Protein amyloid aggregate: Structure and function

Protein amyloid aggregation has been widely observed to occur and plays important roles in both physiological processes and pathological diseases. Remarkably, amyloid aggregates assembled by native proteins gain a variety of different biological activities, which cannot be adopted by the unassembled protein alone. Thus, it is important to investigate the molecular basis of self‐assembly of protein amyloid aggregates and how the aggregated protein structure determines its function. In the review, we firstly introduce our structural knowledge on how different amyloid proteins undergo conformational transition and assemble into amyloid aggregate, with the main focus on amyloid fibril, which is the major species of amyloid aggregate. Then, we elaborate how different structures of amyloid fibrils enable them to fulfill highly diverse functions in either physiological or pathological condition. Furthermore, we discuss the structural polymorph which is a very unique feature of amyloid fibril, and its implication in understanding the structure‐function relationship of amyloid fibrils. Finally, we point out the importance of applying and integrating new approaches for deepening the structure‐function study of amyloid fibrils and highlight the potential of designing amyloid fibril‐based functional bio‐nanomaterials for application.


INTRODUCTION
Protein amyloid aggregation was originally discovered to form in the diseased brains of different neurodegenerative diseases (NDs) including Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). [1][2] Pathological amyloid fibril is normally the endproduct on the amyloid protein self-assembly and aggregation pathway. Under diseased condition, a variety of amyloid proteins including amyloid β (Aβ), Tau, TAR DNA binding protein (TDP- 43), and α-synuclein (α-syn), which fulfill different biological functions can assemble into amyloid fibrils, respectively. [3][4][5][6][7] Once formed, amyloid fibril is hard to be disassembled and may gain distinct pathological activity, and self-assemble into curli amyloid fibrils to form biofilm, which plays an essential role in bacteria attachment, cell invasion, and protection against phage attacks. [24][25][26] Receptorinteracting serine/threonine kinase 3 (RIPK3) from human can form amyloid fibrils for mediating signal transduction to induce necroptosis. [27,28] Sup35 from yeast may aggregate into fibril as the cytoplasmic determinant of inherited (PSI+). [29,30] Intriguingly, a very recent work demonstrates that the low-complexity domain (LCD) of nucleocapsid protein of SARS-CoV-2 can form amyloid fibril which is accelerated by viral RNA. SARS-CoV-2 infection can be diminished by blocking fibrillation of nucleocapsid protein. [31] Recently, rapid technical development in structural biology methods including cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) leads to structural determination of over 100 amyloid fibrils at atomic level, [32] which were formed by different proteins with diverse pathological activities and physiological functions. [32][33][34][35][36] This greatly advances our knowledge on how different amyloid fibril structures determine their highly diverse functions. Of note, prior to fibril formation, amyloid proteins may interact with different binding proteins (e.g., chaperone and receptor) and membranes, [37][38][39][40] and form different types of amyloid oligomers in the protein amyloid aggregation pathways. [41][42][43][44] In general, as intermediate states, amyloid oligomers are highly heterogenous and intrinsically unstable, which makes it extremely challenging to determine their structures at atomic level. [45][46][47][48][49] Notably, amyloid oligomers formed by Aβ was found to exhibit more potent neurotoxicity than Aβ fibrils, [41,44] implying its important role in AD. Indeed, in some AD cases, the burden of fibrillar deposits of Aβ was found to be not well correlated with the severity of cognitive impairment of patients. [50,51] However, given the very limited structural information of amyloid oligomers, how amyloid oligomer determines their pathology remains poorly understood and will not be discussed here.
In this review, we mainly summarize recent advances in understanding the structure and function relationship of protein amyloid aggregates, with the main focus on amyloid fibrils. Firstly, the common characteristics of structural organization shared by different amyloid fibrils will be introduced. Meanwhile, we will decipher the key molecular interaction and major driving force for triggering amyloid fibril formation as well as regulating the stability and reversibility of amyloid fibril. Second, we will discuss how different architectures of amyloid fibrils enable fibrils to fulfill a wide range of distinct biological activities under different circumstances. Last, we will discuss how one amyloid protein can form different polymorphic fibril structures with distinct physiochemical and functional activities.

2.1
The overall architecture of amyloid fibril Proteins with different native structures, either well-folded or intrinsically discorded, can undergo conformational changes and assemble into morphologically similar amyloid fibrils. [52,53] A single amyloid fibril is normally assembled by several hundreds to thousands of subunits of the same protein. Of note, different proteins may co-assemble into one single fibril. [28] Remarkably, different amyloid fibrils share several common features. For example, amyloid fibrils normally exhibit unbranched and elongated fibrillar morphology with the fibril thickness of ∼10-30 nm and the fibril length of ∼50-500 nm [54] ; fibrils display superior mechanical property with the rigidity ranging from 10 8 to 10 10 Pa [55] ; fibrils feature β-sheet conformation formed by different amyloid proteins and share a characteristic crossβ architecture as measured by X-ray fibril diffraction [56,57] ; different amyloid fibrils can be stained by several amyloiddye molecules such as Thioflavin T and Congo Red [58][59][60] ; the fibrillation kinetics of amyloid fibrils exhibits a typical nucleation-dependent characteristic. [61,62] These common features enable some general functional activities shared by different amyloid fibrils which will be detailed in the next section.
In general, amyloid fibril is composed of two parts, including a rigid fibril core region and a flexible flanking region ( Figure 1A). Upon fibrillation, a portion of protein sequence (referred as the fibril core sequence) may adopt β-conformation and assemble into a common cross-β fibril spine structure. The cross-β diffraction pattern features 4.8 and 10 Å meridional arc ( Figure 1A). The 4.8 Å arc is generated from the "inter-strand" distance between the neighboring β-strands within each β-sheet layer which is perpendicular to the fibril axis. The 10 Å arc represents the "inter-sheet" distance between the two neighboring β-sheet layers running parallel to the fibril axis. This unique structural arrangement of protein assembly is common to different amyloid fibrils, and is believed to be essential for small molecule amyloid dye binding. [59][60] Despite of sharing several common principles and structural characteristics by different fibrils, the fibril core formed by different proteins may display a high degree of structural diversity ( Figure 1A), including the topology of each protein subunit which is determined by the number and spatial arrangement of β-strands, the protofilament number and packing geometry and the chirality and helical twist of fibrils. These differences lead to the structural diversity of amyloid fibrils.
Besides the fibril core, the rest portion of the protein sequence (referred as the flanking sequence), which may display either a well-folded structure or an intrinsically disordered conformation ( Figure 1A), is aligned on the outer surface of the rigid fibril core and forms a relatively flexible outer layer of fibril. For example: the cryo-EM imaging data clearly demonstrate that the well-folded mCherry, which is fused to the N-terminal of heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2)-LCD retains its native global 3D structure and is intensively decorated on the surface of the rigid fibril core formed by hnRNPA2-LCD [63] (Figure 1B). In contrast, the N-and C-terminal of α-syn, which are intrinsically disordered regions (IDRs) in both native and fibrillar states, form a highly flexible outer layer like a fuzzy coat surrounding the fibril core formed by residues V37-Q99 of α-syn as visualized by cryo-EM ( Figure 1B). Thus, the rigid fibril core provides a scaffold to recruit and display the flanking sequence on the fibril surface for functions. For example, the flexible C-terminal IDR flanking on the fibril surface of α-syn F I G U R E 1 Structural organization and morphology of amyloid fibril. (A) Amyloid fibril is composed of a rigid fibril core region and a flexible flanking region. The core region adopts a common cross-β sheets architecture, while the fibril core formed by different protein subunits displays varsious topology with different number and spatial arrangement of β-strands. The flanking region displays either well-folded structure or intrinsically disordered conformation. (B) The cryo-electron microscopy (cryo-EM) image and 2D classification density map of hnRNPA2 fibril with a well-folded domain (left). Images minimally modified from ref. [63] with permission from Springer Nature. The cryo-EM image and 2D classification density map of α-syn fibril with IDRs (right). fibril mediates fibril binding to different receptors including lymphocyte activation gene 3 (LAG3) and receptor for advanced glycation end products (RAGE) on the membrane of neuron and microglia, respectively. [37][38] Heat shock protein 40 (Hsp40) prefers to bind the C-terminal IDR of α-syn fibril for further recruiting heat shock protein 70 (Hsp70) and heat shock protein 100 (Hsp100) to disassemble α-syn fibril. [64][65][66] In addition, the flanking region of other amyloid fibrils were reported to capture monomers for elongation, interact with mRNA to regulate translation, and interact with membrane. [67] Taken together, the rigid fibril core determines the basic mechanical properties of amyloid fibrils, and the flanking region with different structures and domain organization on the fibril surface may account for the functional activities of different fibrils.

Atomic view of amyloid fibril
Despite that the common cross-β structure of amyloid fibrils was revealed in 1971, [68] we have very limited knowledge of the atomic structures of amyloid fibrils formed by fulllength amyloid proteins until the last decade. [34,[69][70][71][72][73][74][75] So far, we have over 100 different atomic structural models of amyloid fibrils formed by a variety of different amyloid proteins, [32] which provides a wealthy structural database to decipher the molecular grammar of how different proteins assemble into amyloid fibrils. In the fibril structure, each fulllength amyloid protein subunit commonly folds into several β-strands structures interrupted by kinks and coils. The βstructured subunit twists around and stacks on top of each other along the fibril to form a single protofilament. Multiple protofilaments (two or three in most cases) may further assemble together by heterosteric zipper-like interaction and (or) salt-bridge to form mature fibril structures. As for the inter-subunit packing within each protofilament, the β-strands of each subunit normally form in-register β-sheets with the two neighboring subunits parallel to the fibril axis. Importantly, the backbones of β-strands form an intensive and continuous hydrogen-bonding network within β-sheet layer along the fibril axis, which contributes to the overall stability and stiffness of the fibril core structure (Figure 2A). In contrast to the uniformed backbone interaction, different sidechain packing patterns along the fibril axis were observed in different fibril structures, including (1) π-π stacking formed by Phe or Tyr; (2) D-ladder formed by Asp; (3) E-ladder formed by Glu; (4) salt bridge ladder; (5) aliphatic ladder ( Figure 2B). Notably, different sidechain interactions may play different or even opposite roles in determining the structural stability of fibrils. For example, the π-π stacking and salt bridge ladder can both strengthen the stability of fibril structures. While, the D-ladder may destabilize the overall fibril structures by charge repulsion which results in the formation of reversible fibrils. [76] Within each subunit, the hydrophobic interaction, electrostatic interaction and π-π & π-cation interaction serves as the major driving force for subunit folding and for determining the topology of each subunit ( Figure 2C). Moreover, subunits from different protofilament can further interact with each other to form mature fibril. The inter-protofilament interaction can be formed by highly complementary steric zipper-like interaction ( Figure 2D), which was originally observed to be dominantly formed in the crystal structures of amyloidogenic peptides, and is believed to be important in maintaining the thermostability of fibrils. [77] In addition, other interactions such as electrostatics interaction, hydrophilic interaction, or even water-mediated interaction (hydrous interface) were identified ( Figure 2D). This broadens our knowledge of the diverse driving forces mediating inter-protofilament assembly which in turn fine-tuning the stability and reversibility of fibrils formed by different proteins. To evaluate how different types of residues and interaction contribute to the stability of fibril structure, Eisenberg and coworkers developed the stabilization energy map by calculating the solvation standard free energy of each fibril. [32,63,78] Compared to the reversible fibril, the F I G U R E 3 The templating and seeding activity of amyloid fibril. (A) Different preformed fibril seeds can template native protein to form different fibril strains with similar structures as the seeds. The seeded growth process is zoomed in, where fibril seed seeds the native protein for rapid fibril formation by open-end templating or secondary nucleation. (B) Amyloid fibrils may self-template and spread in brain through cell-to-cell transmission. (C) The top panel is the cryo-electron microscopy (cryo-EM) image of Ac-wild-type (WT) and Ac-E46K α-syn fibrils after sonication, showing Ac-E46K α-syn is more prone to be fragmented. The middle panel is ThT kinetic assay of Ac-WT and Ac-E46K α-syn fibril formation. The bottom panel is the distinct fibril core structure of Ac-WT and Ac-E46K α-syn fibrils. Reprinted from ref. [90] with permission from Springer Nature. (D) Immunofluorescence images of dSTR of mouse brains injected with PBS, hWT and hE46K α-syn fibrils at 3 mpi. Scale bar, 50 μm. Zoom-in of the merged images are shown on the Right. Scale bar, 10 μm. Reprinted from ref. [89] with permission from National Academy of Science. stabilization energy of irreversible fibril structures generally features a more negative value per chain and per residue. [32] Taken together, the combination of different types of intraand inter-subunit interactions results in an extremely high diversity of subunit topology and the subunit packing in the fibrils, leading to the structural diversity of amyloid fibrils.

STRUCTURE AND FUNCTION RELATIONSHIP OF AMYLOID FIBRIL
Amyloid fibrils formed by different proteins exhibit a wide spectrum of biological activities ranging from biofilm formation to signal transduction in cell death and inflammation. [27,[79][80] Moreover, amyloid fibrils assembled by disease-associated proteins display a variety of different pathological activities, which are crucial in initiation and progression of different NDs. [81][82][83][84][85][86] Importantly, different biological activity is emerging only once the protein assembles into amyloid fibril but not possessed by the same protein in its native conformation. Therefore, how the structure of amyloid fibril determines the emerging new biological functions is one of the key questions in the amyloid field. In this section, we will discuss the structure and function relationship of amyloid fibrils by classifying the different pathological and physiological activities of fibrils into five categories and elucidate the role of different regions of fibril in mediating biological functions, including (1) templating and seeding activity determined by the rigid fibril core; (2) mechanical and surface charge-mediated activity by the rigid fibril core; (3) tunable activity in response to different environmental stimuli by the rigid core; (4) scaffolding and signaling amplification activity by combination of the rigid core and flanking region; (5) boosted partner binding activity mediated by the flanking region. We will evaluate how structural knowledge of amyloid fibrils may help to understand the distinct functions of fibrils.

Templating and seeding activity of amyloid fibril
The most well-known and unique property commonly shared by different amyloid fibrils is that fibril exhibits strong activity to template or seed the native protein for rapid fibril formation [87][88] (Figure 3A). Since the native protein, especially the fibril core sequence, is required to undergo a series of conformational changes in order to assembling into the βstructured fibril core, energy barriers do exist in the protein fibrillation pathway. Addition of preformed amyloid fibril, which serves as a seed, can effectively lower the energy barrier, diminish, or even bypass the nucleation process to accelerate fibrillation of the same native protein by providing the open ends of the growing fibril core for native protein attachment ( Figure 3A). Additionally, the fibril surface can also template protein fibrillation via the secondary nucleation process ( Figure 3A). The superior templating activity of the rigid fibril core is important for inducing rapid conformational switch of amyloid protein from the native state to fibrillation state, which can be spread within the same cell, between different cells and tissues, and even among different living organisms ( Figure 3B).
Importantly, mounting evidence demonstrates that the newly formed amyloid fibril may faithfully inherits the rigid fibril core structure as well as the pathological activity of its template. For example, in the presence of preformed E46K (a PD-associated mutation) α-syn fibril seeds, both E46K α-syn and wild-type (WT) α-syn monomer form E46K-like fibril core structure, which exhibits potent neuronal activity in the PD mouse model. [89][90] While, upon adding WT α-syn fibril (polymorph 1a) seed, WT α-syn monomer rapidly assembles into fibril with polymorph 1a structure. Therefore, templating activity of the amyloid fibril core includes not only accelerating structural conversion of amyloid proteins from native to fibrillar state, but also passing on the structure and function of the fibrils during propagation and spreading process. Of note, in some cases, templating process may result in amplification of fibril structure distinct from the template due to lacking of co-factors or posttranslational modification (PTM). [45,91] Moreover, amyloid fibrils with different core structures may exhibit distinct seeding activity in vitro and cell-tocell transmission capability in vivo. The biochemical and mechanical nature of fibril core such as fibril fragmentation rate, stability and surface property may determine the fibril seeding activity. For example, compared to WT α-syn polymorph 1a fibril, the E46K α-syn fibril features a smaller and instable core structure which is easier to be fragmented [90] ( Figure 3C). Accordingly, E46K fibril displays stronger capability for seeding in vitro and spreads more rapidly in mouse brains [89][90] (Figure 3D). Therefore, the fibril core structure can accelerate conformational transition and fibrillation of amyloid proteins, and different fibril core structure may feature different seeding activity which can be faithfully replicated by self-amplification both in vitro and in vivo.
The pathological significance of the fibril templating activity was originally identified from the transmissible prion proteins which is causative to prion diseases including madcow disease in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and so on. [92][93][94] Strikingly, more and more evidences reveal that amyloid fibrils formed by different proteins such as α-syn, Tau, and TDP-43 can all rapidly spread and propagate in the diseased brains via templatedepend cell-to-cell transmission ( Figure 3B), which plays an important role in disease progression of AD, PD and ALS. [10,12,95] Accordingly, targeting the templating activity of amyloid fibril may provide a potential strategy for developing therapeutic approaches for preventing NDs. [12][13]

Mechanical and surface charge-mediated activity
Compared to the native conformation of amyloid protein, amyloid fibrils as a bio-macromolecular polymer possess unique mechanical properties such as rigidity and stiffness achieved by the extensive hydrogen-bonded cross-β architecture commonly adopted by the fibril cores of different proteins ( Figure 4A). Interestingly, the superior mechanical activity can be used by different amyloid fibrils to either fulfil important physiological function or cause devastating pathological consequence. For example, Saibil and co-workers revealed by using cryo-EM that the fibril formed by β2m, which was found to be massively formed and served as a pathological entity in dialysis-related amyloidosis (DRA), can efficiently disrupt cell membrane by providing strong mechanical force for cell deformation, causing cellular damage [96] (Figure 4B). This study provides direct evidence on how amyloid fibril destroys cells by mechanical force, which may be shared by other pathological amyloid fibrils. More interestingly, cells can probe the rigid and exogenous pathological fibril by membrane receptor and trigger fibril degradation for protection. A very recent study reveals that mechanosensitive ion channel-Piezo1 on the microglial membrane can sense the stiffness of Aβ fibril to promote phagocytosis and clearance of toxic Aβ fibrils by microglia [97] (Figure 4C).
Beside the mechanical activity, fibril can be endowed with additional new activity derived from the extensively elongated fibril surface along the rigid fibril core ( Figure 4A), which doesn't exist in native proteins. One excellent example is the semen-derived amyloid fibril called as semen-derived enhancer of viral infection (SEVI). SEVI fibril is formed by proteolytic peptide fragments from prostatic acid phosphatase (PAP) in human semen and is important in mediating human immunodeficiency virus (HIV) virion infection. [98] Interestingly, the fibril core sequence of SEVI contains an unexpected high percentage of positively charged residues such as Arg and Lys. Thus, the SEVI fibril surface is highly positive-charged, which enable SEVI fibril to efficiently capture HIV virion via electrostatic interaction between the positive fibril surface and the negative membrane of HIV [99] (Figure 4D). While, disrupting this electrostatic interaction by molecular tweezers CLR01 can block HIV infection. [100,101] Another example of using fibril surface property for fulfilling biological function is the curli fibril generated from bacterial. The curli fibril surface exhibits strong adhesion property, which enable curli fibril to function in adhesion to surfaces, cell aggregation, biofilm formation to promote cell community behavior, and increase resistance to environmental stress. [102][103]

Dynamic disassembly activity fine-tuned by different environmental factors
Amyloid fibrils, especially the disease-related pathological fibrils, were initially found to feature high thermostability. [104] Once formed, most of the fibril cores could hardly disassemble both in vitro and in vivo. However, recent studies revealed that several RNA-binding proteins including fused in sarcoma (FUS), heterogrneous nuclear ribonucleoprotein A1 (hnRNPA1) and TDP-43 can assemble into amyloid fibrils with high reversibility which can be fine-tuned by different environmental factors, such as PTM, temperature, and salt concentration [105][106][107] (Figure 5A).
For example, FUS can form reversible fibrils in a temperature and phosphorylation-dependent manner. Several reversible amyloid core (RAC) segments were identified in FUS. [105] As for FUS-RAC1 ( 37 SYSGYS 42 ), it can self-assemble into an ordered-coil structure via hydrophilic interaction of the aromatic rings from the two neighboring Tyr residues ( Figure 5B). This structural arrangement is less stable than the typic dry steric zipper commonly observed in the irreversible fibril core. Accordingly, FUS-RAC1 fibril formed at 4 • C gradually disassembles upon increasing the temperature and reassembles once the temperature goes back to 4 • C ( Figure 5C). More interestingly, a phosphorylation site was identified at the Ser42 of FUS-RAC1, installation of phosphate on Ser42 can abolish the formation of coiledcore structure of FUS-RAC1 fibril core by disrupting the hydrogen bond formed by Ser42 and Tyr38, and effectively halts FUS-RAC1 fibrillation ( Figure 5D). Similar less stable hydrophilic interfaces were also observation in the fibril core formed by FUS-RAC2 ( 54 SYSSYG 59 ) and hnRNPA1-RAC1 ( 209 GFGGNDNFG 217 ). [76] In contrast to the thermostable fibril core structure where hydrophobic, zipper-like dry and highly complementary interaction is in general dominated, these reversible fibril core contains more hydrophilic or even hydrous interface, which significantly decreases the overall stability of fibril core. [105] The dynamic and tunable dissociation of reversible amyloid fibril is believed to play a role in regulating the dynamic assembly/disassembly and material properties of stress granule. [76,108,109] While mutation, which reinforms the fibril stability by strengthening the hydrophobic interaction within the fibril core structure, was found to harden stress granules and lead to formation of irreversible fibrillar aggregates which are closely associated with ALS. [105,[110][111][112][113] This part will be detailly discussed in the next section.

Scaffolding and signaling amplification activity
In addition to the fibril core sequence, many amyloid proteins contain well-folded functional domains within the flanking region sequence, which may play an important role in mediating fibril function. Upon fibril core assembly, a large number of well-folded domains could be condensed and aligned on the fibril surface in a proximity to each other, which otherwise remains separated in the protein native conformation ( Figure 6A). This condensed spatial arrangement can efficiently stimulate protein self-activation and signal amplification which is adopted by proteins in different signal transduction pathways [27] (Figure 6A). Moreover, co-assembly of different proteins together into fibril may provide a scaffold for facilitating protein-protein interaction and enzymatic reaction.
For example, RIPK1 and RIPK3 were previously reported to assemble into amyloid fibrils which serve as the key node in signal transduction to induce necroptosis-one major type of cell death. [114][115] RIPK1 contains a fibril core sequence, a kinase domain (KD) and a death domain. While, RIPK3 is composed of a fibril core sequence with high sequence similarity with that of RIPK1, and a KD. Each protein can form homotypical fibril, where the KDs of either RIPK1 or RIPK3 could gather together on the fibril surface for triggering self-and cross-phosphorylation of KD, which is essential for amplifying the signaling transduction to the downstream effectors. Moreover, RIPK1 and RIPK3 can co-assemble via the conserved fibril core sequence into heterotypical fibrils, where RIPK1 KD can phosphorylate not only its own KD but also RIPK3 KD to efficiently pass on the cell death signals [116] (Figue 6B). Once got fully activated via KD phosphorylation, RIPK3 KD on the fibril surface may further recruit its downstream protein mixed lineage kinase domain-like protein (MLKL) for signal transduction [116] ( Figure 6C).
Interestingly, the fibril core structure of RIPK3 exhibits a unique small S-shaped fold with an extremely short fibril pitch (23 nm) compared to the other fibrils formed by different pathological proteins with a fibril pitch normally ranging from 50 to 250 nm [117] (Figure 6D,E). This unique arrangement of fibril core may provide appropriate geometry and space for accommodating KDs on the fibril surface to promote effective phosphorylation without steric clash. [117] Thus, amyloid fibril can serve as a superior scaffold for recruiting a large number of functional domains arranged in certain geometry and proximity for promoting protein interaction and enzymatic reactivity in signal amplification and transduction.

Boosted partner binding activity
Protein-protein interaction plays an essential role in mediating protein function. [118] Amyloid fibril can interact with a variety of different protein binding partners for fulfilling their biological functions ( Figure 7A). Remarkably, despite of containing the identical primary sequence, amyloid protein in the native form and fibrillar form may exhibit largely distinct binding profile to different protein binding partners. [119] For example, recent studies showed that several receptors such as LAG3, RAGE, and amyloid precursor-like protein 1(APLP1) bind α-syn fibril with over 100-fold higher affinity compared to that of α-syn monomer. [37][38] This indicates that amyloid protein may dramatically enhance its binding affinity to certain protein binding partner by self-assembling into fibril. Structural characterization of the interaction between α-syn fibril and different receptors allows us to appreciate how amyloid fibril structure acquires this activity. Nuclear magnetic resonance (NMR) study showed that LAG3 used its positively charged pocket to bind the C-terminal negatively charged flanking region of α-syn both in monomeric and . Yellow arrow shows MLKL oligomer in a rod-shaped necrosome. Reprinted from ref. [116] with permission from Springer Nature. (D) Cryo-EM reconstruction density map and the density map with a structure model of RIPK3 C-terminal domain fibril. Reprinted from ref. [117] with permission from National Academy of Science. (E) Statistics of the fibril pitch and the number of residues within fibril core per 4.8 Å layer for 50 different cryo-EM fibril structures. Reprinted from ref. [117] with permission from National Academy of Science. fibrillar states ( Figure 7B). Interestingly, the C-terminal flanking region is self-shielded in α-syn monomer by transiently interacting with the central fibril core sequence. Upon forming α-syn fibril, the center core sequence self-assembles into fibril and unleashes the C-terminal flanking region.
Meanwhile, the C-terminal flanking region is decorated and highly decondensed on the fibril surface ( Figure 7B). Increased local concentration of the C-terminal flanking region on the fibril may in turn recruit plenty of LAG3 molecules on the membrane to jointly bind the entire α-syn fibril for endocytosis. Therefore, the structural arrangement of α-syn in fibril greatly enhances its binding of the Cterminal flanking region with LAG3. The other receptors including APLP1 and RAGE use similar mechanism for capturing α-syn fibrils. Moreover, binding of α-syn fibril to LAG3 and APLP1 initiates endocytosis of α-syn fibril and cell-to-cell transmission of α-syn fibril in neurons. [37] While, binding of RAGE to α-syn fibril on the microglia membrane triggers neuroinflammation [38] ( Figure 7C). Together, conformational transition upon protein fibrillation may not only release the potential protein binding sequence but also display the binding sequence with high local concentration on the fibril surface for boosting its interaction with other proteins ( Figure 7A).

STRUCTURAL POLYMORPHISM OF AMYLOID FIBRILS
As we summarized above, different amyloid proteins may assembly into fibrils with distinct fibril structures with the fibril core and the flanking region assembled in certain spatial arrangement, which enables fibrils to fulfil diverse biological functions. As for protein folding, the thermodynamic F I G U R E 7 The boosted partner binding activity of amyloid fibril. (A) Formation of amyloid fibrils releases the potential protein binding domain and display the binding domain with high local concentration on the fibril surface for boosting its interaction with receptors. (B) Schematic illustration of the structural mechanism by which LAG3 domain 1 (L3D1) preferentially binds with α-syn fibrils over the monomer. The α-syn monomer adopts a self-shielded conformation. As forming amyloid fibrils, α-syn exposes and condenses the C termini, which significantly enhance the binding with L3D1. Reprinted from ref. [37] with permission from National Academy of Science. (C) Images and qPCR analysis of α-syn monomer or α-syn PFFs binding to wild-type (WT) or mRAGE −/− primary microglial cells. Scale bar, 50 mm. Reprinted from ref. [38] with permission from Elsevier. hypothesis by Christian Anfinsen demonstrates that the 3D protein structure is determined by its primary sequence. [120] Most of the native proteins normally have only one or very few thermodynamic stable native structures, except for the intrinsically disordered protein. However, it seems that the Anfinsen's dogma needs to be expanded for protein amyloid aggregate, since one amyloid protein can fold into a variety of different fibril structures, which is termed as structural polymorphism of amyloid fibril. [32] As we proposed, protein amyloid aggregation in the fibrillar state is more like a 2D folding with less restrains for confining the subunit in one or very few conformations as achieved by 3D folding in protein folding of native structure. [45] Moreover, the different fibril structures formed by the same protein may display largely distinct activities, which increases the complexity of the structure-function relationship of amyloid fibrils and broadens the usage of amyloid fibril for various biological functions. Thus, in this section, we will discuss how the same protein may form different types of amyloid fibrils with diverse structural polymorphs either during aggregation process or under different aggregation conditions. We will further discuss the corresponding biological significances of different fibril structures formed by the same protein under different conditions.

Different fibril structures formed during aggregation process
As mentioned above, RNA-binding proteins including FUS, hnRNPA1, and TDP-43 can form highly reversible amyloid fibrils either de novo or within the dynamic liquid-like droplet formed by the same protein. [113,[121][122][123][124] Moreover, the association and disassociation of these reversible fibrils can be dynamically regulated by phosphorylation and temperature fluctuation. [76,105] The formation of hnRNPA1 reversible fibrils is involved in fine-tunning the material properties and interior dynamics of the liquid-droplet in vitro and stress granule formation in cells [76] (Figure 8A). In these reversible fibrils, RACs or LARKS, short for LC, aromatic-rich, kinked segments, derived from the LCD of these three proteins were identified to mediate reversible fibril formation. [123] Structurally, these reversible fibril cores exhibit diverse structural arrangement including ordered-coil with hydrophilic interface (FUS-RAC1), zipper-like structure with hydrous interface (FUS-RAC2), and stacking-D structure with hydrophilic interface (hnRNPA1-RAC1), which is distinct from typical hydrophobic zipper-like structure of irreversible fibrils [76,105] (Figure 8B). These highly diverse core structures enable reversible fibril to serve as a functional fibril to assemble and disassemble in response to different regulatory stimulus.
FUS, hnRNPA1 and TDP-43 were also previously identified to form stable fibrillar aggregates in the diseased brains of ALS and frontotemporal dementia (FTD) patients. Moreover, ALS-associated mutations were identified to accelerate the formation of irreversible and pathological fibrils of these proteins, reinforming the important role of the pathological fibrils in diseases. [111][112] Further structural investigation revealed that the stable and detergent-resistant TDP-43 fibrils directly extracted from ALS patients' brains features typical cross-β architecture with the TDP-43 subunit forming multiple β-strand arranged in a relatively large doublespiral-shaped fold. [125] Meanwhile, the atomic structures of Reprinted from ref. [36] with permission from Elsevier. The structure of FUS monomer is from Alphafold protein structure database. [143,144] irreversible amyloid fibrils formed by the LCD of the other two ALS-associated protein hnRNPA1 and FUS were also determined by cryo-EM [36,106] (Figure 8B). Similar to the TDP-43 irreversible fibril structure, these two irreversible amyloid fibrils feature typical cross-β structures with multiple β-strands forming enlarged fibril cores. [125,126] However, unlike the traditional pathological fibril structure where the main driving force for intramolecular β-strand assembly is hydrophobic interaction, hydrophilic interaction and ππ stacking are dominate in these three irreversible fibril structures. [32,36,106] Notably, an ALS-associated mutation D262V of hnRNPA1 can effectively disrupt the key stacking-D structure of the reversible fibril core. Replacement of Asp by Val induces hnRNPA1 to form a more stable fibril core and promote the structural transition of hnRNPA1 from reversible fibril to irreversible fibrils, thus convert functional fibril to pathological fibril. [105,127] Therefore, one protein can use different regions to form amyloid fibrils with different structures, which may play either functional and pathological roles ( Figure 8B). These RNA-binding proteins can form reversible amyloid fibrils with physiological function, which may further convert into irreversible pathological fibrils during maturation and aging process, which is closely associated with disease ( Figure 8A).

Different fibril structures formed under different conditions
In addition to forming different structural polymorphs during aggregation process, it is more commonly observed that one protein can form a high diversity of polymorphic fibrils under different in vitro or in vivo aggregation F I G U R E 9 Different α-syn fibril structures formed under different conditions. (A) Schematic depicting the 140 amino acids of human α-syn and intrinsically disordered conformations of α-syn. Top views of four cryo-electron microscopy (cryo-EM) structures of α-syn fibrils extracted from patients' brains and prepared in vitro are shown. Co-factors and pY39 modification are indicated. PDB ID of structures: 8A9L (Lewy α-syn strain), [139] 6XYO (MSA α-syn fibrils), [138] 6A6B (WT α-syn strain), [72] and 6L1T (pY39 α-syn strain). [145] (B) Schematic depicting pathological features of PD and MSA associated with Lewy strain and MSA strain, respectively. α-Syn inclusion types, affected cell type and brain areas of neuron loss are shown. Created with BioRender.com. LB, Lewy body; GCI, glial cytoplasmic inclusion. (C) Comparison of the stability and cell toxicity of WT strain and pY39 strain. Reprinted from ref. [145] with permission from National Academy of Science.
conditions. [35,45,128,129] One of the most intensively studied amyloid proteins is α-syn, which is closely related to various synucleinopathies including PD, dementia with Lewy body (DLB) and multiple system atrophy (MSA). [130,131] α-Syn is a small protein highly enriched in presynaptic terminal of neuron, which contains only 140 amino acids and features an intrinsically disordered conformation in solution. [132,133] Strikingly, once assembling into amyloid fibril, over 50 different structural polymorphs of α-syn fibrils were determined at atomic level so far. A variety of different factors including PTM, fibrillation buffer, and unidentified endogenous co-factor may induce α-syn to form different structural polymorphs of fibrils. [72][73][134][135][136][137][138][139] WT α-syn can form a Greek key-like structure in the fibril core of polymorph 1a. [72] While one single phosphorylation at Y39 may completely re-configurate α-syn to form a new large hook-like structure of the fibril core [137] ( Figure 9A). This entire conformational reconfiguration was barely observed to be achieved by a single PTM in the native protein. Moreover, α-syn fibrils directly extracted from different regions of PD and MSA patients' brains are structurally distinct from each other [138,139] and from the fibrils prepared in vitro ( Figure 9A,B). Remarkably, several unidentified nonproteinaceous molecules presumably as endogenous cofactors of α-syn were observed to settle in the central of the fibril cores, which may play an essential role in holding the entire fibril structures [138,139] (Figure 9A). Thus, co-factors from different types of cells (e.g., neuron and oligodendrocyte) and organs (e.g., brain and gut) could be important in inducing different fibril structures of α-syn. More importantly, different structures endow distinct physiochemical properties of α-syn fibril, which results in distinct pathological activities of α-syn fibril counting for the high pathological heterogeneity of disease. For example, due to the extremely large fibril core structure, pY39 α-syn fibril is highly stable and resistant to protease cleavage, and thus exhibits potent neurotoxicity [137] (Figure 9C). While the E46K α-syn fibril structure as mentioned above is prone to be fragmented and feature enhanced self-propagation capability in both in vitro fibrillation assay and in vivo cell-to-cell transmission and spreading assay. [89][90]

PERSPECTIVE
Amyloid fibril serves as a unique protein assemble scaffold, which can be commonly adopted by a variety of different proteins. Technical advances in structural determination of amyloid fibrils have greatly pushed forward our molecular understanding on how proteins assemble into different amyloid fibril structures for carrying out distinct biological activities. In this review, we summarized five different categories of amyloid fibrils for using their distinct assembled architecture to acquire new functions and discussed the extremely high structural polymorphism of amyloid fibril formed by the same protein.
In the end, we sought to discuss further directions for developing new approaches to investigate the structure and function relationship of amyloid fibrils and highlight the potential of amyloid fibrils for design of functional bio-nanomaterial with diverse applications.
1. As mentioned above, bioactive amyloid fibrils normally contain both the amyloid core region and the flanking region. In many cases, the flanking region decorated on the fibril surface plays an essential role in mediating fibril function. However, so far, most of the structural studies on amyloid fibrils focus on the structure determination of the fibril core but no the flanking region due to the technical limitation. As the flanking region on the fibril generally exhibits high local flexibility which excludes it from being directly visualized by cryo-EM and crosspolarization-based ssNMR. Other biophysical approaches such as the insensitive nuclei enhanced by polarization transfer-based ssNMR, single molecule fluorescence resonance energy transfer, mass spectrometry-based methods and super-resolution microscopy need to be developed and jointly applied to explore the atomic structure of the flanking region on the fibril, as well as its interplay with other binding partners such as protein, metabolite, and lipid membrane. 2. Currently, our fundamental knowledges on how amyloid fibrils fulfill their activities at atomic level were mostly attributed from the in vitro model systems. It will be important to directly visualize amyloid fibril structures and their cross-talk with other endogenous players in cells. Cryo-electron tomography (Cryo-ET) provides a powerful approach to decipher the structure-function relationship of different amyloid fibrils in the context of cellular environment. Indeed, recent studies by using cryo-ET to investigate polyQ fibrils in intact neurons demonstrated that polyQ fibrils can deform endoplasmic reticulum (ER) membranes and alter ER organization and dynamics. [140] Further structural characterization of different structural polymorphs of amyloid fibrils deposited in tissues from diseased patients will provide valuable information on how different fibrils induce distinct pathology in diseases. 3. As a unique form of protein assembly designed and used by nature, amyloid fibrillar structure once assembled can acquire numerous different activities which cannot be adopted by native-folded protein. Therefore, amyloid fibril may serve as a superior proteinaceous template for development of functional bio-nanomaterials. [141,142] By integrating different functional groups/domains with the fibril core, one may generate amyloid fibrils with highly diverse biological activities. Moreover, our deeper understanding on the structure-function relationship of amyloid fibril will greatly prompt rational design of functional amyloid fibrils.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflicts of interest.