Specific DNA recognition mediated by a type IV pilin Cehovin, Ana; Simpson, Peter J.; McDowell, Melanie A. ...
Proceedings of the National Academy of Sciences - PNAS,
02/2013, Letnik:
110, Številka:
8
Journal Article
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Natural transformation is a dominant force in bacterial evolution by promoting horizontal gene transfer. This process may have devastating consequences, such as the spread of antibiotic resistance or ...the emergence of highly virulent clones. However, uptake and recombination of foreign DNA are most often deleterious to competent species. Therefore, model naturally transformable Gram-negative bacteria, including the human pathogen Neisseria meningitidis , have evolved means to preferentially take up homotypic DNA containing short and genus-specific sequence motifs. Despite decades of intense investigations, the DNA uptake sequence receptor in Neisseria species has remained elusive. We show here, using a multidisciplinary approach combining biochemistry, molecular genetics, and structural biology, that meningococcal type IV pili bind DNA through the minor pilin ComP via an electropositive stripe that is predicted to be exposed on the filaments surface and that ComP displays an exquisite binding preference for DNA uptake sequence. Our findings illuminate the earliest step in natural transformation, reveal an unconventional mechanism for DNA binding, and suggest that selective DNA uptake is more widespread than previously thought.
The Notch signaling pathway is essential for many aspects of development, cell fate determination, and tissue homeostasis. Notch signaling can be modulated by posttranslational modifications to the ...Notch receptor, which are known to alter both ligand binding and receptor activation. We have modified the ligand-binding region (EGF domains 11–13) of human Notch1 (hN1) with O -fucose and O -glucose glycans and shown by flow cytometry and surface plasmon resonance that the Fringe-catalyzed addition of GlcNAc to the O -fucose at T466 in EGF12 substantially increases binding to Jagged1 and Delta-like 1 (DLL1) ligands. We have subsequently determined the crystal structures of EGF domains 11–13 of hN1 modified with either the O -fucose monosaccharide or the GlcNAc–fucose disaccharide at T466 of EGF12 and observed no change in backbone structure for each variant. Collectively, these data demonstrate a role for GlcNAc in modulating the ligand-binding site in hN1 EGF12, resulting in an increased affinity of this region for ligands Jagged1 and DLL1. We propose that this finding explains the Fringe-catalyzed enhancement of Notch–Delta signaling observed in flies and humans, but suggest that the inhibitory effect of Fringe on Jagged/Serrate mediated signaling involves other regions of Notch.
The type VII protein secretion system (T7SS) is found in many Gram-positive bacteria and in pathogenic mycobacteria. All T7SS substrate proteins described to date share a common helical domain ...architecture at the N-terminus that typically interacts with other helical partner proteins, forming a composite signal sequence for targeting to the T7SS. The C-terminal domains are functionally diverse and in Gram-positive bacteria such as Staphylococcus aureus often specify toxic anti-bacterial activity. Here we describe the first example of a class of T7 substrate, TslA, that has a reverse domain organisation. TslA is widely found across Bacillota including Staphylococcus, Enterococcus and Listeria. We show that the S. aureus TslA N-terminal domain is a phospholipase A with anti-staphylococcal activity that is neutralised by the immunity lipoprotein TilA. Two small helical partner proteins, TlaA1 and TlaA2 are essential for T7-dependent secretion of TslA and at least one of these interacts with the TslA C-terminal domain to form a helical stack. Cryo-EM analysis of purified TslA complexes indicate that they share structural similarity with canonical T7 substrates. Our findings suggest that the T7SS has the capacity to recognise a secretion signal present at either end of a substrate.
The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant ...chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism.
The Notch receptor is a key component of a core metazoan signaling pathway activated by Delta/Serrate/Lag-2 ligands expressed on an adjacent cell. This results in a short-range signal with profound ...effects on cell-fate determination, cell proliferation, and cell death. Key to understanding receptor function is structural knowledge of the large extracellular portion of Notch which contains multiple repeats of epidermal growth factor (EGF)-like domains. Here we investigate the EGF4-13 region of human Notch1 (hN1) using a multidisciplinary approach. Ca2+-binding measurements, X-ray crystallography, {1H}-15N heteronuclear nuclear Overhauser effects, and residual dipolar couplings support a non-linear organization for the EGF4-13 region with a rigid, bent conformation for EGF4-7 and a single flexible linkage between EGF9 and EGF10. These data allow us to construct an informed model for EGF10-13 which, in conjunction with comparative binding studies, demonstrates that EGF10 has an important role in determining Notch receptor sensitivity to Dll-4.
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•The EGF4-13 region of human Notch1 contains both bent and flexible interfaces•This changes the classical view of the receptor as a rigid linear structure•Extra interaction sites of Notch with ligand may occur along its longitudinal axis•Interfaces next to ligand-binding site differentially modulate ligand interactions
The structure of the extracellular domain of Notch receptor is key to understanding Notch biology. Weisshuhn et al. identify both bent and flexible regions within the receptor, changing the classical view of the receptor as a rigid linear structure extending from the cell surface.
Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune ...responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches.
The bacterial flagellum is a macromolecular protein complex that enables motility in many species. Bacterial flagella self-assemble a strong, multicomponent drive shaft that couples rotation in the ...inner membrane to the micrometre-long flagellar filament that powers bacterial swimming in viscous fluids
. Here, we present structures of the intact Salmonella flagellar basal body
, encompassing the inner membrane rotor, drive shaft and outer-membrane bushing, solved using cryo-electron microscopy to resolutions of 2.2-3.7 Å. The structures reveal molecular details of how 173 protein molecules of 13 different types assemble into a complex spanning two membranes and a cell wall. The helical drive shaft at one end is intricately interwoven with the rotor component with both the export gate complex and the proximal rod forming interactions with the MS-ring. At the other end, the drive shaft distal rod passes through the LP-ring bushing complex, which functions as a molecular bearing anchored in the outer membrane through interactions with the lipopolysaccharide. The in situ structure of a protein complex capping the drive shaft provides molecular insights into the assembly process of this molecular machine.
•Flagellar and non-flagellar T3SS are built assembling homologous protein machineries.•Unified nomenclature for non-flagellar T3SS.•New model of the T3SS needle is consistent with the flagellar ...filament, both in terms of helical parameters and orientation.•Structural and functional implication of the new architecture of the T3SS export apparatus and ATPase complex.
To fulfill complex biological tasks, such as locomotion and protein translocation, bacteria assemble macromolecular nanomachines. One such nanodevice, the type III secretion system (T3SS), has evolved to provide a means of transporting proteins from the bacterial cytoplasm across the periplasmic and extracellular spaces. T3SS can be broadly classified into two highly homologous families: the flagellar T3SS which drive cell motility, and the non-flagellar T3SS (NF-T3SS) that inject effector proteins into eukaryotic host cells, a trait frequently associated with virulence. Although the structures and symmetries of ancillary components of the T3SS have diversified to match requirements of different species adapted to different niches, recent genetic, molecular and structural studies demonstrate that these systems are built by arranging homologous modular protein assemblies.
Gliding motility using cell surface adhesins, and export of proteins by the type IX secretion system (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are ...energized by the GldLM motor complex, which transduces the proton motive force at the inner membrane into mechanical work at the outer membrane. We previously used cryo-electron microscopy to solve the structure of the GldLM motor core from Flavobacterium johnsoniae at 3.9-Å resolution (R. Hennell James, J. C. Deme, A. Kjaer, F. Alcock, et al., Nat Microbiol 6:221-233, 2021, https://dx.doi.org/10.1038/s41564-020-00823-6). Here, we present structures of homologous complexes from a range of pathogenic and environmental
species at up to 3.0-Å resolution. These structures show that the architecture of the GldLM motor core is conserved across the
phylum, although there are species-specific differences at the N terminus of GldL. The resolution improvements reveal a cage-like structure that ties together the membrane-proximal cytoplasmic region of GldL and influences gliding function. These findings add detail to our structural understanding of bacterial ion-driven motors that drive the T9SS and gliding motility.
Many bacteria in the
phylum use the type IX secretion system to secrete proteins across their outer membrane. Most of these bacteria can also glide across surfaces using adhesin proteins that are propelled across the cell surface. Both secretion and gliding motility are driven by the GldLM protein complex, which forms a nanoscale electrochemical motor. We used cryo-electron microscopy to study the structure of the GldLM protein complex from different species, including the human pathogens Porphyromonas gingivalis and Capnocytophaga canimorsus. The organization of the motor is conserved across species, but we find species-specific structural differences and resolve motor features at higher resolution. This work improves our understanding of the type IX secretion system, which is a virulence determinant in human and animal diseases.
Recent data have expanded our understanding of Notch signalling by identifying a C2 domain at the N‐terminus of Notch ligands, which has both lipid‐ and receptor‐binding properties. We present novel ...structures of human ligands Jagged2 and Delta‐like4 and human Notch2, together with functional assays, which suggest that ligand‐mediated coupling of membrane recognition and Notch binding is likely to be critical in establishing the optimal context for Notch signalling. Comparisons between the Jagged and Delta family show a huge diversity in the structures of the loops at the apex of the C2 domain implicated in membrane recognition and Jagged1 missense mutations, which affect these loops and are associated with extrahepatic biliary atresia, lead to a loss of membrane recognition, but do not alter Notch binding. Taken together, these data suggest that C2 domain binding to membranes is an important element in tuning ligand‐dependent Notch signalling in different physiological contexts.
Synopsis
Notch ligands possess variable lipid‐binding domains that mediate interaction with membranes of diverse lipid composition. A complex formation between Notch ligands, membrane lipids and Notch is required for efficient Notch signalling, and is disrupted in Jagged1 mutations associated with extrahepatic biliary atresia.
New crystal structures for human Notch ligands Jagged2 and Delta‐like4 show variation in the ligand C2 domain lipid‐binding region, which is reflected by their binding preferences to liposomes of different compositions.
Liposomes show enhanced binding to most ligands in the presence of Notch, suggesting a crosstalk between lipid and Notch binding.
Selective loss of membrane binding appears to underlie defective Notch activation associated with a subset of Jagged1 C2 domain disease‐causing variants.
The data suggest an important role for membrane binding in fine‐tuning the Notch signal in specific physiological contexts.
Structural variability in the C2 domain of Notch ligands determines membrane binding and regulates Notch activation, a role that is lost in disease‐associated Jagged1 variants.
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