Search

The mitochondrial Complex I assembly (MCIA) complex is an essential player in the biogenesis of respiratory Complex I (CI), the multiprotein complex responsible for the initiation of oxidative phosphorylation (OXPHOS). It is not well understood how MCIA facilitates the assembly of CI. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, resulting in efflux of the FAD cofactor and redeployment of ACAD9 from fatty acid β-oxidation (FAO) to CI assembly. We identify an adjacent α-helix as a key structural element that specifically enables the CI assembly functionality of ACAD9, distinguishing it from its closely related VLCAD counterpart. Furthermore, we show that ECSIT is phosphorylatedin vitroandex celluloand provide evidence that phosphorylation downregulates its association with ACAD9. Interestingly, ECSIT has previously been linked to the pathogenesis of Alzheimer’s disease and here we show that ECSIT phosphorylation in neuronal cells is reduced upon exposure to amyloid-β (Aβ) oligomers.These findings shed light on the assembly of the MCIA complex and implicate ECSIT as a potential reprogrammer of bioenergetic metabolic pathways that can be altered when mitochondria are affected by Aβ toxicity, a hallmark of Alzheimer’s disease.

Lipid mesophases are lyotropic liquid crystalline systems which differ from liposomes and other globular aggregates in dilute regimes due to their inner ordering. It is known that natural lipids enable to obtain a rich variety of nanosystems and many of them have been proposed as delivery agents for bioactive compounds. Due to their packing parameters, several classes of lipids found in natural sources are able to self-assemble into nonlamellar structures. Among lipids occurring in plants and algae, triglycerides display this tendency. In the present study we examine new nanosystems built with lipids extracted from the marine microalga Nannochloropsis sp and their use as carriers for lipophilic antioxidants. The antioxidants studied, curcumin and tocopherol were encapsulated with high rate in the carriers. The physico-chemical characterization of plain and loaded vectors showed their structure and localization site, as well as the structure-functionality relationship related to potential drug delivery. The results show that the cargo molecules play an active role in driving the interactions which characterize the overall structure of the aggregates. The systems studied showed several coexisting mesophases, the most predominant structure being of cubic symmetry.

Lipid-based soft nanovectors are widely used for delivery purposes in many fields of applications. Although it is generally considered a class of molecules with superior biocompatibility, in recent times researchers have expressed concerns with regards to the sustainability of preparation procedures. In particular, when large-scale production is involved, as in the case of agro-nanotechnology applications, this becomes a most pressing issue. Recently, we proposed novel green nanoformulations for the delivery of phytohormones (i.e., indole-3-butyric acid and 1-naphthaleneacetic acid) to Olea europaea L., obtained from agricultural waste derived from the plant itself, which yielded promising results, especially when engineered by addition of pure phospholipids in small amounts (10% w/w). Here we have performed an advanced analysis of high-resolution structural data from X-ray and neutron scattering (SAXS/SANS) and cryo-transmission electron microscopy (cryo-TEM). The results obtained from these combined techniques were complementary and fully consistent, evidencing the fundamental role of the employed adjuvant phospholipid in dictating the structural arrangement.

A likelihood-based method is improved to correct for dynamical scattering in weakly diffracting protein nanocrystals. The method has been tested on three protein crystals and yielded better refinement statistics and structure interpretations, including charge estimations of bound ions., Electron diffraction allows protein structure determination when only nanosized crystals are available. Nevertheless, multiple elastic (or dynamical) scattering, which is prominent in electron diffraction, is a concern. Current methods for modeling dynamical scattering by multi-slice or Bloch wave approaches are not suitable for protein crystals because they are not designed to cope with large molecules. Here, dynamical scattering of nanocrystals of insulin, thermolysin and thaumatin was limited by collecting data from thin crystals. To accurately measure the weak diffraction signal from the few unit cells in the thin crystals, a low-noise hybrid pixel Timepix electron-counting detector was used. The remaining dynamical component was further reduced in refinement using a likelihood-based correction, which was introduced previously for analyzing electron diffraction data of small-molecule nanocrystals and was adapted here for protein crystals. The procedure is shown to notably improve the structural refinement, in one case allowing the location of solvent molecules. It also allowed refinement of the charge states of bound metal atoms, an important element in protein function, through B-factor analysis of the metal atoms and their ligands. These results clearly increase the value of macromolecular electron crystallography as a complementary structural biology technique.

International audience; Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid stress-response regulation of Escherichia coli LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed E. coli cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-EM and reveal the molecular determinants of LdcI polymerization, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organization in the acid stress response.

Significance Bacteria possess a sophisticated arsenal of defense mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, Escherichia coli and many other enterobacteria activate a proton-consuming resistance system based on the acid stress-inducible lysine decarboxylase LdcI. Here we develop generally applicable tools to uncover the spatial localization of LdcI inside the cell by superresolution fluorescence microscopy and investigate the in vitro supramolecular organization of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations., Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid stress-response regulation of Escherichia coli LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed E. coli cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-EM and reveal the molecular determinants of LdcI polymerization, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organization in the acid stress response.

Electron diffraction allows protein structure determination when only nanosized crystals are available. Nevertheless, multiple elastic (or dynamical) scattering, prominent in electron diffraction, is a concern. Current methods for modeling dynamical scattering by multi-slice or Bloch wave approaches are not suitable for protein crystals because they are not designed to cope with large molecules. Here, we limited dynamical scattering of nanocrystals of insulin, thermolysin, and thaumatin by collecting data from thin crystals. To accurately measure the weak diffraction signal from the few unit cells in the thin crystals, we used a low-noise hybrid-pixel Timepix electron counting detector. The remaining dynamical component was further reduced in refinement using a likelihood-based correction, which we introduced previously for analyzing electron diffraction data of small molecule nanocrystals and adapted here for protein crystals. We show that the procedure notably improved the structural refinement, allowing in one case the location of solvent molecules. It also allowed the refinement of the charge state of bound metal atoms, an important element in protein functions, through B-factor analysis of the metal atoms and their ligands. Our results clearly increase the value of macromolecular electron crystallography as a complementary structural biology technique.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysical characterisation with negative stain and cryo-electron microscopy, and wide-field and super-resolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localisation of nanobody-labelled endogenous wild-type LdcI in acid-stressed E. coli cells, and show that it organises into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerisation as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-electron microscopy, and reveal the molecular determinants of LdcI polymerisation, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organisation in the acid stress response.Significance statementBacteria possess a sophisticated arsenal of defence mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, E. coli and many other enterobacteria activate a proton-consuming resistance system based on the acid-stress inducible lysine decarboxylase LdcI. Here we develop generally-applicable tools to uncover the spatial localisation of LdcI inside the cell by super-resolution fluorescence microscopy, and investigate the in vitro supramolecular organisation of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations.

The phAPEC6 genome encodes 551 predicted gene products, with the vast majority (83%) of unknown function. Of these, 62 have been identified as virion-associated proteins by mass spectrometry (ESI-MS/MS), including the major capsid protein (Gp225, present in 1620 copies), which shows a HK97 capsid protein-based fold. Cryo-electron microscopy experiments showed that the 350-kbp DNA molecule of Escherichia coli virus phAPEC6 is packaged in at least 15 concentric layers in the phage capsid. A capsid inner body rod is also present, measuring about 91 nm by 18 nm and oriented along the portal axis. In the phAPEC6 contractile tail, 25 hexameric stacked rings can be distinguished, built of the identified tail sheath protein (Gp277). Cryo-EM reconstruction reveals the base of the unique hairy fibers observed during an initial transmission electron microscopy (TEM) analysis. These very unusual filaments are ordered at three annular positions along the contractile sheath, as well as around the capsid, and may be involved in host interaction.

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array., Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Here authors introduce an E. coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays that are visualized by cryo-electron tomography.

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays.1,2Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit (CSU). Here we introduce a novelE. colistrain that forms small minicells possessing extended and highly ordered chemosensory arrays. We provide a three-dimensional (3D) map of a complete CSU at ~16 Å resolution by cryo-electron tomography (cryo-ET) and subtomogram averaging. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields an atomistic model of the membrane-bound CSU and enables spatial localisation of its signalling domains. Our work thus offers a solid structural basis for interpretation of existing data and design of new experiments to elucidate signalling mechanisms within the CSU and larger array.

International audience; The only enzyme responsible for cadaverine production in the major multidrug-resistant human pathogen Pseudomonas aeruginosa is the lysine decarboxylase LdcA. This enzyme modulates the general polyamine homeostasis, promotes growth, and reduces bacterial persistence during carbenicillin treatment. Here we present a 3.7-Å resolution cryoelectron microscopy structure of LdcA. We introduce an original approach correlating phylogenetic signal with structural information and reveal possible recombination among LdcA and arginine decarboxylase subfamilies within structural domain boundaries. We show that LdcA is involved in full virulence in an insect pathogenesis model. Furthermore, unlike its enterobacterial counterparts, LdcA is regulated neither by the stringent response alarmone ppGpp nor by the AAA+ ATPase RavA. Instead, the P. aeruginosa ravA gene seems to play a defensive role. Altogether, our study identifies LdcA as an important player in P. aeruginosa physiology and virulence and as a potential drug target.

International audience; Abstract Imogolite is known to form bundles when dried; but these nanotubes can also be perfectly dispersed in water. When the silicon precursor in the synthesis of imogolite nanotubes is changed from tetraethoxysilane (TEOS) to methyltriethoxysilane (TEMS), internal wall hydroxyls are replaced with methyl groups. This chemical modification also happens to change their dispersion properties; the nanotubes then form small bundles in solution. In this work, we explore modifications of the synthesis procedure in order to enhance the dispersion of such hybrid nanotubes. The study focuses on the effects of [Al]/[Si] ratio, acid types, ethanol addition or choice of silicon precursor. Using small-angle X-ray scattering (SAXS), infra-red spectroscopy (IR) and cryo-TEM, we show that it is possible to improve yield and length. However, none of the tested modifications allowed the prevention of bundle formation in solution.

Imogolites are natural aluminosilicate nanotubes displaying an astonishing monodispersity in diameter. The diameter is controlled by the structure and composition of the nanotube wall and can be tuned by several chemical manipulations. It has recently been discovered that the structure of imogolite nanotubes can change from single-walled (SW) to double-walled (DW) when Si is replaced by Ge during synthesis. Starting from the pure Ge composition, we show that the transition between DW and SW structures can be induced by the incorporation of a small quantity of Si in the synthesis. At that point, the suspension contains a mixture of structures with a nearly constant average diameter. In particular, we found evidence for the presence of a few nanoscrolls. Above 25% Si, SW nanotubes become more stable and present a continuously decreasing diameter with increasing Si. A model is proposed to explain the stability of these different nanotubes and, more generally, the structures of other organic or inorganic nano...

International audience; Imogolite nanotubes are promising building blocks for nanotechnologies with potential applications in molecular separation, molecular storage, or catalysis. We present an experimental study of the structure of germanium-based imogolite nanotubes Al2O3Ge(OH)4 arranged in bundles. It combines cryo-transmission electron microscopy, infrared spectroscopy, thermogravimetric measurements, and X-ray scattering experiments. Thanks to a systematic method developed to analyze X-ray scattering diagrams as a function of the nanotube shape, single-walled germanium-based imogolite nanotubes, known as cylindrical for more than 30 years, are shown to take an hexagonal base shape when arranged in bundles. Physical and chemical properties of hexagonal imogolite nanotubes should markedly differ from those of cylindrical ones, making hexagonal basis nanotubes a ?new? member, of particular interest, of the rich family of imogolites.

International audience; Micron-long germanium-based double-walled imogolite nanotubes were synthesized at high concentrations, as evidenced by cryo-TEM, AFM, SAXS and IR characterization methods. In addition, the spontaneous formation of a liquid-crystalline phase was observed. The novel synthesis route made it possible for the first time to obtain both long and concentrated germanium-based imogolite-like nanotubes in a single step.