The structural line between prion and "prion-like": Insights from prion protein and tau.

The concept of 'prion-like' behavior has emerged in the study of diseases involving protein misfolding where fibrillar structures, called amyloids, self-propagate and induce disease in a fashion similar to prions. From a biological standpoint, in order to be considered 'prion-like,' a protein must traverse cells and tissues and further propagate via a templated conformational change. Since 2017, cryo-electron microscopy structures from patient-derived 'prion-like' amyloids, in particular tau, have been presented and revealed structural similarities shared across amyloids. Since 2021, cryo-EM structures from prions of known infectivity have been added to the ex vivo amyloid structure family. In this review, we discuss current proposals for the 'prion-like' mechanisms of spread for tau and prion protein as well as discuss different influencers on structures of aggregates from tauopathies and prion diseases. Lastly, we discuss some of the current hypotheses for what may distinguish structures that are 'prion-like' from transmissible prion structures.

Alzheimer proteopathic tau seeds are biochemically a forme fruste of mature paired helical filaments.

Aggregation prone molecules, such as tau, form both historically well characterized fibrillar deposits (neurofibrillary tangles) and recently identified phosphate-buffered saline (PBS) extract species called proteopathic seeds. Both can cause normal endogenous tau to undergo templated misfolding. The relationship of these seeds to the fibrils that define tau-related diseases is unknown. We characterized the aqueous extractable and sarkosyl insoluble fibrillar tau species derived from human Alzheimer brain using mass spectrometry and in vitro bioassays. Post-translational modifications (PTMs) including phosphorylation, acetylation and ubiquitination are identified in both preparations. PBS extract seed competent tau can be distinguished from sarkosyl insoluble tau by the presence of overlapping, but less abundant, PTMs and an absence of some PTMs unique to the latter. The presence of ubiquitin and other PTMs on the PBS-extracted tau species correlates with the amount of tau in the seed competent size exclusion fractions, with the bioactivity and with the aggressiveness of clinical disease. These results demonstrate that the PTMs present on bioactive, seed competent PBS extract tau species are closely related to, but distinct from, the PTMs of mature paired helical filaments, consistent with the idea that they are a forme fruste of tau species that ultimately form fibrils.

Reconstitution of the Alzheimer's Disease Tau Core Structure from Recombinant Tau297-391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM.

The protein tau misfolds into disease-specific fibrillar structures in more than 20 neurodegenerative diseases collectively referred to as tauopathies. To understand and prevent disease-specific mechanisms of filament formation, in vitro models for aggregation that robustly yield these different end point structures will be necessary. Here, we used cryo-electron microscopy (cryo-EM) to reconstruct fibril polymorphs taken on by residues 297-391 of tau under conditions previously shown to give rise to the core structure found in Alzheimer's disease (AD). While we were able to reconstitute the AD tau core fold, the proportion of these paired helical filaments (PHFs) was highly variable, and a majority of filaments were composed of PHFs with an additional identical C-shaped protofilament attached near the PHF interface, termed triple helical filaments (THFs). Since the impact of filament layer quaternary structure on the biological properties of tau and other amyloid filaments is not known, the applications for samples of this morphology are presently uncertain. We further demonstrate the variation in the proportion of PHFs and PHF-like fibrils compared to other morphologies as a function of shaking time and AD polymorph-favoring cofactor concentration. This variation in polymorph abundance, even under identical experimental conditions, highlights the variation that can arise both within a lab and in different laboratory settings when reconstituting specific fibril polymorphs in vitro.

Tau seeding and spreading in vivo is supported by both AD-derived fibrillar and oligomeric tau.

Insoluble fibrillar tau, the primary constituent of neurofibrillary tangles, has traditionally been thought to be the biologically active, toxic form of tau mediating neurodegeneration in Alzheimer's disease. More recent studies have implicated soluble oligomeric tau species, referred to as high molecular weight (HMW), due to their properties on size-exclusion chromatography, in tau propagation across neural systems. These two forms of tau have never been directly compared. We prepared sarkosyl-insoluble and HMW tau from the frontal cortex of Alzheimer patients and compared their properties using a variety of biophysical and bioactivity assays. Sarkosyl-insoluble fibrillar tau comprises abundant paired-helical filaments (PHF) as quantified by electron microscopy (EM) and is more resistant to proteinase K, compared to HMW tau, which is mostly in an oligomeric form. Sarkosyl-insoluble and HMW tau are nearly equivalent in potency in HEK cell bioactivity assay for seeding aggregates, and their injection reveals similar local uptake into hippocampal neurons in PS19 Tau transgenic mice. However, the HMW preparation appears to be far more potent in inducing a glial response including Clec7a-positive rod microglia in the absence of neurodegeneration or synapse loss and promotes more rapid propagation of misfolded tau to distal, anatomically connected regions, such as entorhinal and perirhinal cortices. These data suggest that soluble HMW tau has similar properties to fibrillar sarkosyl-insoluble tau with regard to tau seeding potential, but may be equal or even more bioactive with respect to propagation across neural systems and activation of glial responses, both relevant to tau-related Alzheimer phenotypes.

Tau seeding and spreading in vivo is supported by both AD-derived fibrillar and oligomeric tau.

Insoluble fibrillar tau, the primary constituent of neurofibrillary tangles, has traditionally been thought to be the biologically active, toxic form of tau mediating neurodegeneration in Alzheimer's disease. More recent studies have implicated soluble oligomeric tau species, referred to as high molecular weight (HMW) due to its properties on size exclusion chromatography, in tau propagation across neural systems. These two forms of tau have never been directly compared. We prepared sarkosyl insoluble and HMW tau from the frontal cortex of Alzheimer patients and compared their properties using a variety of biophysical and bioactivity assays. Sarkosyl insoluble fibrillar tau is comprised of abundant paired helical filaments (PHF) as quantified by electron microscopy (EM), and is more resistant to proteinase K, compared to HMW tau which is mostly in an oligomeric form. Sarkosyl insoluble and HMW tau are nearly equivalent in potency in a HEK cell bioactivity assay for seeding aggregates and their injection reveals similar local uptake into hippocampal neurons in PS19 Tau transgenic mice. However, the HMW preparation appears to be far more potent in inducing a glial response including Clec7a-positive rod-microglia in the absence of neurodegeneration or synapse loss and promotes more rapid propagation of misfolded tau to distal, anatomically connected regions, such as entorhinal and perirhinal cortices. These data suggest that soluble HMW tau has similar properties to fibrillar sarkosyl insoluble tau with regard to tau seeding potential but may be equal or even more bioactive with respect to propagation across neural systems and activation of glial responses, both relevant tau-related Alzheimer phenotypes.

Fragment-Based Ab Initio Phasing of Peptidic Nanocrystals by MicroED.

Electron diffraction (MicroED/3DED) can render the three-dimensional atomic structures of molecules from previously unamenable samples. The approach has been particularly transformative for peptidic structures, where MicroED has revealed novel structures of naturally occurring peptides, synthetic protein fragments, and peptide-based natural products. Despite its transformative potential, MicroED is beholden to the crystallographic phase problem, which challenges its de novo determination of structures. ARCIMBOLDO, an automated, fragment-based approach to structure determination, eliminates the need for atomic resolution, instead enforcing stereochemical constraints through libraries of small model fragments, and discerning congruent motifs in solution space to ensure validation. This approach expands the reach of MicroED to presently inaccessible peptide structures including fragments of human amyloids, and yeast and mammalian prions. For electron diffraction, fragment-based phasing portends a more general phasing solution with limited model bias for a wider set of chemical structures.

Structural consequences of sequence variation in mammalian prion ß2α2 loop segments.

Sequence variation in the ß2α2 loop, residues 165-175 of the mammalian prion protein (PrP), influences its structure. To better understand the consequences of sequence variation in this region of the protein, we biochemically and biophysically interrogate natural and artificial sequence variants of the ß2α2 loop of mammalian PrP. Using microcrystal electron diffraction (MicroED), we determine atomic resolution structures of segments encompassing residues 168-176 from the ß2α2 loop of PrP with sequences corresponding to human, mouse/cow, bank vole/hamster, rabbit/pig/guinea pig, and naked mole rat (elk-T174S) ß2α2 loops, as well as synthetic ß2α2 loop sequences. This collection of structures presents two dominant amyloid packing polymorphisms. In the first polymorph, denoted "clasped", side chains within a sheet form polar clasps by facing each other on the same strand, exemplified by the mouse/cow, human, and bank vole/hamster sequences. Because its stability is derived from within a strand and through polar ladders within a sheet, the sequence requirements for the mating strand are less restrictive. A second polymorph, denoted "interdigitated," has sidechains interdigitate across mating sheets, exemplified by the elk, naked mole rat (elk T174S), and rabbit sequences. The two types of packing present distinct networks of stabilizing hydrogen bonds. The identity of residue 174 appears to strongly influence the packing adopted in these peptides, but consideration of the overall sequence of a given segment is needed to understand the stability of its assemblies. Incorporation of these ß2α2 loop sequences into an 85 residue recombinant segment encoding wild-type bank vole PrP94-178 demonstrates that even single residue substitutions could impact fibril morphology as evaluated by negative stain electron microscopy. This is in line with recent findings supporting the accessibility of different structural geometries by varied mammalian prion sequences, and indicates that sequence-specific polymorphisms may be influenced by residues in the ß2α2 loop.

Accurate de novo design of membrane-traversing macrocycles.

We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6-12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6-12 residue size range cross membranes with an apparent permeability greater than 1 × 10-6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.

Intrinsic electronic conductivity of individual atomically resolved amyloid crystals reveals micrometer-long hole hopping via tyrosines.

Proteins are commonly known to transfer electrons over distances limited to a few nanometers. However, many biological processes require electron transport over far longer distances. For example, soil and sediment bacteria transport electrons, over hundreds of micrometers to even centimeters, via putative filamentous proteins rich in aromatic residues. However, measurements of true protein conductivity have been hampered by artifacts due to large contact resistances between proteins and electrodes. Using individual amyloid protein crystals with atomic-resolution structures as a model system, we perform contact-free measurements of intrinsic electronic conductivity using a four-electrode approach. We find hole transport through micrometer-long stacked tyrosines at physiologically relevant potentials. Notably, the transport rate through tyrosines (105 s-1) is comparable to cytochromes. Our studies therefore show that amyloid proteins can efficiently transport charges, under ordinary thermal conditions, without any need for redox-active metal cofactors, large driving force, or photosensitizers to generate a high oxidation state for charge injection. By measuring conductivity as a function of molecular length, voltage, and temperature, while eliminating the dominant contribution of contact resistances, we show that a multistep hopping mechanism (composed of multiple tunneling steps), not single-step tunneling, explains the measured conductivity. Combined experimental and computational studies reveal that proton-coupled electron transfer confers conductivity; both the energetics of the proton acceptor, a neighboring glutamine, and its proximity to tyrosine influence the hole transport rate through a proton rocking mechanism. Surprisingly, conductivity increases 200-fold upon cooling due to higher availability of the proton acceptor by increased hydrogen bonding.

Cryo-EM structure of a human prion fibril with a hydrophobic, protease-resistant core.

Self-templating assemblies of the human prion protein are clinically associated with transmissible spongiform encephalopathies. Here we present the cryo-EM structure of a denaturant- and protease-resistant fibril formed in vitro spontaneously by a 9.7-kDa unglycosylated fragment of the human prion protein. This human prion fibril contains two protofilaments intertwined with screw symmetry and linked by a tightly packed hydrophobic interface. Each protofilament consists of an extended beta arch formed by residues 106 to 145 of the prion protein, a hydrophobic and highly fibrillogenic disease-associated segment. Such structures of prion polymorphs serve as blueprints on which to evaluate the potential impact of sequence variants on prion disease.

Homochiral and racemic MicroED structures of a peptide repeat from the ice-nucleation protein InaZ.

The ice-nucleation protein InaZ from Pseudomonas syringae contains a large number of degenerate repeats that span more than a quarter of its sequence and include the segment GSTSTA. Ab initio structures of this repeat segment, resolved to 1.1 Šby microfocus X-ray crystallography and to 0.9 Šby the cryo-EM method MicroED, were determined from both racemic and homochiral crystals. The benefits of racemic protein crystals for structure determination by MicroED were evaluated and it was confirmed that the phase restriction introduced by crystal centrosymmetry increases the number of successful trials during the ab initio phasing of the electron diffraction data. Both homochiral and racemic GSTSTA form amyloid-like protofibrils with labile, corrugated antiparallel ß-sheets that mate face to back. The racemic GSTSTA protofibril represents a new class of amyloid assembly in which all-left-handed sheets mate with their all-right-handed counterparts. This determination of racemic amyloid assemblies by MicroED reveals complex amyloid architectures and illustrates the racemic advantage in macromolecular crystallography, now with submicrometre-sized crystals.

The Cfd1 Subunit of the Nbp35-Cfd1 Iron Sulfur Cluster Scaffolding Complex Controls Nucleotide Binding.

The cytosolic iron sulfur cluster assembly (CIA) scaffold biosynthesizes iron sulfur cluster cofactors for enzymes residing in the cytosol and the nucleus. In fungi and animals, it comprises two homologous ATPases, called Nbp35 and Cfd1 in yeast, which can form homodimeric and heterodimeric complexes. Both proteins are required for CIA function, but their individual roles are not well understood. Here we investigate the nucleotide affinity of each form of the scaffold for ATP and ADP to reveal any differences that could shed light on the functions of the different oligomeric forms of the protein or any distinct roles of the individual subunits. All forms of the CIA scaffold are specific for adenosine nucleotides and not guanosine nucleotides. Although the Cfd1 homodimer has no detectable ATPase activity, it binds ATP with an affinity comparable to that of the hydrolysis competent forms, Nbp352 and Nbp35-Cfd1. Titrations to determine the number of nucleotide binding sites combined with site-directed mutagenesis demonstrate that the nucleotide must bind to the Cfd1 subunit of the heterodimer before it can bind to Nbp35 and that the Cfd1 subunit is hydrolysis competent when bound to Nbp35 in the heterodimer. Altogether, our work reveals the distinct roles of the Nbp35 and Cfd1 subunits in their heterodimeric complex. Cfd1 controls nucleotide binding, and the Nbp35 subunit is required to activate nucleotide hydrolysis.

Nanoscale mosaicity revealed in peptide microcrystals by scanning electron nanodiffraction.

Changes in lattice structure across sub-regions of protein crystals are challenging to assess when relying on whole crystal measurements. Because of this difficulty, macromolecular structure determination from protein micro and nanocrystals requires assumptions of bulk crystallinity and domain block substructure. Here we map lattice structure across micron size areas of cryogenically preserved three-dimensional peptide crystals using a nano-focused electron beam. This approach produces diffraction from as few as 1500 molecules in a crystal, is sensitive to crystal thickness and three-dimensional lattice orientation. Real-space maps reconstructed from unsupervised classification of diffraction patterns across a crystal reveal regions of crystal order/disorder and three-dimensional lattice tilts on the sub-100nm scale. The nanoscale lattice reorientation observed in the micron-sized peptide crystal lattices studied here provides a direct view of their plasticity. Knowledge of these features facilitates an improved understanding of peptide assemblies that could aid in the determination of structures from nano- and microcrystals by single or serial crystal electron diffraction.

Data-driven challenges and opportunities in crystallography.

Structural biology is in the midst of a revolution fueled by faster and more powerful instruments capable of delivering orders of magnitude more data than their predecessors. This increased pace in data gathering introduces new experimental and computational challenges, frustrating real-time processing and interpretation of data and requiring long-term solutions for data archival and retrieval. This combination of challenges and opportunities is driving the exploration of new areas of structural biology, including studies of macromolecular dynamics and the investigation of molecular ensembles in search of a better understanding of conformational landscapes. The next generation of instruments promises to yield even greater data rates, requiring a concerted effort by institutions, centers and individuals to extract meaning from every bit and make data accessible to the community at large, facilitating data mining efforts by individuals or groups as analysis tools improve.

Analysis of Global and Site-Specific Radiation Damage in Cryo-EM.

Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.

Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp.

The atomic structure of the infectious, protease-resistant, ß-sheet-rich and fibrillar mammalian prion remains unknown. Through the cryo-EM method MicroED, we reveal the sub-ångström-resolution structure of a protofibril formed by a wild-type segment from the ß2-α2 loop of the bank vole prion protein. The structure of this protofibril reveals a stabilizing network of hydrogen bonds that link polar zippers within a sheet, producing motifs we have named 'polar clasps'.

Identifying the Protein Interactions of the Cytosolic Iron-Sulfur Cluster Targeting Complex Essential for Its Assembly and Recognition of Apo-Targets.

The cytosolic iron-sulfur cluster assembly (CIA) system assembles iron-sulfur (FeS) cluster cofactors and inserts them into >20 apoprotein targets residing in the cytosol and nucleus. Three CIA proteins, called Cia1, Cia2, and Met18 in yeast, form the targeting complex responsible for apo-target recognition. There is little information about the structure of this complex or its mechanism of CIA substrate recognition. Herein, we exploit affinity co-purification and size exclusion chromatography to determine the subunit connectivity and stoichiometry of the CIA targeting complex. We conclude that Cia2 is the organizing center of the targeting complex, which contains one Met18, two Cia1, and four Cia2 polypeptides. To probe target recognition specificity, we utilize the CIA substrates Leu1 and Rad3 as well as the Escherichia coli FeS-binding transcription factor FNR (fumerate nitrate reductase). We demonstrate that both of the yeast CIA substrates are recognized, whereas the bacterial protein is not. Thus, while the targeting complex exhibits flexible target recognition in vitro, it cannot promiscuously recognize any FeS protein. Additionally, we demonstrate that the full CIA targeting complex is required to stably bind Leu1 in vitro, whereas the Met18-Cia2 subcomplex is sufficient to recognize Rad3. Together, these results allow us to propose a unifying model for the architecture of this highly conserved complex and demonstrate what component or subcomplexes are vital for target identification.