NMR Dynamic View of the Stabilization of the WW4 Domain by Neutral NaCl and Kosmotropic Na2SO4 and NaH2PO4.

The Hofmeister series categorizes ions based on their effects on protein stability, yet the microscopic mechanism remains a mystery. In this series, NaCl is neutral, Na2SO4 and Na2HPO4 are kosmotropic, while GdmCl and NaSCN are chaotropic. This study employs CD and NMR to investigate the effects of NaCl, Na2SO4, and Na2HPO4 on the conformation, stability, binding, and backbone dynamics (ps-ns and µs-ms time scales) of the WW4 domain with a high stability and accessible side chains at concentrations ≤ 200 mM. The results indicated that none of the three salts altered the conformation of WW4 or showed significant binding to the four aliphatic hydrophobic side chains. NaCl had no effect on its thermal stability, while Na2SO4 and Na2HPO4 enhanced the stability by ~5 °C. Interestingly, NaCl only weakly interacted with the Arg27 amide proton, whereas Na2SO4 bound to Arg27 and Phe31 amide protons with Kd of 32.7 and 41.6 mM, respectively. Na2HPO4, however, bound in a non-saturable manner to Trp9, His24, and Asn36 amide protons. While the three salts had negligible effects on ps-ns backbone dynamics, NaCl and Na2SO4 displayed no effect while Na2HPO4 significantly increased the µs-ms backbone dynamics. These findings, combined with our recent results with GdmCl and NaSCN, suggest a microscopic mechanism for the Hofmeister series. Additionally, the data revealed a lack of simple correlation between thermodynamic stability and backbone dynamics, most likely due to enthalpy-entropy compensation. Our study rationalizes the selection of chloride and phosphate as the primary anions in extracellular and intracellular spaces, as well as polyphosphate as a primitive chaperone in certain single-cell organisms.

NMR Dynamic View of the Destabilization of WW4 Domain by Chaotropic GdmCl and NaSCN.

GdmCl and NaSCN are two strong chaotropic salts commonly used in protein folding and stability studies, but their microscopic mechanisms remain enigmatic. Here, by CD and NMR, we investigated their effects on conformations, stability, binding and backbone dynamics on ps-ns and µs-ms time scales of a 39-residue but well-folded WW4 domain at salt concentrations ≤200 mM. Up to 200 mM, both denaturants did not alter the tertiary packing of WW4, but GdmCl exerted more severe destabilization than NaSCN. Intriguingly, GdmCl had only weak binding to amide protons, while NaSCN showed extensive binding to both hydrophobic side chains and amide protons. Neither denaturant significantly affected the overall ps-ns backbone dynamics, but they distinctively altered µs-ms backbone dynamics. This study unveils that GdmCl and NaSCN destabilize a protein before the global unfolding occurs with differential binding properties and µs-ms backbone dynamics, implying the absence of a simple correlation between thermodynamic stability and backbone dynamics of WW4 at both ps-ns and µs-ms time scales.

Extreme diversity of 12 cations in folding ALS-linked hSOD1 unveils novel hSOD1-dependent mechanisms for Fe2+/Cu2+-induced cytotoxicity.

153-Residue copper-zinc superoxide dismutase 1 (hSOD1) is the first gene whose mutation was linked to FALS. To date, > 180 ALS-causing mutations have been identified within hSOD1, yet the underlying mechanism still remains mysterious. Mature hSOD1 is exceptionally stable constrained by a disulfide bridge to adopt a Greek-key ß-barrel fold that accommodates copper/zinc cofactors. Conversely, nascent hSOD1 is unfolded and susceptible to aggregation and amyloid formation, requiring Zn2+ to initiate folding to a coexistence of folded and unfolded states. Recent studies demonstrate mutations that disrupt Zn2+-binding correlate with their ability to form toxic aggregates. Therefore, to decode the role of cations in hSOD1 folding provides not only mechanistic insights, but may bear therapeutic implications for hSOD1-linked ALS. Here by NMR, we visualized the effect of 12 cations: 8 essential for humans (Na+, K+, Ca2+, Zn2+, Mg2+, Mn2+, Cu2+, Fe2+), 3 mimicking zinc (Ni2+, Cd2+, Co2+), and environmentally abundant Al3+. Surprisingly, most cations, including Zn2+-mimics, showed negligible binding or induction for folding of nascent hSOD1. Cu2+ exhibited extensive binding to the unfolded state but led to severe aggregation. Unexpectedly, for the first time Fe2+ was deciphered to have Zn2+-like folding-inducing capacity. Zn2+ was unable to induce folding of H80S/D83S-hSOD1, while Fe2+ could. In contrast, Zn2+ could trigger folding of G93A-hSOD1, but Fe2+ failed. Notably, pre-existing Fe2+ disrupted the Zn2+-induced folding of G93A-hSOD1. Comparing with the ATP-induced folded state, our findings delineate that hSOD1 maturation requires: (1) intrinsic folding capacity encoded by the sequence; (2) specific Zn2+-coordination; (3) disulfide formation and Cu-load catalyzed by hCCS. This study unveils a previously-unknown interplay of cations in governing the initial folding of hSOD1, emphasizing the pivotal role of Zn2+ in hSOD1-related ALS and implying new hSOD1-dependent mechanisms for Cu2+/Fe2+-induced cytotoxicity, likely relevant to aging and other diseases.

ATP induces folding of ALS-causing C71G-hPFN1 and nascent hSOD1.

ALS-causing C71G-hPFN1 coexists in both folded and unfolded states, while nascent hSOD1 is unfolded. So far, the mechanisms underlying their ALS-triggering potential remain enigmatic. Here we show by NMR that ATP completely converts C71G-hPFN1 into the folded state at a 1:2 ratio, while inducing nascent hSOD1 into two co-existing states at a 1:8 ratio. Surprisingly, the inducing capacity of ATP comes from its triphosphate, but free triphosphate triggers aggregation. The inducing capacity ranks as: ATP = ATPP = PPP > ADP = AMP-PNP = AMP-PCP = PP, while AMP, adenosine, P, and NaCl show no conversion. Mechanistically, ATP and triphosphate appear to enhance the intrinsic folding capacity encoded in the sequences, as unveiled by comparing conformations and dynamics of ATP- and Zn2+-induced hSOD1 folded states. Our study provides a mechanism for the finding that some single-cell organisms employ polyphosphates as primordial chaperones, and sheds light on the enigma of age-related onset of familial ALS and risk increase of neurodegenerative diseases.

ALS-causing hPFN1 mutants differentially disrupt LLPS of FUS prion-like domain.

hPFN1 mutations including C71G cause ALS by gain of toxicity but the mechanism still remains unknown. Stress granules (SGs) are formed by phase separation of the prion-like domain (PLD) of RNA-binding proteins including FUS, whose inclusion was also associated with ALS. C71G-hPFN1 triggers seed-dependent co-aggregation with FUS/TDP-43 to manifest the prion-like propagandation but its biophysical basis remains unexplored. Here by DIC imaging we first showed that three hPFN1 mutants have differential capacity in disrupting the dynamics of liquid droplets formed by phase separation of FUS prion-like domain (PLD). C71G-hPFN1 co-exists with the folded and unfolded states, thus allowing to simultaneously characterize conformations, hydrodynamics and dynamics of the interactions of both states with the phase separated FUS PLD by NMR. The results reveal that the folded state is not significantly affected while by contrast, the unfolded state has extensive interactions with FUS PLD. As a consequence, the dynamics of FUS liquid droplets become significantly reduced. Such interactions might act to recruit C71G-hPFN1 into the droplets, thus leading to the increase of the local concentrations and subsequent co-aggregation of C71G-hPFN1 with FUS. Our study sheds the first light on the biophysical basis by which hPFN1 mutants gain toxicity to cause ALS. As other aggregation-prone proteins have no fundamental difference from hPFN1 mutants, aggregation-prone proteins might share a common capacity in disrupting phase separation responsible for organizing various membrane-less organelles. As such, the mechanism for C71G-hPFN1 might also be utilized by other aggregation-prone proteins for gain of toxicity to trigger diseases and aging.

Myricetin Allosterically Inhibits the Dengue NS2B-NS3 Protease by Disrupting the Active and Locking the Inactive Conformations.

The dengue NS2B-NS3 protease existing in equilibrium between the active and inactive forms is essential for virus replication, thus representing a key drug target. Here, myricetin, a plant flavonoid, was characterized to noncompetitively inhibit the dengue protease. Further NMR study identified the protease residues perturbed by binding to myricetin, which were utilized to construct the myricetin-protease complexes. Strikingly, in the active form, myricetin binds to a new allosteric site (AS2) far away from the active site pocket and the allosteric site (AS1) for binding curcumin, while in the inactive form, it binds to both AS1 and AS2. To decipher the mechanism for the allosteric inhibition by myricetin, we conducted molecular dynamics simulations on different forms of dengue NS2B-NS3 proteases. Unexpectedly, the binding of myricetin to AS2 is sufficient to disrupt the active conformation by displacing the characteristic NS2B C-terminal ß-hairpin from the active site pocket. By contrast, the binding of myricetin to AS1 and AS2 results in locking the inactive conformation. Therefore, myricetin represents the first small molecule, which allosterically inhibits the dengue protease by both disrupting the active conformation and locking the inactive conformation. The results enforce the notion that a global allosteric network exists in the dengue NS2B-NS3 protease, which is susceptible to allosteric inhibition by small molecules such as myricetin and curcumin. As myricetin has been extensively used as a food additive, it might be directly utilized to fight the dengue infections and as a promising starting material for further design of potent allosteric inhibitors.

ATP biphasically modulates LLPS of TDP-43 PLD by specifically binding arginine residues.

Mysteriously neurons maintain ATP concentrations of ~3 mM but whether ATP modulates TDP-43 LLPS remains completely unexplored. Here we characterized the effect of ATP on LLPS of TDP-43 PLD and seven mutants by DIC and NMR. The results revealed: 1) ATP induces and subsequently dissolves LLPS of TDP-43 PLD by specifically binding Arg saturated at 1:100. 2) ATP modifies the conformation-specific electrostatic property beyond just imposing screening effect. 3) Reversibility of LLPS of TDP-43 PLD and further exaggeration into aggregation appear to be controlled by a delicate network composed of both attractive and inhibitory interactions. Results together establish that ATP might be a universal but specific regulator for most, if not all, R-containing intrinsically-disordered regions by altering physicochemical properties, conformations, dynamics, LLPS and aggregation. Under physiological conditions, TDP-43 is highly bound with ATP and thus inhibited for LLPS, highlighting a central role of ATP in cell physiology, pathology and aging.

Curcumin Allosterically Inhibits the Dengue NS2B-NS3 Protease by Disrupting Its Active Conformation.

Flaviviruses including dengue virus and Zika virus encode a unique two-component NS2B-NS3 protease essential for maturation/infectivity, thus representing a key target for designing antiflavivirus drugs. Here, for the first time, by NMR and molecular docking, we reveal that curcumin allosterically inhibits the dengue protease by binding to a cavity with no overlap with the active site. Further molecular dynamics simulations decode that the binding of curcumin leads to unfolding/displacing the characteristic ß-hairpin of the C-terminal NS2B and consequently disrupting the closed (active) conformation of the protease. Our study identified a cavity most likely conserved in all flaviviral NS2B-NS3 proteases, which could thus serve as a therapeutic target for the discovery/design of small-molecule allosteric inhibitors. Moreover, as curcumin has been used as a food additive for thousands of years in many counties, it can be directly utilized to fight the flaviviral infections and as a promising starting for further design of potent allosteric inhibitors.

ATP is a cryptic binder of TDP-43 RRM domains to enhance stability and inhibit ALS/AD-associated fibrillation.

ATP is the universal energy currency for all cells but has cellular concentrations of 2-12 mM, much higher than required for its classic functions. RNA-recognition motif (RRM) constitutes one of the most abundant domains in eukaryotes and most heterogeneous nuclear ribonucleoproteins (hnRNP) contain RRM domains which not only mediate direct interactions with nucleic acids, but whose aggregation/fibrillation is the pathological hallmark of various human diseases. Here, by NMR and molecular docking, ATP has been decoded to bind TDP-43 two tandem RRM domains with distinctive types of interactions, thus resulting in diverse affinities. Most strikingly, the binding of ATP enhances thermodynamic stability of TDP-43 RRM domains and inhibits ALS-/AD-associated fibrillation. Together, ATP is a cryptic binder of RRM-containing proteins which generally safeguards functional phase separation from transforming into pathological aggregation/fibrillation associated with various diseases and ageing. Our study thus reveals a mechanism of ATP to control protein homeostasis by specific binding.

ATP binds nucleic-acid-binding domains beyond RRM fold.

It has remained a mystery why cells maintain ATP concentrations of 2-12 mM, much higher than required for its known functions, until ATP is decoded to act as a hydrotrope to non-specifically control protein homeostasis above 5 mM. Unexpectedly, our NMR studies further reveal that by specific binding, ATP also mediates liquid-liquid phase separation in a two-stage style and inhibits fibrillation of RRM domains of FUS and TDP-43, implying that ATP might have a second category of functions previously unknown. So can ATP also bind nucleic-acid-binding proteins without RRM fold? Here we characterized the interaction between ATP and SYNCRIP acidic domain (AcD), a non-canonical RNA-binding domain with no similarity to RRM fold in sequence and structure. The results reveal that ATP does bind AcD at physiologically-relevant concentrations with the affinity determinants generally underlying protein-nucleic acid interactions. Therefore, at concentrations above mM, ATP might bind most, if not all, nucleic-acid-binding proteins.

ATP binds and inhibits the neurodegeneration-associated fibrillization of the FUS RRM domain.

Adenosine triphosphate (ATP) provides energy for cellular processes but has recently been found to act also as a hydrotrope to maintain protein homeostasis. ATP bivalently binds the disordered domain of FUS containing the RG/RGG sequence motif and thereby affects FUS liquid-liquid phase separation. Here, using NMR spectroscopy and molecular docking studies, we report that ATP specifically binds also to the well-folded RRM domain of FUS at physiologically relevant concentrations and with the binding interface overlapping with that of its physiological ssDNA ligand. Importantly, although ATP has little effect on the thermodynamic stability of the RRM domain or its binding to ssDNA, ATP kinetically inhibits the RRM fibrillization that is critical for the gain of cytotoxicity associated with ALS and FTD. Our study provides a previously unappreciated mechanism for ATP to inhibit fibrillization by specific binding, and suggests that ATP may bind additional proteins other than the classic ATP-dependent enzymes.

A unified mechanism for LLPS of ALS/FTLD-causing FUS as well as its modulation by ATP and oligonucleic acids.

526-residue Fused in sarcoma (FUS) undergoes liquid-liquid phase separation (LLPS) for its functions, which can further transit into pathological aggregation. ATP and nucleic acids, the universal cellular actors, were shown to modulate LLPS of FUS in a unique manner: enhancement and then dissolution. Currently, the driving force for LLPS of FUS is still under debate, while the mechanism for the modulation remains completely undefined. Here, by NMR and differential interference contrast (DIC) imaging, we characterized conformations, dynamics, and LLPS of FUS and its domains and subsequently their molecular interactions with oligonucleic acids, including one RNA and two single-stranded DNA (ssDNA) molecules, as well as ATP, Adenosine monophosphate (AMP), and adenosine. The results reveal 1) both a prion-like domain (PLD) rich in Tyr but absent of Arg/Lys and a C-terminal domain (CTD) abundant in Arg/Lys fail to phase separate. By contrast, the entire N-terminal domain (NTD) containing the PLD and an Arg-Gly (RG)-rich region efficiently phase separate, indicating that the π-cation interaction is the major driving force; 2) despite manifesting distinctive NMR observations, ATP has been characterized to modulate LLPS by specific binding as oligonucleic acids but with much lower affinity. Our results together establish a unified mechanism in which the π-cation interaction acts as the major driving force for LLPS of FUS and also serves as the target for modulation by ATP and oligonucleic acids through specific binding. This mechanism predicts that a myriad of proteins unrelated to RNA-binding proteins (RBPs) but with Arg/Lys-rich disordered regions could be modulated by ATP and nucleic acids, thus rationalizing the pathological association of Amyotrophic lateral sclerosis (ALS)-causing C9ORF72 dipeptides with any nucleic acids to manifest cytotoxicity.

A novel mechanism for ATP to enhance the functional oligomerization of TDP-43 by specific binding.

Pathological TDP-43 aggregation has been found in ∼98% ALS and other neurodegenerative diseases including Alzheimer's. TDP-43 N-terminal domain (NTD) was recently shown to form a tubular super-helical filament by oligomerization in vivo, which functions to prevent its pathological aggregation. ATP, the universal energy currency with very high concentrations in all living cells, was recently decoded to act as a biological hydrotrope to maintain protein homeostasis. Here by NMR spectroscopy, we reveal for the first time that at physiological concentrations ATP binds the TDP-43 NTD to enhance its oligomerization. Most strikingly, this binding is specifically coupled with oligomerization because three mutants with the capacity of oligomerization eliminated lose the ability to bind ATP. Our study thus provides a novel mechanism for ATP to prevent pathological aggregation by specific binding; and further implies that ATP might have many previously-unknown functions in cells by binding to proteins other than the classic ATP-dependent proteins/enzymes.

Structurally- and dynamically-driven allostery of the chymotrypsin-like proteases of SARS, Dengue and Zika viruses.

Coronavirus 3C-like and Flavivirus NS2B-NS3 proteases utilize the chymotrypsin fold to harbor their catalytic machineries but also contain additional domains/co-factors. Over the past decade, we aimed to decipher how the extra domains/co-factors mediate the catalytic machineries of SARS 3C-like, Dengue and Zika NS2B-NS3 proteases by characterizing their folding, structures, dynamics and inhibition with NMR, X-ray crystallography and MD simulations, and the results revealed: 1) the chymotrypsin fold of the SARS 3C-like protease can independently fold, while, by contrast, those of Dengue and Zika proteases lack the intrinsic capacity to fold without co-factors. 2) Mutations on the extra domain of SARS 3C-like protease can transform the active catalytic machinery into the inactive collapsed state by structurally-driven allostery. 3) Amazingly, even without detectable structural changes, mutations on the extra domain are sufficient to either inactivate or enhance the catalytic machinery of SARS 3C-like protease by dynamically-driven allostery. 4) Global networks of correlated motions have been identified: for SARS 3C-like protease, N214A inactivates the catalytic machinery by decoupling the network, while STI/A and STIF/A enhance by altering the patterns of the network. The global networks of Dengue and Zika proteases are coordinated by their NS2B-cofactors. 5) Natural products were identified to allosterically inhibit Zika and Dengue proteases through binding a pocket on the back of the active site. Therefore, by introducing extra domains/cofactors, nature develops diverse strategies to regulate the catalytic machinery embedded on the chymotrypsin fold through folding, structurally- and dynamically-driven allostery, all of which might be exploited to develop antiviral drugs.

TMEM106B, a risk factor for FTLD and aging, has an intrinsically disordered cytoplasmic domain.

TMEM106B was initially identified as a risk factor for FTLD, but recent studies highlighted its general role in neurodegenerative diseases. Very recently TMEM106B has also been characterized to regulate aging phenotypes. TMEM106B is a 274-residue lysosomal protein whose cytoplasmic domain functions in the endosomal/autophagy pathway by dynamically and transiently interacting with diverse categories of proteins but the underlying structural basis remains completely unknown. Here we conducted bioinformatics analysis and biophysical characterization by CD and NMR spectroscopy, and obtained results reveal that the TMEM106B cytoplasmic domain is intrinsically disordered with no well-defined three-dimensional structure. Nevertheless, detailed analysis of various multi-dimensional NMR spectra allowed defining residue-specific conformations and dynamics. Overall, the TMEM106B cytoplasmic domain is lacking of any tight tertiary packing and relatively flexible. However, several segments are populated with dynamic/nascent secondary structures and have relatively restricted backbone motions on ps-ns time scale, as indicated by their positive {1H}-15N steady-state NOE. Our study thus decodes that being intrinsically disordered may allow the TMEM106B cytoplasmic domain to dynamically and transiently interact with a variety of distinct partners.

ATP enhances at low concentrations but dissolves at high concentrations liquid-liquid phase separation (LLPS) of ALS/FTD-causing FUS.

ATP is the universal energy currency but mysteriously its cellular concentration is much higher than that needed for providing energy. Recently ATP was decoded to act as a hydrotrope to dissolve liquid-liquid phase separation (LLPS) of FUS whose aggregation leads to ALS/FTD. By DIC microscopy and NMR, here we characterized the effect of ATP on LLPS of FUS and its N-/C-terminal domains. Very unexpectedly, we found that like nucleic acids, ATP enhances LLPS of FUS at low but dissolves at high concentrations. Intriguingly, ATP monotonically dissolves LLPS of NTD, while it induces LLPS of CTD at low but dissolves at high concentrations. Our study reveals for the first time that ATP can enhance LLPS most likely by behaving as a bivalent binder. Most importantly, our results imply that age-dependent reduction of ATP concentrations may not only result in decreasing its capacity in preventing protein aggregation, but also in enhancing aggregation.

TDP-43 NTD can be induced while CTD is significantly enhanced by ssDNA to undergo liquid-liquid phase separation.

TDP-43 inclusions are characterized by a large spectrum of neurodegenerative diseases such as ALS and Alzheimer's. Functionally, TDP-43 is engaged in forming dynamic granules via liquid-liquid phase separation (LLPS), which is now recognized to be a general principle for organizing a variety of cellular membrane-less organelles. TDP-43 is composed of the N-terminal domain (NTD) adopting an ubiquitin-like fold, two RRMs and C-terminal domain (CTD) with the low-complexity (LC) prion-like sequences. Previously, only the CTD was found to undergo LLPS to form dynamic liquid droplets with relatively small numbers and sizes. Here we found for the first time that ssDNA can induce the NTD as well as significantly enhance the CTD to undergo LLPS. Further systematic investigations with 10 ssDNA of different sequences and lengths reveal that two distinct mechanisms exist respectively for the ssDNA-mediated LLPS of the NTD and CTD. As most, if not all functions of TDP-43, are involved in contacting nucleic acids including ssDNA, our results imply that nucleic acids might mediate the physiological functions and pathological roles of TDP-43 by previously-unappreciated mechanisms.

ALS-causing profilin-1-mutant forms a non-native helical structure in membrane environments.

Despite having physiological functions completely different from superoxide dismutase 1 (SOD1), profilin 1 (PFN1) also carries mutations causing amyotrophic lateral sclerosis (ALS) with a striking similarity to that triggered by SOD1 mutants. Very recently, the C71G-PFN1 has been demonstrated to cause ALS by a gain of toxicity and the acceleration of motor neuron degeneration preceded the accumulation of its aggregates. Here by atomic-resolution NMR determination of conformations and dynamics of WT-PFN1 and C71G-PFN1 in aqueous buffers and in membrane mimetics DMPC/DHPC bicelle and DPC micelle, we deciphered that: 1) the thermodynamic destabilization by C71G transforms PFN1 into coexistence with the unfolded state, which is lacking of any stable tertiary/secondary structures as well as restricted ps-ns backbone motions, thus fundamentally indistinguishable from ALS-causing SOD1 mutants. 2) Most strikingly, while WT-PFN1 only weakly interacts with DMPC/DHPC bicelle without altering the native structure, C71G-PFN1 acquires abnormal capacity in strongly interacting with DMPC/DHPC bicelle and DPC micelle, energetically driven by transforming the highly disordered unfolded state into a non-native helical structure, similar to what has been previously observed on ALS-causing SOD1 mutants. Our results imply that one potential mechanism for C71G-PFN1 to initiate ALS might be the abnormal interaction with membranes as recently established for SOD1 mutants.

Solution conformations of Zika NS2B-NS3pro and its inhibition by natural products from edible plants.

The recent Zika viral (ZIKV) epidemic has been associated with severe neurological pathologies such as neonatal microcephaly and Guillain-Barre syndrome but unfortunately no vaccine or medication is effectively available yet. Zika NS2B-NS3pro is essential for the proteolysis of the viral polyprotein and thereby viral replication. Thus NS2B-NS3pro represents an attractive target for anti-Zika drug discovery/design. Here, we have characterized the solution conformations and catalytic parameters of both linked and unlinked Zika NS2B-NS3pro complexes and found that the unlinked complex manifested well-dispersed NMR spectra. Subsequently with selective isotope-labeling using NMR spectroscopy, we demonstrated that C-terminal residues (R73-K100) of NS2B is highly disordered without any stable tertiary and secondary structures in the Zika NS2B-NS3pro complex in the free state. Upon binding to the well-characterized serine protease inhibitor, bovine pancreatic trypsin inhibitor (BPTI), only the extreme C-terminal residues (L86-K100) remain disordered. Additionally, we have identified five flavonoids and one natural phenol rich in edible plants including fruits and vegetables, which inhibit Zika NS2B-NS3pro in a non-competitive mode, with Ki ranging from 770 nM for Myricetin to 34.02 µM for Apigenin. Molecular docking showed that they all bind to a pocket on the back of the active site and their structure-activity relationship was elucidated. Our study provides valuable insights into the solution conformation of Zika NS2B-NS3pro and further deciphers its susceptibility towards allosteric inhibition by natural products. As these natural product inhibitors fundamentally differ from the currently-known active site inhibitors in terms of both inhibitory mode and chemical scaffold, our finding might open a new avenue for development of better allosteric inhibitors to fight ZIKV infection.

RRM domain of ALS/FTD-causing FUS characteristic of irreversible unfolding spontaneously self-assembles into amyloid fibrils.

526-residue FUS functions to self-assemble into reversible droplets/hydrogels, which could be further solidified into pathological fibrils. FUS is intrinsically prone to aggregation, composed of N-terminal low-sequence complexity (LC); RNA-recognition motif (RRM) and C-terminal LC domains. Intriguingly, previous in vivo studies revealed that its RRM is required for manifesting FUS cytotoxicity but the underlying mechanism remains unknown. Here, we characterized solution conformations of FUS and its five differentially dissected fragments, followed by detailed investigations on thermal unfolding, NMR dynamics and self-assembly of RRM. The results decipher: (1) the N- and C-terminal LC domains are intrinsically disordered, while RRM is folded. Intriguingly, well-dispersed HSQC peaks of RRM disappear in the full-length FUS, reminiscent of the previous observation on TDP-43. (2) FUS RRM is characteristic of irreversible unfolding. "Model-free" analysis of NMR relaxation data decodes that RRM has high ps-ns conformational dynamics even over some residues within secondary structure regions. (3) RRM spontaneously self-assembles into amyloid fibrils. Therefore, in addition to the well-established prion-like region, FUS RRM is also prone to self-assembly to form amyloid fibrils. Taken together, FUS RRM appears to play a crucial role in exaggerating the physiological/reversible self-assembly into pathological/irreversible fibrillization, thus contributing to manifestation of FUS cytotoxicity.