Analytical Framework to Understand the Origins of Methyl Side-Chain Dynamics in Protein Assemblies.

Side-chain motions play an important role in understanding protein structure, dynamics, protein-protein, and protein-ligand interactions. However, our understanding of protein side-chain dynamics is currently limited by the lack of analytical tools. Here, we present a novel analytical framework employing experimental nuclear magnetic resonance (NMR) relaxation measurements at atomic resolution combined with molecular dynamics (MD) simulation to characterize with a high level of detail the methyl side-chain dynamics in insoluble protein assemblies, using amyloid fibrils formed by the prion HET-s. We use MD simulation to interpret experimental results, where rotameric hops, including methyl group rotation and χ1/χ2 rotations, cannot be completely described with a single correlation time but rather sample a broad distribution of correlation times, resulting from continuously changing local structure in the fibril. Backbone motion similarly samples a broad range of correlation times, from ∼100 ps to µs, although resulting from mostly different dynamic processes; nonetheless, we find that the backbone is not fully decoupled from the side-chain motion, where changes in side-chain dynamics influence backbone motion and vice versa. While the complexity of side-chain motion in protein assemblies makes it very challenging to obtain perfect agreement between experiment and simulation, our analytical framework improves the interpretation of experimental dynamics measurements for complex protein assemblies.

Structural and molecular basis of cross-seeding barriers in amyloids.

Neurodegenerative disorders are frequently associated with ß-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 ß-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.

Efficient 18.8 T MAS-DNP NMR reveals hidden side chains in amyloid fibrils.

Amyloid fibrils are large and insoluble protein assemblies composed of a rigid core associated with a cross-ß arrangement rich in ß-sheet structural elements. It has been widely observed in solid-state NMR experiments that semi-rigid protein segments or side chains do not yield easily observable NMR signals at room temperature. The reasons for the missing peaks may be due to the presence of unfavorable dynamics that interfere with NMR experiments, which result in very weak or unobservable NMR signals. Therefore, for amyloid fibrils, semi-rigid and dynamically disordered segments flanking the amyloid core are very challenging to study. Here, we show that high-field dynamic nuclear polarization (DNP), an NMR hyperpolarization technique typically performed at low temperatures, can circumvent this issue because (i) the low-temperature environment (~ 100 K) slows down the protein dynamics to escape unfavorable detection regime, (ii) DNP improves the overall NMR sensitivity including those of flexible side chains, and (iii) efficient cross-effect DNP biradicals (SNAPol-1) optimized for high-field DNP (≥ 18.8 T) are employed to offer high sensitivity and resolution suitable for biomolecular NMR applications. By combining these factors, we have successfully established an impressive enhancement factor of ε ~ 50 on amyloid fibrils using an 18.8 T/ 800 MHz magnet. We have compared the DNP efficiencies of M-TinyPol, NATriPol-3, and SNAPol-1 biradicals on amyloid fibrils. We found that SNAPol-1 (with ε ~ 50) outperformed the other two radicals. The MAS DNP experiments revealed signals of flexible side chains previously inaccessible at conventional room-temperature experiments. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains and dynamically disordered segments otherwise hidden at room temperature.

The Rigid Core and Flexible Surface of Amyloid Fibrils Probed by Magic-Angle-Spinning NMR Spectroscopy of Aromatic Residues.

Aromatic side chains are important reporters of the plasticity of proteins, and often form important contacts in protein-protein interactions. We studied aromatic residues in the two structurally homologous cross-ß amyloid fibrils HET-s, and HELLF by employing a specific isotope-labeling approach and magic-angle-spinning NMR. The dynamic behavior of the aromatic residues Phe and Tyr indicates that the hydrophobic amyloid core is rigid, without any sign of "breathing motions" over hundreds of milliseconds at least. Aromatic residues exposed at the fibril surface have a rigid ring axis but undergo ring flips on a variety of time scales from nanoseconds to microseconds. Our approach provides direct insight into hydrophobic-core motions, enabling a better evaluation of the conformational heterogeneity generated from an NMR structural ensemble of such amyloid cross-ß architecture.

A polymorphic helix of a Salmonella needle protein relays signals defining distinct steps in type III secretion.

Type III protein-secretion machines are essential for the interactions of many pathogenic or symbiotic bacterial species with their respective eukaryotic hosts. The core component of these machines is the injectisome, a multiprotein complex that mediates the selection of substrates, their passage through the bacterial envelope, and ultimately their delivery into eukaryotic target cells. The injectisome is composed of a large cytoplasmic complex or sorting platform, a multiring base embedded in the bacterial envelope, and a needle-like filament that protrudes several nanometers from the bacterial surface and is capped at its distal end by the tip complex. A characteristic feature of these machines is that their activity is stimulated by contact with target host cells. The sensing of target cells, thought to be mediated by the distal tip of the needle filament, generates an activating signal that must be transduced to the secretion machine by the needle filament. Here, through a multidisciplinary approach, including solid-state NMR (SSNMR) and cryo electron microscopy (cryo-EM) analyses, we have identified critical residues of the needle filament protein of a Salmonella Typhimurium type III secretion system that are involved in the regulation of the activity of the secretion machine. We found that mutations in the needle filament protein result in various specific phenotypes associated with different steps in the type III secretion process. More specifically, these studies reveal an important role for a polymorphic helix of the needle filament protein and the residues that line the lumen of its central channel in the control of type III secretion.

Protein resonance assignment by solid-state NMR based on 1H-detected 13C double-quantum spectroscopy at fast MAS.

Solid-state NMR spectroscopy is a powerful technique to study insoluble and non-crystalline proteins and protein complexes at atomic resolution. The development of proton (1H) detection at fast magic-angle spinning (MAS) has considerably increased the analytical capabilities of the technique, enabling the acquisition of 1H-detected fingerprint experiments in few hours. Here an approach based on double-quantum (DQ) 13C spectroscopy, detected on 1H, is proposed for fast MAS regime (> 60 kHz) to perform the sequential assignment of insoluble proteins of small size, without any specific deuteration requirement. By combining two three-dimensional 1H detected experiments correlating a 13C DQ dimension respectively to its intra-residue and sequential 15 N-1H pairs, a sequential walk through DQ (Ca + CO) resonance is obtained. The approach takes advantage of fast MAS to achieve an efficient sensitivity and the addition of a DQ dimension provides spectral features useful for the resonance assignment process.

Structures of Type III Secretion System Needle Filaments.

Among the Gram-negative bacterial secretion systems, type III secretion systems (T3SS) possess a unique extracellular molecular apparatus called the needle. This macromolecular protein assembly is a nanometre-size filament formed by the helical arrangement of hundreds of copies of a single, small protein, which is highly conserved between T3SSs from animal to plant bacterial pathogens. The needle filament forms a hollow tube with a channel ~20 Å in diameter that serves as a conduit for proteins secreted into the targeted host cell. In the past ten years, technical breakthroughs in biophysical techniques such as cryo-electron microscopy (cryo-EM) and solid-state NMR (SSNMR) spectroscopy have uncovered atomic resolution details about the T3SS needle assembly. Several high-resolution structures of Salmonella typhimurium and Shigella flexneri T3SS needles have been reported demonstrating a common structural fold. These structural models have been used to explain the active role of the needle in transmitting the host-cell contact signal from the tip to the base of the T3SS through conformational changes as well as during the injection of effector proteins. In this chapter, we summarize the current knowledge about the structure and the role of the T3SS needle during T3SS assembly and effector secretion.

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 ß-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.

Coiled-coil oligomerization controls localization of the plasma membrane REMORINs.

REMORINs are nanodomain-organized proteins located in the plasma membrane and involved in cellular responses in plants. The dynamic assembly of the membrane nanodomains represents an essential tool of the versatile membrane barriers to control and modulate cellular functions. Nevertheless, the assembly mechanisms and protein organization strategies of nanodomains are poorly understood and many structural aspects are difficult to visualize. Using an ensemble of biophysical approaches, including solid-state nuclear magnetic resonance, cryo-electron microscopy and in vivo confocal imaging, we provide first insights on the role and the structural mechanisms of REMORIN trimerization. Our results suggest that the formation of REMORIN coiled-coil trimers is essential for membrane recruitment and promotes REMORIN assembly in vitro into long filaments by trimer-trimer interactions that might participate in nanoclustering into membrane domains in vivo.

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

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

Identification of a novel cell death-inducing domain reveals that fungal amyloid-controlled programmed cell death is related to necroptosis.

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 ß-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.

Detection of side-chain proton resonances of fully protonated biosolids in nano-litre volumes by magic angle spinning solid-state NMR.

We present a new solid-state NMR proton-detected three-dimensional experiment dedicated to the observation of protein proton side chain resonances in nano-liter volumes. The experiment takes advantage of very fast magic angle spinning and double quantum 13C-13C transfer to establish efficient (H)CCH correlations detected on side chain protons. Our approach is demonstrated on the HET-s prion domain in its functional amyloid fibrillar form, fully protonated, with a sample amount of less than 500 µg using a MAS frequency of 70 kHz. The majority of aliphatic and aromatic side chain protons (70%) are observable, in addition to Hα resonances, in a single experiment providing a complementary approach to the established proton-detected amide-based multidimensional solid-state NMR experiments for the study and resonance assignment of biosolid samples, in particular for aromatic side chain resonances.

NMR Spectroscopic Assignment of Backbone and Side-Chain Protons in Fully Protonated Proteins: Microcrystals, Sedimented Assemblies, and Amyloid Fibrils.

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.

Cryo-EM observation of the amyloid key structure of polymorphic TDP-43 amyloid fibrils.

The transactive response DNA-binding protein-43 (TDP-43) is a multi-facet protein involved in phase separation, RNA-binding, and alternative splicing. In the context of neurodegenerative diseases, abnormal aggregation of TDP-43 has been linked to amyotrophic lateral sclerosis and frontotemporal lobar degeneration through the aggregation of its C-terminal domain. Here, we report a cryo-electron microscopy (cryo-EM)-based structural characterization of TDP-43 fibrils obtained from the full-length protein. We find that the fibrils are polymorphic and contain three different amyloid structures. The structures differ in the number and relative orientation of the protofilaments, although they share a similar fold containing an amyloid key motif. The observed fibril structures differ from previously described conformations of TDP-43 fibrils and help to better understand the structural landscape of the amyloid fibril structures derived from this protein.

NMR resonance assignment of the cell death execution domain BELL2 from multicellular bacterial signalosomes.

Signalosomes are high-order protein machineries involved in complex mechanisms controlling regulated immune defense and cell death execution. The immune response is initiated by the recognition of exogeneous or endogenous signals, triggering the signalosome assembly process. The final step of signalosome fate often involves membrane-targeting and activation of pore-forming execution domains, leading to membrane disruption and ultimately cell death. Such cell death-inducing domains have been thoroughly characterized in plants, mammals and fungi, notably for the fungal cell death execution protein domain HeLo. However, little is known on the mechanisms of signalosome-based immune response in bacteria, and the conformation of cell death executors in bacterial signalosomes is still poorly characterized. We recently uncovered the existence of NLR signalosomes in various multicellular bacteria and used genome mining approaches to identify putative cell death executors in Streptomyces olivochromogenes. These proteins contain a C-terminal amyloid domain involved in signal transmission and a N-terminal domain, termed BELL for Bacteria analogous to fungal HeLL (HeLo-like), presumably responsible for membrane-targeting, pore-forming and cell death execution. In the present study, we report the high yield expression of S. olivochromogenes BELL2 and its characterization by solution NMR spectroscopy. BELL is folded in solution and we report backbone and sidechain assignments. We identified five α-helical secondary structure elements and a folded core much smaller than its fungal homolog HeLo. This study constitutes the first step toward the NMR investigation of the full-length protein assembly and its membrane targeting.

Hfq C-terminal region forms a ß-rich amyloid-like motif without perturbing the N-terminal Sm-like structure.

Hfq is a pleitropic actor that serves as stress response and virulence factor in the bacterial cell. To execute its multiple functions, Hfq assembles into symmetric torus-shaped hexamers. Extending outward from the hexameric core, Hfq presents a C-terminal region, described as intrinsically disordered in solution. Many aspects of the role and the structure of this region remain unclear. For instance, in its truncated form it can promote amyloid-like filament assembly. Here, we show that a minimal 11-residue motif at the C-terminal end of Hfq assembles into filaments with amyloid characteristics. Our data suggest that the full-length Hfq in its filamentous state contains a similar molecular fingerprint than that of the short ß-strand peptide, and that the Sm-core structure is not affected by filament formation. Hfq proteins might thus co-exist in two forms in vivo, either as isolated, soluble hexamers or as self-assembled hexamers through amyloid-reminiscent interactions, modulating Hfq cellular functions.

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-43300-414), TDP-11 (TDP-43300-399), and TDP-10 (TDP-43314-414), 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.

Cell-free synthesis of amyloid fibrils with infectious properties and amenable to sub-milligram magic-angle spinning NMR analysis.

Structural investigations of amyloid fibrils often rely on heterologous bacterial overexpression of the protein of interest. Due to their inherent hydrophobicity and tendency to aggregate as inclusion bodies, many amyloid proteins are challenging to express in bacterial systems. Cell-free protein expression is a promising alternative to classical bacterial expression to produce hydrophobic proteins and introduce NMR-active isotopes that can improve and speed up the NMR analysis. Here we implement the cell-free synthesis of the functional amyloid prion HET-s(218-289). We present an interesting case where HET-s(218-289) directly assembles into infectious fibril in the cell-free expression mixture without the requirement of denaturation procedures and purification. By introducing tailored 13C and 15N isotopes or CF3 and 13CH2F labels at strategic amino-acid positions, we demonstrate that cell-free synthesized amyloid fibrils are readily amenable to high-resolution magic-angle spinning NMR at sub-milligram quantity.

Structural characterization of the EmrAB-TolC efflux complex from E. coli.

Gram-negative bacteria export a large variety of antimicrobial compounds by forming two-membrane spanning tripartite multidrug efflux systems composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. Here we present the co-expression, purification and first electron microscopy insights of the Escherichia coli EmrAB-TolC tripartite Major Facilitator Superfamily (MSF) efflux system as a whole complex stabilized by Amphipol polymer. The structure reveals a 33 nm long complex delineated by the Amphipol belt at both extremities. Comparison of projection structures of EmrAB-TolC and AcrAB-TolC indicates that the outer membrane protein TolC linked to the periplasmic adaptor EmrA protein form an extended periplasmic canal. The overall length of EmrAB-TolC complex is similar to that of AcrAB-TolC with a probable tip-to-tip interaction between EmrA and TolC unveiling how the adaptor protein connects TolC and EmrB embedded in the inner membrane.

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

Infectious 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.