Recent findings have revealed the role of prion-like mechanisms in the control of host defense and programmed cell death cascades. In fungi, HET-S, a cell death-inducing protein containing a HeLo pore-forming domain, is activated through amyloid templating by a Nod-like receptor (NLR). Here we characterize the HELLP protein behaving analogously to HET-S and bearing a new type of N-terminal cell death-inducing domain termed HeLo-like (HELL) and a C-terminal regulatory amyloid motif known as PP. The gene encoding HELLP is part of a three-gene cluster also encoding a lipase (SBP) and a Nod-like receptor, both of which display the PP motif. The PP motif is similar to the RHIM amyloid motif directing formation of the RIP1/RIP3 necrosome in humans. The C-terminal region of HELLP, HELLP(215-278), encompassing the motif, allows prion propagation and assembles into amyloid fibrils, as demonstrated by X-ray diffraction and FTIR analyses. Solid-state NMR studies reveal a well-ordered local structure of the amyloid core residues and a primary sequence that is almost entirely arranged in a rigid conformation, and confirm a β-sheet structure in an assigned stretch of three amino acids. HELLP is activated by amyloid templating and displays membrane-targeting and cell death-inducing activity. HELLP targets the SBP lipase to the membrane, suggesting a synergy between HELLP and SBP in membrane dismantling. Remarkably, the HeLo-like domain of HELLP is homologous to the pore-forming domain of MLKL, the cell death-execution protein in necroptosis, revealing a transkingdom evolutionary relationship between amyloid-controlled fungal programmed cell death and mammalian necroptosis.amyloid | prion | programmed cell death | incompatibility | necroptosis

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR spectroscopy with 100-111 kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα-Hα detection block was developed and applied for the sequence-specific backbone and aliphatic side-chain resonance assignment using only 500 μg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.

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 β‐sheet and α‐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., Cámara‐Almirón, J., Caro‐Astorga, J., Álvarez‐Mena, A., Dutour, A., Berbon, M., Shenoy, J., Morvan, E., Grélard, A., Kauffmann, B., Lecomte, S., de Vicente, A., Habenstein, B., Romero, D., Loquet, A. Molecular architecture of bacterial amyloids in Bacillus biofilms. FASEB J. 33, 12146‐12163 (2019). http://www.fasebj.org

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Neurodegenerative 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 fungusPodospora anserinarepresents 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.

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 ∆GaroS2), in their fibrillar state. Although the different protein constructs exhibit similar fibril morphology and a typical cross‐β 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.

Despite breakthroughs in MAS NMR hardware and experimental methodologies sensitivity remains a major challenge for large and complex biological systems. Here, we report 3-4 fold higher sensitivities obtained in heteronuclear-detected experiments, using a novel HCN CPMAS probe, where the sample coil and the electronics operate at cryogenic temperatures, while the sample is maintained at ambient temperatures (BioSolids CryoProbe™). Such intensity enhancements permit recording 2D and 3D experiments for large assemblies that are otherwise time-prohibitive, such as 2D 15 N-15 N proton-driven spin diffusion and 15 N-13 C double cross polarization to natural abundance carbon experiments. The benefits of CPMAS CryoProbe-based experiments are illustrated for assemblies of kinesin Kif5b with microtubules, HIV-1 capsid protein assemblies, and fibrils of human Y145Stop and fungal HET-s prion proteins -demanding systems for conventional MAS solid-state NMR and excellent reference systems in terms of spectral quality. We envision that this probe technology will be beneficial for a wide range of Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Significant, 3-4 fold sensitivity gains are obtained experiments with a novel CPMAS CryoProbe.The benefits of CPMAS CryoProbe are demonstrated in heteronuclear-detected 2D and 3D experiments on five challenging biological assemblies.High-quality 2D NN and NCA/NCO spectra are obtained on U-15 N-CA tubular capsid assembly containing carbons at natural abundance.

The amyloid fold is structurally characterized by a typical cross-β 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-β 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.