Supramolecular biological protein assemblies study using solid-state NMR

Ma thèse a pour buts l'élucidation fonctionnelle et l'étude structurale de nanomachines biologiques en utilisant principalement la RMN du solide : (i) La protéine SesB trouvée chez Nectria haematococca, dont l'assemblage en fibrilles amyloïdes, serait impliquée dans les mécanismes de signalisation de mort cellulaire programmée. En utilisant la RMN du solide avec rotation à l’angle magique, un nouveau modèle structural de cet amyloïde a été établi pour les fibrilles amyloïdes SesB de Nectria haematococca. (ii) Des protéines appelées TasA, TapA et CalY dont l'assemblage en fibrilles amyloïdes est impliqué dans la formation et l'intégrité des biofilms bactériens chez Bacillus subtilis et Bacillus cereus. Des différences structurales ont été observées entre ces deux espèces. Bacillus subtilis et quelques mutants (mutations de gènes impliqués dans la formation de biofilms) ont été étudiés en utilisant la RMN du solide sur cellules entières pour comprendre l'impact de la délétion de ces composants sur la paroi cellulaire globale et la composition de la matrice. (iii) HET-s, l'amyloïde fonctionnelle fongique aujourd’hui très étudié, est utilisé ici comme système modèle dans une stratégie visant à utiliser les avantages combinés de la DNP, de la rotation très rapide à l’angle magique et de la dilution de spin dans le contexte d'études de biologie structurale. Il a été prouvé qu'une utilisation pertinente de ces techniques constitue une stratégie potentiellement robuste pour la caractérisation structurale des assemblages supramoléculaires pour augmenter la sensibilité des analyses par RMN. (iv) Des polymères protéiques inhabituels, en forme de ruban, appelés R-bodies (« refractile bodies » de type 51), que l'on trouve chez de nombreuses espèces bactériennes, comme Caedibacter et Pseudomonas. Une attribution complète des signaux RMN des R-bodies a été réalisée en utilisant la RMN du solide avec rotation à l’angle magique à très grande vitesse. Un modèle structurel des monomères est établi et tient compte de leur changement conformationnel en fonction du pH, ainsi que de leur capacité à relarguer des biomolécules.I have worked on elucidating and studying biological nanomachines using mainly solid-state NMR (SSNMR): (i) The protein SesB found in Nectria haematococca whose assembly into amyloid fibrils is thought to be involved in programmed-cell death signalling mechanisms. Using magic-angle spinning SSNMR, a novel structural amyloid fold model for Nectria haematococca amyloid fibrils was established. (ii) Proteins called TasA, TapA, and CalY found in Bacillus subtilis and Bacillus cereus whose assembly into amyloid fibrils is involved in biofilm formation and integrity. Bacillus subtilis and its mutants have been studied using whole-cell SSNMR to understand the impact of biofilm matrix components deletion in the overall cell wall, and matrix composition. The protein TasA found in Bacillus subtilis has been observed to perturb liposomes as membrane models. (iii) HET-s, the well documented fungal functional amyloid, used here as a model system in a scheme to use the combined advantages of DNP, fast MAS, and spin dilution in the context of structural biology studies. A relevant use of DNP in combination with fast MAS and specific labelling has been proven to be a potential robust strategy for structural characterization of supramolecular assemblies. (iv) Unusual, ribbon-like protein polymers called R-bodies (Type 51 refractile bodies) found in many bacterial species, such as Caedibacter and Pseudomonas. A complete resonance assignment of R- bodies was achieved using very fast MAS SSNMR. A structural model of the monomers was established and accounts for their interesting pH-dependent switch, as well as the ability to deliver biomolecule

Supramolecular biological protein assemblies study using solid-state NMR

Ma thèse a pour buts l'élucidation fonctionnelle et l'étude structurale de nanomachines biologiques en utilisant principalement la RMN du solide : (i) La protéine SesB trouvée chez Nectria haematococca, dont l'assemblage en fibrilles amyloïdes, serait impliquée dans les mécanismes de signalisation de mort cellulaire programmée. En utilisant la RMN du solide avec rotation à l’angle magique, un nouveau modèle structural de cet amyloïde a été établi pour les fibrilles amyloïdes SesB de Nectria haematococca. (ii) Des protéines appelées TasA, TapA et CalY dont l'assemblage en fibrilles amyloïdes est impliqué dans la formation et l'intégrité des biofilms bactériens chez Bacillus subtilis et Bacillus cereus. Des différences structurales ont été observées entre ces deux espèces. Bacillus subtilis et quelques mutants (mutations de gènes impliqués dans la formation de biofilms) ont été étudiés en utilisant la RMN du solide sur cellules entières pour comprendre l'impact de la délétion de ces composants sur la paroi cellulaire globale et la composition de la matrice. (iii) HET-s, l'amyloïde fonctionnelle fongique aujourd’hui très étudié, est utilisé ici comme système modèle dans une stratégie visant à utiliser les avantages combinés de la DNP, de la rotation très rapide à l’angle magique et de la dilution de spin dans le contexte d'études de biologie structurale. Il a été prouvé qu'une utilisation pertinente de ces techniques constitue une stratégie potentiellement robuste pour la caractérisation structurale des assemblages supramoléculaires pour augmenter la sensibilité des analyses par RMN. (iv) Des polymères protéiques inhabituels, en forme de ruban, appelés R-bodies (« refractile bodies » de type 51), que l'on trouve chez de nombreuses espèces bactériennes, comme Caedibacter et Pseudomonas. Une attribution complète des signaux RMN des R-bodies a été réalisée en utilisant la RMN du solide avec rotation à l’angle magique à très grande vitesse. Un modèle structurel des monomères est établi et tient compte de leur changement conformationnel en fonction du pH, ainsi que de leur capacité à relarguer des biomolécules.I have worked on elucidating and studying biological nanomachines using mainly solid-state NMR (SSNMR): (i) The protein SesB found in Nectria haematococca whose assembly into amyloid fibrils is thought to be involved in programmed-cell death signalling mechanisms. Using magic-angle spinning SSNMR, a novel structural amyloid fold model for Nectria haematococca amyloid fibrils was established. (ii) Proteins called TasA, TapA, and CalY found in Bacillus subtilis and Bacillus cereus whose assembly into amyloid fibrils is involved in biofilm formation and integrity. Bacillus subtilis and its mutants have been studied using whole-cell SSNMR to understand the impact of biofilm matrix components deletion in the overall cell wall, and matrix composition. The protein TasA found in Bacillus subtilis has been observed to perturb liposomes as membrane models. (iii) HET-s, the well documented fungal functional amyloid, used here as a model system in a scheme to use the combined advantages of DNP, fast MAS, and spin dilution in the context of structural biology studies. A relevant use of DNP in combination with fast MAS and specific labelling has been proven to be a potential robust strategy for structural characterization of supramolecular assemblies. (iv) Unusual, ribbon-like protein polymers called R-bodies (Type 51 refractile bodies) found in many bacterial species, such as Caedibacter and Pseudomonas. A complete resonance assignment of R- bodies was achieved using very fast MAS SSNMR. A structural model of the monomers was established and accounts for their interesting pH-dependent switch, as well as the ability to deliver biomolecule

Etude d'assemblages protéiques biologiques supramoléculaires par RMN du solide

I have worked on elucidating and studying biological nanomachines using mainly solid-state NMR (SSNMR): (i) The protein SesB found in Nectria haematococca whose assembly into amyloid fibrils is thought to be involved in programmed-cell death signalling mechanisms. Using magic-angle spinning SSNMR, a novel structural amyloid fold model for Nectria haematococca amyloid fibrils was established. (ii) Proteins called TasA, TapA, and CalY found in Bacillus subtilis and Bacillus cereus whose assembly into amyloid fibrils is involved in biofilm formation and integrity. Bacillus subtilis and its mutants have been studied using whole-cell SSNMR to understand the impact of biofilm matrix components deletion in the overall cell wall, and matrix composition. The protein TasA found in Bacillus subtilis has been observed to perturb liposomes as membrane models. (iii) HET-s, the well documented fungal functional amyloid, used here as a model system in a scheme to use the combined advantages of DNP, fast MAS, and spin dilution in the context of structural biology studies. A relevant use of DNP in combination with fast MAS and specific labelling has been proven to be a potential robust strategy for structural characterization of supramolecular assemblies. (iv) Unusual, ribbon-like protein polymers called R-bodies (Type 51 refractile bodies) found in many bacterial species, such as Caedibacter and Pseudomonas. A complete resonance assignment of R- bodies was achieved using very fast MAS SSNMR. A structural model of the monomers was established and accounts for their interesting pH-dependent switch, as well as the ability to deliver biomoleculesMa thèse a pour buts l'élucidation fonctionnelle et l'étude structurale de nanomachines biologiques en utilisant principalement la RMN du solide : (i) La protéine SesB trouvée chez Nectria haematococca, dont l'assemblage en fibrilles amyloïdes, serait impliquée dans les mécanismes de signalisation de mort cellulaire programmée. En utilisant la RMN du solide avec rotation à l’angle magique, un nouveau modèle structural de cet amyloïde a été établi pour les fibrilles amyloïdes SesB de Nectria haematococca. (ii) Des protéines appelées TasA, TapA et CalY dont l'assemblage en fibrilles amyloïdes est impliqué dans la formation et l'intégrité des biofilms bactériens chez Bacillus subtilis et Bacillus cereus. Des différences structurales ont été observées entre ces deux espèces. Bacillus subtilis et quelques mutants (mutations de gènes impliqués dans la formation de biofilms) ont été étudiés en utilisant la RMN du solide sur cellules entières pour comprendre l'impact de la délétion de ces composants sur la paroi cellulaire globale et la composition de la matrice. (iii) HET-s, l'amyloïde fonctionnelle fongique aujourd’hui très étudié, est utilisé ici comme système modèle dans une stratégie visant à utiliser les avantages combinés de la DNP, de la rotation très rapide à l’angle magique et de la dilution de spin dans le contexte d'études de biologie structurale. Il a été prouvé qu'une utilisation pertinente de ces techniques constitue une stratégie potentiellement robuste pour la caractérisation structurale des assemblages supramoléculaires pour augmenter la sensibilité des analyses par RMN. (iv) Des polymères protéiques inhabituels, en forme de ruban, appelés R-bodies (« refractile bodies » de type 51), que l'on trouve chez de nombreuses espèces bactériennes, comme Caedibacter et Pseudomonas. Une attribution complète des signaux RMN des R-bodies a été réalisée en utilisant la RMN du solide avec rotation à l’angle magique à très grande vitesse. Un modèle structurel des monomères est établi et tient compte de leur changement conformationnel en fonction du pH, ainsi que de leur capacité à relarguer des biomolécules

Membrane-induced tau amyloid fibrils

Abstract The intrinsically disordered protein tau aggregates into β-sheet amyloid fibrils that spread in human brains afflicted with Alzheimer’s disease and other neurodegenerative diseases. Tau interaction with lipid membranes might play a role in the formation and spreading of these pathological aggregates. Here we investigate the conformation and assembly of membrane-induced tau aggregates using solid-state NMR and transmission electron microscopy. A tau construct that encompasses the microtubule-binding repeats and a proline-rich domain is reconstituted into cholesterol-containing phospholipid membranes. 2D 13C-13C correlation spectra indicate that tau converted from a random coil to a β-sheet conformation over weeks. Small unilamellar vesicles (SUVs) cause different equilibrium conformations from large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). Importantly, SUV-bound tau developed long fibrils that exhibit the characteristic β-sheet chemical shifts of Tyr310 in heparin-fibrillized tau. In comparison, LUVs and MLVs do not induce fibrils but cause different β-sheet aggregates. Lipid-protein correlation spectra indicate that these tau aggregates reside at the membrane-water interface, without inserting into the middle of the lipid bilayer. Removal of cholesterol from the SUVs abolished the fibrils, indicating that both membrane curvature and cholesterol are required for tau fibril formation. These results have implications for how lipid membranes might nucleate tau aggregates

3D structure determination of amyloid fibrils using solid-state NMR spectroscopy

The amyloid fold is structurally characterized by a typical cross-beta architecture, which is under debate to represent an energy-favourable folding state that many globular or natively unfolded proteins can adopt. Being initially solely associated with amyloid fibrils observed in the propagation of several neurodegenerative disorders, the discovery of non-pathological (or "functional") amyloids in many native biological processes has recently further intensified the general interest invested in those cross-beta supramolecular assemblies. The insoluble and non-crystalline nature of amyloid fibrils and their usually inhomogeneous appearance on the mesoscopic level pose a challenge to biophysical techniques aiming at an atomic-level structural characterization. Solid-state NMR spectroscopy (SSNMR) has granted breakthroughs in structural investigations on amyloid fibrils ranging from the assessment of the impact of polymorphism in disease development to the 3D atomic structure determination of amyloid fibrils. First landmark studies towards the characterization of atomic structures and interactions involving functional amyloids have provided new impulses in the understanding of the role of the amyloid fold in native biological functions. Over the last decade many strategies have been developed in protein isotope labelling, NMR resonance assignment, distance restraint determination and 3D structure calculation of amyloid fibrils based on SSNMR approaches. We will here discuss the emerging concepts and state-of-the-art methods related to the assessment of amyloid structures and interactions involving amyloid entities by SSNMR.Nanostructures biologiques et synthétiques étudiées par Résonance Magnétique Nucléaire du SolideStructures d'Assemblages Supramoléculaires par RMN du Solide : le Pseudopilus du Système de Sécrétion de Type II et le Tube de Queue du Bactériophag

Structural dissection of amyloid aggregates of TDP-43 and its C-terminal fragments TDP-35 and TDP-16

The TAR DNA-binding protein (TDP-43) self-assembles into prion-like aggregates considered to be the structural hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. Here, we use a combination of electron microscopy, X-ray fiber diffraction, Fourier-transform infrared spectroscopy analysis, and solid-state NMR spectroscopy to investigate the molecular organization of different TDP constructs, namely the full-length TDP-43 (1-414), two C-terminal fragments [TDP-35 (90-414) and TDP-16 (267-414)], and a C-terminal truncated fragment (TDP-43 increment GaroS2), in their fibrillar state. Although the different protein constructs exhibit similar fibril morphology and a typical cross-beta signature by X-ray diffraction, solid-state NMR indicates that TDP-43 and TDP-35 share the same polymorphic molecular structure, while TDP-16 encompasses a well-ordered amyloid core. We identified several residues in the so-called C-terminal GaroS2 (368-414) domain that participates in the rigid core of TDP-16 fibrils, underlining its importance during the aggregation process. Our findings demonstrate that C-terminal fragments can adopt a different molecular conformation in isolation or in the context of the full-length assembly, suggesting that the N-terminal domain and RRM domains play an important role in the TDP-43 amyloid transition.Advanced Materials by DesignNanostructures biologiques et synthétiques étudiées par Résonance Magnétique Nucléaire du Solid

Structural polymorphism of the low-complexity C-terminal domain of TDP-43 amyloid aggregates revealed by solid-state NMR

Aberrant aggregation of the transactive response DNA-binding protein (TDP-43) is associated with several lethal neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. Cytoplasmic neuronal inclusions of TDP-43 are enriched in various fragments of the low-complexity C-terminal domain and are associated with different neurotoxicity. Here we dissect the structural basis of TDP-43 polymorphism using magic-angle spinning solid-state NMR spectroscopy in combination with electron microscopy and Fourier-transform infrared spectroscopy. We demonstrate that various low-complexity C-terminal fragments, namely TDP-13 (TDP-43 300–414 ), TDP-11 (TDP-43 300–399 ), and TDP-10 (TDP-43 314–414 ), adopt distinct polymorphic structures in their amyloid fibrillar state. Our work demonstrates that the removal of less than 10% of the low-complexity sequence at N- and C-termini generates amyloid fibrils with comparable macroscopic features but different local structural arrangement. It highlights that the assembly mechanism of TDP-43, in addition to the aggregation of the hydrophobic region, is also driven by complex interactions involving low-complexity aggregation-prone segments that are a potential source of structural polymorphism

Structural and molecular basis of cross-seeding barriers in amyloids

International audienceNeurodegenerative disorders are frequently associated with β-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behavior and distinct pathophenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical β-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and sheds light on key molecular features concerning an important class of pathogenic agents

Structures of Pathological and Functional Amyloids and Prions, a Solid-State NMR Perspective

International audienceInfectious proteins or prions are a remarkable class of pathogens, where pathogenicity and infectious state correspond to conformational transition of a protein fold. The conformational change translates into the formation by the protein of insoluble amyloid aggregates, associated in humans with various neurodegenerative disorders and systemic protein-deposition diseases. The prion principle, however, is not limited to pathogenicity. While pathological amyloids (and prions) emerge from protein misfolding, a class of functional amyloids has been defined, consisting of amyloid-forming domains under natural selection and with diverse biological roles. Although of great importance, prion amyloid structures remain challenging for conventional structural biology techniques. Solid-state nuclear magnetic resonance (SSNMR) has been preferentially used to investigate these insoluble, morphologically heterogeneous aggregates with poor crystallinity. SSNMR methods have yielded a wealth of knowledge regarding the fundamentals of prion biology and have helped to solve the structures of several prion and prion-like fibrils. Here, we will review pathological and functional amyloid structures and will discuss some of the obtained structural models. We will finish the review with a perspective on integrative approaches combining solid-state NMR, electron paramagnetic resonance and cryo-electron microscopy, which can complement and extend our toolkit to structurally explore various facets of prion biology

Molecular architecture of bacterial amyloids in Bacillus biofilms

The formation of biofilms provides structural and adaptive bacterial response to the environment. In Bacillus species, the biofilm extracellular matrix is composed of exopolysaccharides, hydrophobins, and several functional amyloid proteins. We report, using multiscale approaches such as solid-state NMR (SSNMR), electron microscopy, X-ray diffraction, dynamic light scattering, attenuated total reflection Fourier transform infrared (FTIR), and immune-gold labeling, the molecular architecture of B. subtilis and pathogenic B. cereus functional amyloids. SSNMR data reveal that the major amyloid component TasA in its fibrillar amyloid form contain beta-sheet and alpha-helical secondary structure, suggesting a nontypical amyloid architecture in B. subtilis. Proteinase K digestion experiments indicate the amyloid moiety is 100 aa long, and subsequent SSNMR and FTIR signatures for B. subtilis and B. cereus TasA filaments highlight a conserved amyloid fold, albeit with substantial differences in structural polymorphism and secondary structure composition. Structural analysis and coassembly data on the accessory protein TapA in B. subtilis and its counterpart camelysin in B. cereus reveal a catalyzing effect between the functional amyloid proteins and a common structural architecture, suggesting a coassembly in the context of biofilm formation. Our findings highlight nontypical amyloid behavior of these bacterial functional amyloids, underlining structural variations between biofilms even in closely related bacterial species.-El Mammeri, N., Hierrezuelo, J., Tolchard, J., Camara-Almiron, J., Caro-Astorga, J., Alvarez-Mena, A., Dutour, A., Berbon, M., Shenoy, J., Morvan, E., Grelard, A., Kauffmann, B., Lecomte, S., de Vicente, A., Habenstein, B., Romero, D., Loquet, A. Molecular architecture of bacterial amyloids in Bacillus biofilms