Explore the vast range of titles available.

Bacillus thuringiensis Vip3 ( V egetative I nsecticidal P rotein 3 ) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins. Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. Here authors present the cryo-EM structures of the protoxin and the protease-activated state of Vip3Aa which shed light on the molecular basis for Vip3 activation.

Reversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. Autophosphorylation in a dimeric sensor histidine kinase is the first step in two-component signalling, the predominant signal-transduction device in bacteria. Despite being the most abundant sensor kinases in nature, the molecular bases of the histidine kinase autophosphorylation mechanism are still unknown. Furthermore, it has been demonstrated that autophosphorylation can occur in two directions, cis (intrasubunit) or trans (intersubunit) within the dimeric histidine kinase. Here, we present the crystal structure of the complete catalytic machinery of a chimeric histidine kinase. The structure shows an asymmetric histidine kinase dimer where one subunit is caught performing the autophosphorylation reaction. A structure-guided functional analysis on HK853 and EnvZ, two prototypical cis- and trans -phosphorylating histidine kinases, has allowed us to decipher the catalytic mechanism of histidine kinase autophosphorylation, which seems to be common independently of the reaction directionality. The phosphorylation of proteins is a common mechanism for signal transduction. Here, the authors present a structural analysis of a histidine kinase in the process of autophosphorylation, helping to elucidate the catalytic mechanism.

Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853–RR468 and EnvZ–OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH. Bacterial two-component systems are composed of a sensor histidine kinase (HK) and an effector response regulator and upon signal detection, the HK autophosphorylates a conserved His residue. Here the authors structurally and functionally characterise two HKs, HK853–RR468 and EnvZ–OmpR, and find that the rotamer of the phosphorylatable catalytic His is not influenced by the environmental pH, ruling out an earlier proposed pH-gated model.

Vip3 proteins are produced by Bacillus thuringiensis and are toxic against lepidopterans, reason why the vip3Aa gene has been introduced into cotton and corn to control agricultural pests. Recently, the structure of Vip3 proteins has been determined and consists of a tetramer where each monomer is composed of five structural domains. The transition from protoxin to the trypsin‐activated form involves a major conformational change of the N‐terminal Domain I, which is remodelled into a tetrameric coiled‐coil structure that is thought to insert into the apical membrane of the midgut cells. To better understand the relevance of this major change in Domain I for the insecticidal activity, we have generated several mutants aimed to alter the activity and remodelling capacity of this central region to understand its function. These mutants have been characterized by proteolytic processing, negative staining electron microscopy, and toxicity bioassays against Spodoptera exigua. The results show the crucial role of helix α1 for the insecticidal activity and in restraining the Domain I in the protoxin conformation, the importance of the remodelling of helices α2 and α3, the proteolytic processing that takes place between Domains I and II, and the role of the C‐t Domains IV and V to sustain the conformational change necessary for toxicity. To better understand the relevance of the major change in Domain I of Vip3 proteins in the transition from protoxin to activated toxin for the insecticidal activity, we have generated several Vip3Aa mutants aimed to alter the activity and remodelling capacity of this central region to understand its function. These mutants have been characterized by proteolytic processing, negative staining electron microscopy, and toxicity bioassays against Spodoptera exigua. The results show the crucial role of helix α1 for the insecticidal activity and for restraining the Domain I in the protoxin conformation, the importance of the remodelling of helices α2 and α3, the proteolytic processing that takes place between Domains I and II, and the role of the C‐t Domains IV and V to sustain the conformational change necessary for toxicity.

Summary Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P. acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6‐DMP, and non‐conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well‐studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties. Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P. acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6‐DMP, and non‐conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well‐studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties.

Protein phosphorylation is a universal regulatory mechanism, controlling virtually all aspects of bacterial physiology and pathogenesis, yet histidine phosphorylation remains among the least understood. The histidine phosphocarrier protein HPr not only drives bacterial glucose transmembrane uptake through the phosphotransferase system (PTS), but also controls key enzymes for central carbon metabolism, including glycogen phosphorylase (GP). Here we report cryoEM structures of multimeric Escherichia coli Ec GP and their complexes with Ec HPr. The EM maps reveal an unanticipated density at H806 of Ec GP, consistent with histidine phosphorylation within a histidine-rich pocket at the N-terminal domain. Enzymatic assays reveal that Ec HPr transfers a phosphoryl group to the N 1 position of a histidine residue in Ec GP. Through an integrative structural, mutational and functional approach, we uncover the molecular basis of Ec HPr- Ec GP selectivity and define the allosteric mechanism by which Ec HPr regulates Ec GP. We establish histidine phosphorylation as a mechanism of GP regulation, expanding the traditional paradigm of glycogen metabolism control in bacteria. Glycogen serves as an intracellular energy storage polymer while some bacteria also produce structurally diverse extracellular α-glucans. Here, the authors establish the molecular basis of phosphorylation and allosteric regulation of glycogen phosphorylase by histidine phosphocarrier protein HPr.

n human pathogenic fungi, receiver domains from hybrid histidine kinases (hHK) have to recognize one HPt. To understand the recognition mechanism, we have assessed phosphorelay from receiver domains of five hHKs of group III, IV, V, VI, and XI to HPt from Chaetomium thermophilum and obtained the structures of Ct_HPt alone and in complex with the receiver domain of hHK group VI. Our data indicate that receiver domains phosphotransfer to Ct_HPt, show a low affinity for complex formation, and prevent a Leu-Thr switch to stabilize phosphoryl groups, also derived from the structures of the receiver domains of hHK group III and Candida albicans Sln1. Moreover, we have elucidated the envelope structure of C. albicans Ypd1 using small-angle X-ray scattering which reveals an extended flexible conformation of the long loop αD-αE which is not involved in phosphotransfer. Finally, we have analyzed the role of salt bridges in the structure of Ct_HPt alone.

Quality assurance and food safety are of great concern within the food industry because of unknown quantities of allergens often present in food. Therefore, there is an ongoing need to develop rapid, sensitive, and easy to use methods that serve as an alternative to mass spectrometry and enzyme-linked immunosorbent assay (ELISA) for monitoring food safety. Lateral flow immunoassay is one of the most used point-of-need devices for clinical, environmental, and food safety applications. Compared to traditional methods, it appears to be a simple and fast alternative for detecting food allergens. However, its reliability is frequently questioned due to the lack of quantitative information. In this study, a lateral flow microimmunoassay (LFµIA) is presented that integrates up to 36 spots in microarray format in a single strip, providing semi-quantitative information about the level of allergens, positive and negative controls, internal calibration, and hook effect. The LFµIA has been evaluated for the on-site simultaneous and reliable quantification of almond and peanut allergens as a proof of concept, demonstrating high sensitivity (185 and 229 µg/kg, respectively), selectivity (77%), and accuracy (RSD 5–25%) when analyzing commercial allergen-suspicious food consumables.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nature Communications 5: Article number: 3258 (2014); Published: 6 February 2014; Updated: 14 April 2014. Previous work by Diensthuber et al. describing the structure of the YF1 histidine kinase was inadvertently omitted from the reference list of this Article and should have been cited in place of reference 8 at instances where YF1 is referred to.