Crystal Structure of the Minor Pilin FctB Reveals Determinants of Group A Streptococcal Pilus Anchoring

Cell surface pili are polymeric protein assemblies that enable bacteria to adhere to surfaces and to specific host tissues. The pili expressed by Gram-positive bacteria constitute a unique paradigm in which sortase-mediated covalent linkages join successive pilin subunits like beads on a string. These pili are formed from two or three distinct types of pilin subunit, typically encoded in small gene clusters, often with their cognate sortases. In Group A streptococci (GAS), a major pilin forms the polymeric backbone, whereas two minor pilins are located at the tip and the base. Here, we report the 1.9-Å resolution crystal structure of the GAS basal pilin FctB, revealing an immunoglobulin (Ig)-like N-terminal domain with an extended proline-rich tail. Unexpected structural homology between the FctB Ig-like domain and the N-terminal domain of the GAS shaft pilin helps explain the use of the same sortase for polymerization of the shaft and its attachment to FctB. It also enabled the identification, from mass spectral data, of the lysine residue involved in the covalent linkage of FctB to the shaft. The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal (cell wall-anchoring) pilins. Together, our results indicate distinct structural elements in the pilin proteins that play a role in selecting for the appropriate sortases and thereby help orchestrate the ordered assembly of the pilus.     

Cryo-electron Microscopy Reveals the Structure of the Nuclear Pore Complex

The nuclear pore complex (NPC) is a giant protein assembly that penetrates the double layers of the nuclear membrane. The overall structure of the NPC has approximately eightfold symmetry and is formed by approximately 30 nucleoporins. The great size and complexity of the NPC have hindered the study of its structure for many years until recent breakthroughs were achieved by integrating the latest high-resolution cryo-electron microscopy (cryo-EM), the emerging artificial intelligence-based modeling and all other available structural information from crystallography and mass spectrometry. Here, we review our latest knowledge of the NPC architecture and the history of its structural study from in vitro to in situ with progressively improved resolutions by cryo-EM, with a particular focus on the latest subnanometer-resolution structural studies. The future directions for structural studies of NPCs are also discussed.     

Structure of a GRK5-Calmodulin Complex Reveals Molecular Mechanism of GRK Activation and Substrate Targeting

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The Structure of Herpesvirus Fusion Glycoprotein B-Bilayer Complex Reveals the Protein-Membrane and Lateral Protein-Protein Interaction

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Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis

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Distinctive Structure of the EphA3/Ephrin-A5 Complex Reveals a Dual Mode of Eph Receptor Interaction for Ephrin-A5

The Eph receptor tyrosine kinase/ephrin ligand system regulates a wide spectrum of physiological processes, while its dysregulation has been implicated in cancer progression. The human EphA3 receptor is widely upregulated in the tumor microenvironment and is highly expressed in some types of cancer cells. Furthermore, EphA3 is among the most highly mutated genes in lung cancer and it is also frequently mutated in other cancers. We report the structure of the ligand-binding domain of the EphA3 receptor in complex with its preferred ligand, ephrin-A5. The structure of the complex reveals a pronounced tilt of the ephrin-A5 ligand compared to its orientation when bound to the EphA2 and EphB2 receptors and similar to its orientation when bound to EphA4. This tilt brings an additional area of ephrin-A5 into contact with regions of EphA3 outside the ephrin-binding pocket thereby enlarging the size of the interface, which is consistent with the high binding affinity of ephrin-A5 for EphA3. This large variation in the tilt of ephrin-A5 bound to different Eph receptors has not been previously observed for other ephrins.     

The Probabilistic Niche Model Reveals the Niche Structure and Role of Body Size in a Complex Food Web

The niche model has been widely used to model the structure of complex food webs, and yet the ecological meaning of the single niche dimension has not been explored. In the niche model, each species has three traits, niche position, diet position and feeding range. Here, a new probabilistic niche model, which allows the maximum likelihood set of trait values to be estimated for each species, is applied to the food web of the Benguela fishery. We also developed the allometric niche model, in which body size is used as the niche dimension. About 80% of the links in the empirical data are predicted by the probabilistic niche model, a significant improvement over recent models. As in the niche model, species are uniformly distributed on the niche axis. Feeding ranges are exponentially distributed, but diet positions are not uniformly distributed below the predator. Species traits are strongly correlated with body size, but the allometric niche model performs significantly worse than the probabilistic niche model. The best-fit parameter set provides a significantly better model of the structure of the Benguela food web than was previously available. The methodology allows the identification of a number of taxa that stand out as outliers either in the model''s poor performance at predicting their predators or prey or in their parameter values. While important, body size alone does not explain the structure of the one-dimensional niche.     

Structure of the Toxoplasma gondii ROP18 Kinase Domain Reveals a Second Ligand Binding Pocket Required for Acute Virulence

At least a third of the human population is infected with the intracellular parasite Toxoplasma gondii, which contributes significantly to the disease burden in immunocompromised and neutropenic hosts and causes serious congenital complications when vertically transmitted to the fetus. Genetic analyses have identified the Toxoplasma ROP18 Ser/Thr protein kinase as a major factor mediating acute virulence in mice. ROP18 is secreted into the host cell during the invasion process, and its catalytic activity is required for the acute virulence phenotype. However, its precise molecular function and regulation are not fully understood. We have determined the crystal structure of the ROP18 kinase domain, which is inconsistent with a previously proposed autoinhibitory mechanism of regulation. Furthermore, a sucrose molecule bound to our structure identifies an additional ligand-binding pocket outside of the active site cleft. Mutational analysis confirms an important role for this pocket in virulence.     

Structure of an Arrestin2-Clathrin Complex Reveals a Novel Clathrin Binding Domain That Modulates Receptor Trafficking

Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin β-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formed by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I)₂GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.Many transmembrane signaling systems consist of specific G protein-coupled receptors (GPCRs)³ that transduce a diverse array of extracellular stimuli into intracellular signaling events (1). GPCRs modulate the activity of numerous effector molecules and regulate multiple biological functions including neurotransmission, sensory perception, cardiovascular function, development, and cell growth and differentiation (2). To ensure that extracellular stimuli are translated into intracellular signals of appropriate magnitude and duration, these signaling cascades are tightly regulated. GPCRs are subject to three principle modes of regulation; 1) desensitization, in which a receptor becomes refractory to continued stimuli; 2) endocytosis, where receptors are removed from the cell surface; 3) down-regulation, where total receptor levels are decreased (3, 4). Agonist-dependent regulation is primarily mediated by GPCR kinases that specifically phosphorylate activated GPCRs and initiate the recruitment of arrestins. Arrestins are divided into two major classes, visual and non-visual, based on their localization and function. The non-visual arrestins, arrestin2 and 3 (also termed β-arrestin1 and -2, respectively), are broadly distributed and function in multiple processes including GPCR desensitization, trafficking, and signaling (4–6).Initial structural insight on arrestins was provided by the x-ray crystal structure of bovine arrestin1 (7, 8), whereas the crystal structures of C-terminal-truncated (9) and wild type (10) bovine arrestin2 and salamander arrestin4 (11) have also been solved. In general, arrestins are composed of two major domains made up of β strands and connecting loops that are held together by a polar core region consisting of buried salt bridges. It has been proposed that arrestins adopt an active conformation upon binding to phosphorylated receptors, which disrupts the polar core resulting in the release of the C-terminal tail (12). Disruption of the polar core by point mutation of Arg-169 generates a constitutively active arrestin2, which mimics the active state. This mutated arrestin binds to the β₂-adrenergic receptor (β₂AR) in a phosphorylation-independent manner, induces internalization of a δ-opioid receptor lacking phosphorylation sites (13), and has increased binding to clathrin and AP-2 (14).A role for non-visual arrestins in GPCR endocytosis was first described for the β₂AR (15, 16), although it is now evident that arrestins regulate the trafficking of multiple GPCRs as well as additional classes of receptors (4). An early step in this process involves arrestin binding to an activated phosphorylated receptor that enhances arrestin interaction with the endocytic proteins, clathrin, and AP-2 (16, 17). An additional important step in this process involves arrestin interaction with phosphoinositides such as phosphatidylinositol diphosphate and trisphosphate (18). Although the dynamics of these interactions have not been studied, arrestin2 and -3 have been shown to interact specifically and stoichiometrically with clathrin (16). Furthermore, fluorescence microscopy reveals that activated β₂AR, arrestin2, clathrin, and AP-2 all colocalize upon receptor stimulation (16). The primary clathrin binding determinant in arrestin2, LIELD, spans residues 376–380 and is located in an extended disordered loop that immediately precedes the final C-terminal β-strand (10, 19). This region, the clathrin binding box, is consistent with a consensus motif, LϕXϕ(D/E) (where ϕ is a bulky hydrophobic residue, and X represents any polar amino acid), established in other clathrin-binding proteins including AP-2 (20), AP180 (21), amphiphysin (22), and epsin (23). Importantly, the mutation of this motif in arrestin3 and its deletion in arrestin2 significantly disrupts clathrin binding and receptor endocytosis (14, 19). A mutagenesis study of clathrin localized an arrestin binding site to the N-terminal domain of the clathrin heavy chain, specifically residues Glu-89, Lys-96, and Lys-98 (24). Moreover, a crystal structure of clathrin-(1–363) in complex with an arrestin3 peptide (residues 369–381) supports the mutagenesis data and the predicted location of the arrestin-clathrin interaction site (25).To further elucidate the mechanisms involved in mediating arrestin/clathrin interaction, we have determined the crystal structure of clathrin with the short (arrestin2S) and long (arrestin2L) isoforms of arrestin2, which differ by an 8-amino acid insert between β strands 18 and 19 (26). Our results identify an additional and unique interaction encoded in the arrestin2L isoform that is distinct from the previously well characterized interaction involving the LϕXϕ(D/E) motif. Specifically, we observe that the 8 amino acid splice loop in arrestin2L interacts with a pocket formed by blades 4 and 5 in clathrin. Biochemical and cell biological analysis confirm a role for both binding sites in arrestin2L/clathrin interaction and demonstrate an essential role of these interactions in arrestin-mediated GPCR endocytosis.     

The Structure of the Staphylococcus aureus Sortase-Substrate Complex Reveals How the Universally Conserved LPXTG Sorting Signal Is Recognized

In Gram-positive bacteria, sortase enzymes assemble surface proteins and pili in the cell wall envelope. Sortases catalyze a transpeptidation reaction that joins a highly conserved LPXTG sorting signal within their polypeptide substrate to the cell wall or to other pilin subunits. The molecular basis of transpeptidation and sorting signal recognition are not well understood, because the intermediates of catalysis are short lived. We have overcome this problem by synthesizing an analog of the LPXTG signal whose stable covalent complex with the enzyme mimics a key thioacyl catalytic intermediate. Here we report the solution structure and dynamics of its covalent complex with the Staphylococcus aureus SrtA sortase. In marked contrast to a previously reported crystal structure, we show that SrtA adaptively recognizes the LPXTG sorting signal by closing and immobilizing an active site loop. We have also used chemical shift mapping experiments to localize the binding site for the triglycine portion of lipid II, the second substrate to which surface proteins are attached. We propose a unified model of the transpeptidation reaction that explains the functions of key active site residues. Since the sortase-catalyzed anchoring reaction is required for the virulence of a number of bacterial pathogens, the results presented here may facilitate the development of new anti-infective agents.Bacterial surface proteins function as virulence factors that enable pathogens to adhere to sites of infection, evade the immune response, acquire essential nutrients, and enter host cells (1). Gram-positive bacteria use a common mechanism to covalently attach proteins to the cell wall. This process is catalyzed by sortase transpeptidase enzymes, which join proteins bearing a highly conserved Leu-Pro-X-Thr-Gly (LPXTG, where X is any amino acid) sorting signal to the cross-bridge peptide of the peptidylglycan (2–4). Sortases also polymerize proteins containing sorting signals into pili, filamentous surface exposed structures that promote bacterial adhesion (5, 6). The search for small molecule sortase inhibitors is an active area of research, since these enzymes contribute to the virulence of a number of important pathogens, including among others Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, and Streptococcus pneumoniae (reviewed in Refs. 7 and 8). Sortase enzymes are also promising molecular biology reagents that can be used to site-specifically attach proteins to a variety of biomolecules (9–14, 72).The sortase A (SrtA)⁷ enzyme from S. aureus is the prototypical member of the sortase enzyme family (15, 16). It anchors proteins to the murein sacculus that possess a COOH-terminal cell wall sorting signal that consists of a LPXTG motif, followed by a hydrophobic segment of amino acids and a tail composed of mostly positively charged residues (17). SrtA is located on the extracellular side of the membrane. After partial secretion of its protein substrate across the cell membrane, SrtA cleaves the LPXTG motif between the threonine and glycine residues, forming a thioacyl-linked protein-sortase intermediate (16). It then catalyzes the formation of an amide bond between the carboxyl group of the threonine and the cell wall precursor molecule lipid II (undecaprenyl-pyrophosphate-MurNAc(-l-Ala-d-iGln-l-Lys(NH₂-Gly₅)-d-Ala-d-Ala)-β1–4-GlcNAc)), creating a protein-lipid II-linked product that is incorporated into the peptidylglycan via the transglycosylation and transpeptidation reactions of bacterial cell wall synthesis (18–20). Over 900 sortase-attached proteins in 72 different strains of bacteria have thus far been identified (21, 22). The vast majority of these proteins contain a COOH-terminal sorting signal harboring an LPXTG motif and are anchored to the cell wall by enzymes closely related to SrtA.In vitro studies of SrtA have begun to define the mechanism of transpeptidation. SrtA consists of two parts: an unstructured amino-terminal tail that contains a stretch of nonpolar residues that embed it in the membrane and an autonomously folded catalytic domain that competently performs the transpeptidation reaction in vitro (SrtA_ΔN59, residues 60–206) (16, 23–25). Catalysis occurs through a ping-pong mechanism that is initiated when the thiol group of amino acid Cys¹⁸⁴ nucleophilically attacks the carbonyl carbon of the threonine residue within the LPXTG sorting signal (16, 23–25). This forms a transient tetrahedral intermediate that, upon breakage of the threonine-glycine peptide bond, rearranges into a more stable thioacyl enzyme-substrate linkage. SrtA then joins the terminal amine group within the pentaglycine branch of lipid II to the carbonyl carbon of the threonine, creating a second tetrahedral intermediate that is resolved into the lipid II-linked protein product (23).Sortase enzymes contain three conserved residues within their active sites: His¹²⁰, Cys¹⁸⁴, and Arg¹⁹⁷ (SrtA numbering). These residues play a critical role in catalysis, since their mutation in SrtA causes severe reductions in enzyme activity (16, 26–30). Although it is well established that Cys¹⁸⁴ forms a covalent linkage to the sorting signal, the functions of His¹²⁰ and Arg¹⁹⁷ are controversial. A variety of disparate functions have been ascribed to Arg¹⁹⁷. These include deprotonating Cys¹⁸⁴ (28), deprotonating lipid II (31), or stabilizing the binding of either the LPXTG sorting signal (28, 32) or oxyanion intermediates (31, 32). Different functions have also been proposed for His¹²⁰. Originally, it was suggested that it activated Cys¹⁸⁴ by forming an imidazolium-thiolate ion pair (26). However, subsequent pK_a measurements revealed that both His¹²⁰ and Cys¹⁸⁴ are predominantly uncharged at physiological pH values, leading to the suggestion that His¹²⁰ functions as a general base during catalysis (33). Most recently, it has been proposed that the most active form of the enzyme contains His¹²⁰ and Cys¹⁸⁴ in their charged states but that only a small fraction of SrtA exists in this form (∼0.06%) prior to binding to the sorting signal (25).NMR and crystal structures of SrtA_ΔN59 have revealed that it adopts an eight-stranded β-barrel fold (31, 34). Other sortase enzymes have also been shown to possess a similar overall structure, including SrtB from S. aureus (27, 35), SrtB from Bacillus anthracis (27, 36), SrtA from S. pyogenes (37), and the SrtC-1 and SrtC-3 enzymes from S. pneumoniae (38). However, the molecular basis of substrate recognition remains poorly understood, because all of the structures reported to date have not contained a sorting signal bound to the enzyme. The lone exception is the crystal structure of SrtA_ΔN59 bound to an LPETG peptide (31). However, in this structure the peptide substrate is bound nonspecifically (see below) (32, 39).In this paper, we report the structure and dynamics of SrtA covalently bound to an analog of the LPXTG sorting signal. The structure of the complex resembles the thioacyl intermediate of catalysis, providing insights into the molecular basis of binding of the LPXTG sorting signal and the functions of key active site residues. Notably, the mechanism of substrate binding visualized in the NMR structure differs substantially from a previously reported crystal structure of SrtA_ΔN59 non-covalently bound to a LPETG peptide (31). We have also used NMR chemical shift mapping experiments to localize the binding site for a triglycine cell wall substrate analog. A mechanism of transpeptidation compatible with these new data is proposed.     

Crystal Structure of Dinitrogenase Reductase-activating Glycohydrolase (DRAG) Reveals Conservation in the ADP-Ribosylhydrolase Fold and Specific Features in the ADP-Ribose-binding Pocket

Protein-reversible ADP-ribosylation is emerging as an important post-translational modification used to control enzymatic and protein activity in different biological systems. This modification regulates nitrogenase activity in several nitrogen-fixing bacterial species. ADP-ribosylation is catalyzed by ADP-ribosyltransferases and is reversed by ADP-ribosylhydrolases. The structure of the ADP-ribosylhydrolase that acts on Azospirillum brasilense nitrogenase (dinitrogenase reductase-activating glycohydrolase, DraG) has been solved at a resolution of 2.5 Å. This bacterial member of the ADP-ribosylhydrolase family acts specifically towards a mono-ADP-ribosylated substrate. The protein shows an all-α-helix structure with two magnesium ions located in the active site. Comparison of the DraG structure with orthologues deposited in the Protein Data Bank from Archaea and mammals indicates that the ADP-ribosylhydrolase fold is conserved in all domains of life. Modeling of the binding of the substrate ADP-ribosyl moiety to DraG is in excellent agreement with biochemical data.     

The Pilin Protein FimP from Actinomyces oris: Crystal Structure and Sequence Analyses

The Actinomyces oris type-1 pili are important for the initial formation of dental plaque by binding to salivary proteins that adhere to the tooth surface. Here we present the X-ray structure of FimP, the protein that is polymerized into the type-1 pilus stalk, assisted by a pili-specific sortase. FimP consists of three tandem IgG-like domains. The middle and C-terminal domains contain one autocatalyzed intramolecular isopeptide bond each, a feature used by Gram-positive bacteria for stabilization of surface proteins. While the N-terminal domain harbours all the residues necessary for forming an isopeptide bond, no such bond is observed in the crystal structure of this unpolymerized form of FimP. The monomer is further stabilized by one disulfide bond each in the N- and C-terminal domains as well as by a metal-coordinated loop protruding from the C-terminal domain. A lysine, predicted to be crucial for FimP polymerization by covalent attachment to a threonine from another subunit, is located at the rim of a groove lined with conserved residues. The groove may function as a docking site for the sortase-FimP complex. We also present sequence analyses performed on the genes encoding FimP as well as the related FimA, obtained from clinical isolates.     

Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus

Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and β Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/β globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins.     

The Three-dimensional Structure of an Ionotropic Glutamate Receptor Reveals a Dimer-of-dimers Assembly

The ionotropic glutamate receptors (iGluRs) represent a major family of ion channels whose quaternary structure has not yet been defined. Here, we present the three-dimensional structure of a fully assembled iGluR, determined at approximately 20A resolution by electron microscopy. Analysis of negatively stained single-particle images reveals the presence of 2-fold, but not 4-fold, symmetry for these tetrameric channels, providing the first direct structural evidence for a dimer-of-dimers assembly. The receptor appears elongated, measuring approximately 170Ax140Ax110A, with the 2-fold symmetry centered on its longitudinal axis. The overall molecular shape and symmetry suggest an orientation relative to the membrane and permit the identification of a putative transmembrane domain. Internal cavities located along the longitudinal axis may represent components of the ion conduction pathway.     

Study of the Individual Cytochrome b5 and Cytochrome b5 Reductase Domains of Ncb5or Reveals a Unique Heme Pocket and a Possible Role of the CS Domain

NADH cytochrome b₅ oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b₅ (b₅), CHORD-SGT1 (CS), and cytochrome b₅ reductase (b₅R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b₅ and b₅R domains (Ncb5or-b₅ and Ncb5or-b₅R, respectively) and compared them with human microsomal b₅ (Cyb5A) and b₅R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b₅ reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His⁸⁹ and His¹¹², consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b₅ family shown to have such a heme environment. Like other b₅ family members, Ncb5or-b₅ has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b₅ differs from Cyb5A with respect to location of the second heme ligand (His¹¹²) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b₅R to Ncb5or-b₅ is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b₅ and b₅R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b₅R domains suggest that the CS domain facilitates docking of the b₅ and b₅R domains. Trp¹¹⁴ is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b₅R domain to the b₅ domain.     

Genome Re-Sequencing of Semi-Wild Soybean Reveals a Complex Soja Population Structure and Deep Introgression

Semi-wild soybean is a unique type of soybean that retains both wild and domesticated characteristics, which provides an important intermediate type for understanding the evolution of the subgenus Soja population in the Glycine genus. In this study, a semi-wild soybean line (Maliaodou) and a wild line (Lanxi 1) collected from the lower Yangtze regions were deeply sequenced while nine other semi-wild lines were sequenced to a 3-fold genome coverage. Sequence analysis revealed that (1) no independent phylogenetic branch covering all 10 semi-wild lines was observed in the Soja phylogenetic tree; (2) besides two distinct subpopulations of wild and cultivated soybean in the Soja population structure, all semi-wild lines were mixed with some wild lines into a subpopulation rather than an independent one or an intermediate transition type of soybean domestication; (3) high heterozygous rates (0.19–0.49) were observed in several semi-wild lines; and (4) over 100 putative selective regions were identified by selective sweep analysis, including those related to the development of seed size. Our results suggested a hybridization origin for the semi-wild soybean, which makes a complex Soja population structure.     

Crystal Structure of Clostridium botulinum Whole Hemagglutinin Reveals a Huge Triskelion-shaped Molecular Complex

Clostridium botulinum HA is a component of the large botulinum neurotoxin complex and is critical for its oral toxicity. HA plays multiple roles in toxin penetration in the gastrointestinal tract, including protection from the digestive environment, binding to the intestinal mucosal surface, and disruption of the epithelial barrier. At least two properties of HA contribute to these roles: the sugar-binding activity and the barrier-disrupting activity that depends on E-cadherin binding of HA. HA consists of three different proteins, HA1, HA2, and HA3, whose structures have been partially solved and are made up mainly of β-strands. Here, we demonstrate structural and functional reconstitution of whole HA and present the complete structure of HA of serotype B determined by x-ray crystallography at 3.5 Å resolution. This structure reveals whole HA to be a huge triskelion-shaped molecule. Our results suggest that whole HA is functionally and structurally separable into two parts: HA1, involved in recognition of cell-surface carbohydrates, and HA2-HA3, involved in paracellular barrier disruption by E-cadherin binding.