial cells.

Outer membrane protein A (OmpA) is a key outer membrane protein found in Gram-negative bacteria that contributes to several crucial processes in bacterial virulence. In Porphyromonas gingivalis, OmpA is predicted as a heterotrimer of OmpA1 and OmpA2 subunits encoded by adjacent genes. Here we describe the role of OmpA and its individual subunits in the interaction of P. gingivalis with oral cells. Using knockout mutagenesis, we show that OmpA2 plays a significant role in biofilm formation and interaction with human epithelial cells. We used protein structure prediction software to identify extracellular loops of OmpA2, and determined these are involved in interactions with epithelial cells as evidenced by inhibition of adherence and invasion of P. gingivalis by synthetic extracellular loop peptides and the ability of the peptides to mediate interaction of latex beads with human cells. In particular, we observe that OmpA2-loop 4 plays an important role in the interaction with host cells. These data demonstrate for the first time the important role of P. gingivalis OmpA2 extracellular loops in interaction with epithelial cells, which may help design novel peptide-based antimicrobial therapies for periodontal disease.

Structural basis for Myf and Psa fimbriae-mediated tropism of pathogenic strains of Yersinia for host tissues.

Three pathogenic species of the genus Yersinia assemble adhesive fimbriae via the FGL-chaperone/usher pathway. Closely related Y. pestis and Y. pseudotuberculosis elaborate the pH6 antigen (Psa), which mediates bacterial attachment to alveolar cells of the lung. Y. enterocolitica, instead, assembles the homologous fimbriae Myf of unknown function. Here, we discovered that Myf, like Psa, specifically recognizes ß1-3- or ß1-4-linked galactose in glycosphingolipids, but completely lacks affinity for phosphatidylcholine, the main receptor for Psa in alveolar cells. The crystal structure of a subunit of Psa (PsaA) complexed with choline together with mutagenesis experiments revealed that PsaA has four phosphatidylcholine binding pockets that enable super-high-avidity binding of Psa-fibres to cell membranes. The pockets are arranged as six tyrosine residues, which are all missing in the MyfA subunit of Myf. Conversely, the crystal structure of the MyfA-galactose complex revealed that the galactose-binding site is more extended in MyfA, enabling tighter binding to lactosyl moieties. Our results suggest that during evolution, Psa has acquired a tyrosine-rich surface that enables it to bind to phosphatidylcholine and mediate adhesion of Y. pestis/pseudotuberculosis to alveolar cells, whereas Myf has specialized as a carbohydrate-binding adhesin, facilitating the attachment of Y. enterocolitica to intestinal cells.

Structural and functional analysis of BB0689 from Borrelia burgdorferi, a member of the bacterial CAP superfamily.

Spirochete Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted from infected Ixodes ticks to a mammalian host after a tick bite. The outer surface protein BB0689 from B. burgdorferi is up-regulated when the tick feeds, which indicates a potential role for BB0689 in Lyme disease pathogenesis. We have determined the crystal structure of BB0689, which revealed that the protein belongs to the CAP superfamily. Though the CAP domain is widespread in all three cellular domains of life, thus far the CAP domain has been studied only in eukaryotes, in which it is usually linked to certain other domains to form a multi-domain protein and is associated with the mammalian reproductive tract, the plant response to pathogens, venom allergens from insects and reptiles, and the growth of human brain tumors. Though the exact function of the isolated CAP domain remains ambiguous, several functions, including the binding of cholesterol, lipids and heparan sulfate, have been recently attributed to different CAP domain proteins. In this study, the bacterial CAP domain structure was analyzed and compared with the previously solved crystal structures of representative CAPs, and the function of BB0689 was examined. To determine the potential function of BB0689 and ascertain whether the functions that have been attributed to the CAP domain proteins are conserved, the binding of previously reported CAP domain interaction partners was analyzed, and the results suggested that BB0689 has a unique function that is yet to be discovered.

Out but not in: the large transmembrane ß-barrel protein FhuA unfolds but cannot refold via ß-hairpins.

How transmembrane ß-barrel proteins insert and fold into membranes and by which factors they destabilize, unfold, and misfold represents a field of intense studies. Here, we use single-molecule force spectroscopy to characterize the un- and refolding of the ferric hydroxamate uptake receptor (FhuA), which is one of the largest ß-barrel proteins of the outer membrane of Escherichia coli. Applied to mechanical stress, FhuA undergoes a complex unfolding pathway in which each of the 11 ß-hairpins unfolds one after the other until the entire ß-barrel has unfolded. Once unfolded and relaxed, the FhuA polypeptide cannot fold back into the lipid membrane and adopts various misfolded conformations. Such misfolding is in contrast to the reversible refolding behavior of much smaller ß-barrel outer membrane proteins OmpA and OmpG that occurs at similar experimental conditions. The results suggest that large ß-barrel proteins that show more complex (un-)folding pathways require cofactors for proper insertion and folding into the membrane.

Correlation of in situ mechanosensitive responses of the Moraxella catarrhalis adhesin UspA1 with fibronectin and receptor CEACAM1 binding.

Bacterial cell surfaces are commonly decorated with a layer formed from multiple copies of adhesin proteins whose binding interactions initiate colonization and infection processes. In this study, we investigate the physical deformability of the UspA1 adhesin protein from Moraxella catarrhalis, a causative agent of middle-ear infections in humans. UspA1 binds a range of extracellular proteins including fibronectin, and the epithelial cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Electron microscopy indicates that unliganded UspA1 is densely packed at, and extends about 800 Å from, the Moraxella surface. Using a modified atomic force microscope, we show that the adhesive properties and thickness of the UspA1 layer at the cell surface varies on addition of either fibronectin or CEACAM1. This in situ analysis is then correlated with the molecular structure of UspA1. To provide an overall model for UspA1, we have determined crystal structures for two N-terminal fragments which are then combined with a previous structure of the CEACAM1-binding site. We show that the UspA1-fibronectin complex is formed between UspA1 head region and the 13th type-III domain of fibronectin and, using X-ray scattering, that the complex involves an angular association between these two proteins. In combination with a previous study, which showed that the CEACAM1-UspA1 complex is distinctively bent in solution, we correlate these observations on isolated fragments of UspA1 with its in situ response on the cell surface. This study therefore provides a rare direct demonstration of protein conformational change at the cell surface.

Structural insights into the membrane-extracted dimeric form of the ATPase TraB from the Escherichia coli pKM101 conjugation system.

BACKGROUND: Type IV secretion (T4S) systems are involved in secretion of virulence factors such as toxins or transforming molecules, or bacterial conjugation. T4S systems are composed of 12 proteins named VirB1-B11 and VirD4. Among them, three ATPases are involved in the assembly of the T4S system and/or provide energy for substrate transfer, VirB4, VirB11 and VirD4. The X-ray crystal structures of VirB11 and VirD4 have already been solved but VirB4 has proven to be reluctant to any structural investigation so far. RESULTS: Here, we have used small-angle X-ray scattering to obtain the first structural models for the membrane-extracted, dimeric form of the TraB protein, the VirB4 homolog encoded by the E. coli pKM101 plasmid, and for the monomeric soluble form of the LvhB4 protein, the VirB4 homolog of the T4S system encoded by the Legionella pneumophila lvh operon. We have obtained the low resolution structures of the full-length TraB and of its N- and C-terminal halves. From these SAXS models, we derive the internal organisation of TraB. We also show that the two TraB N- and C-terminal domains are independently involved in the dimerisation of the full-length protein. CONCLUSIONS: These models provide the first structural insights into the architecture of VirB4 proteins. In particular, our results highlight the modular arrangement and functional relevance of the dimeric-membrane-bound form of TraB.

Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins.

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, alpha-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins.

Identification of Chlamydia trachomatis outer membrane complex proteins by differential proteomics.

The extracellular chlamydial infectious particle, or elementary body (EB), is enveloped by an intra- and intermolecular cysteine cross-linked protein shell called the chlamydial outer membrane complex (COMC). A few abundant proteins, including the major outer membrane protein and cysteine-rich proteins (OmcA and OmcB), constitute the overwhelming majority of COMC proteins. The identification of less-abundant COMC proteins has been complicated by limitations of proteomic methodologies and the contamination of COMC fractions with abundant EB proteins. Here, we used parallel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses of Chlamydia trachomatis serovar L2 434/Bu EB, COMC, and Sarkosyl-soluble EB fractions to identify proteins enriched or depleted from COMC. All well-described COMC proteins were specifically enriched in the COMC fraction. In contrast, multiple COMC-associated proteins found in previous studies were strongly enriched in the Sarkosyl-soluble fraction, suggesting that these proteins are not COMC components or are not stably associated with COMC. Importantly, we also identified novel proteins enriched in COMC. The list of COMC proteins identified in this study has provided reliable information for further understanding chlamydial protein secretion systems and modeling COMC and EB structures.

Chlamydia trachomatis polymorphic membrane protein D is an oligomeric autotransporter with a higher-order structure.

Chlamydia trachomatis is a globally important obligate intracellular bacterial pathogen that is a leading cause of sexually transmitted disease and blinding trachoma. Effective control of these diseases will likely require a preventative vaccine. C. trachomatis polymorphic membrane protein D (PmpD) is an attractive vaccine candidate as it is conserved among C. trachomatis strains and is a target of broadly cross-reactive neutralizing antibodies. We show here that immunoaffinity-purified native PmpD exists as an oligomer with a distinct 23-nm flower-like structure. Two-dimensional blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the oligomers were composed of full-length PmpD (p155) and two proteolytically processed fragments, the p73 passenger domain (PD) and the p82 translocator domain. We also show that PmpD undergoes an infection-dependent proteolytic processing step late in the growth cycle that yields a soluble extended PD (p111) that was processed into a p73 PD and a novel p30 fragment. Interestingly, soluble PmpD peptides possess putative eukaryote-interacting functional motifs, implying potential secondary functions within or distal to infected cells. Collectively, our findings show that PmpD exists as two distinct forms, a surface-associated oligomer exhibiting a higher-order flower-like structure and a soluble form restricted to infected cells. We hypothesize that PmpD is a multifunctional virulence factor important in chlamydial pathogenesis and could represent novel vaccine or drug targets for the control of human chlamydial infections.

Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin.

Dodecamerization and insertion of the outer membrane secretin PulD is entirely determined by the C-terminal half of the polypeptide (PulD-CS). In the absence of its cognate chaperone PulS, PulD-CS and PulD mislocalize to the inner membrane, from which they are extractable with detergents but not urea. Electron microscopy of PulD-CS purified from the inner membrane revealed apparently normal dodecameric complexes. Electron microscopy of PulD-CS and PulD in inner membrane vesicles revealed inserted secretin complexes. Mislocalization of PulD or PulD-CS to this membrane induces the phage shock response, probably as a result of a decreased membrane electrochemical potential. Production of PulD in the absence of the phage shock response protein PspA and PulS caused a substantial drop in membrane potential and was lethal. Thus, PulD-CS and PulD assemble in the inner membrane if they do not associate with PulS. We propose that PulS prevents premature multimerization of PulD and accompanies it through the periplasm to the outer membrane. PulD is the first bacterial outer membrane protein with demonstrated ability to insert efficiently into the inner membrane.

Determining OMP topology by computation, surface plasmon resonance and cysteine labelling: the test case of OMPG.

Bacterial outer-membrane proteins (OMP) are important in pathogenicity and the recently solved structure of OmpG provides an excellent test case for topological predictions since it is monomeric. Here we compare the results of applying several computerised structure prediction algorithms to the sequence of OmpG. Furthermore, we probe the OmpG topology by both an established chemical labelling approach and a new method which combines epitope insertion and surface plasmon resonance. The computational approaches are broadly accurate but the exact choice of the number of beta strands remains difficult. The algorithms also tend to predict the entire beta strand rather than just the transmembrane region. Epitope insertion clearly pinpoints exposed loops but its utility in defining buried or periplasmic sites is less clear cut. Cysteine-mutant labelling is largely confined to exposed residues but one periplasmic cysteine may be labelled by reagents entering via the OmpG pore.

Structural insights into the secretin PulD and its trypsin-resistant core.

Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc. The PulD polypeptide comprises two major, structurally quite distinct domains; an N domain, which forms the walls of one of the chambers, and a trypsin-resistant C domain, which contributes to the outer chamber, the central disc, and the plug. The C domain contains a lower proportion of potentially transmembrane beta-structure than classical outer membrane proteins, suggesting that only a small part of it is embedded within the outer membrane. Indeed, the C domain probably extends well beyond the confines of the outer membrane bilayer, forming a centrally plugged channel that penetrates both the peptidoglycan on the periplasmic side and the lipopolysaccharide and capsule layers on the cell surface. The inner chamber is proposed to constitute a docking site for the secreted exoprotein pullulanase, whereas the outer chamber could allow displacement of the plug to open the channel and permit the exoprotein to escape.

Dimeric configuration of SeqA protein bound to a pair of hemi-methylated GATC sequences.

The binding of SeqA protein to hemi-methylated GATC sequences (hemi-sites) regulates chromosome initiation and the segregation of replicated chromosome in Escherichia coli. We have used atomic force microscopy to examine the architecture of SeqA and the mode of binding of one molecule of SeqA to a pair of hemi-sites in aqueous solution. SeqA has a bipartite structure composed of a large and a small lobe. Upon binding of a SeqA molecule to a pair of hemi-sites, the larger lobe becomes visibly separated into two DNA binding domains, each of which binds to one hemi-site. The two DNA binding domains are held together by association between the two multimerization domains that make up the smaller lobe. The binding of each DNA binding domain to a hemi-site leads to bending of the bound DNA inwards toward the bound protein. In this way, SeqA adopts a dimeric configuration when bound to a pair of hemi-sites. Mutational analysis of the multimerization domain indicates that, in addition to multimerization of SeqA polypeptides, this domain contributes to the ability of SeqA to bind to a pair of hemi-sites and to its cooperative behavior.