TAR-DNA-binding protein-43 (TDP-43) C-terminus encodes a prion-like domain widely presented in RNA-binding proteins, which functions to form dynamic oligomers and also, amazingly, hosts most amyotrophic lateral sclerosis (ALS)-causing mutations. Here, as facilitated by our previous discovery, circular dichroism (CD), fluorescence nuclear magnetic resonance (NMR) spectroscopy, we have successfully determined conformations, dynamics, self-associations of the full-length domains wild type three...

BgK is a K+ channel-blocking toxin from the sea anemone Bunodosoma granulifera It 37-residue protein that adopts novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed functional importance of five residues, which include critical lysine an aromatic residue separated 6.6 ± 1.0 Å. The same diad found in three known homologous toxins anemones. More strikingly, similar present all scorpions, although these adopt distinct scaffold. Moreover, diads potassium...

The severe acute respiratory syndrome (SARS) 3C-like protease consists of two distinct folds, namely the N-terminal chymotrypsin fold containing domains I and II hosting complete catalytic machinery C-terminal extra helical domain III unique for coronavirus 3CL proteases. Previously functional role this has been completely unknown, it was believed that proteases share same enzymatic mechanism with picornavirus 3C proteases, which contain but have no domain. To understand to characterize...

Unlike 3C protease, the severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CLpro) is only enzymatically active as a homodimer and its catalysis under extensive regulation by unique extra domain. Despite intense studies, two puzzles still remain: (i) how dimer-monomer switch controlled (ii) why dimerization absolutely required for catalysis. Here we report monomeric crystal structure of SARS-CoV 3CLpro mutant R298A at resolution 1.75 A. Detailed analysis reveals that...

The 3C-like protease of the severe acute respiratory syndrome (SARS) coronavirus has a C-terminal extra domain in addition to chymotrypsin-fold adopted by picornavirus 3C proteases hosting complete catalytic machinery. Previously we identified be involved enzyme dimerization which been considered essential for activity. In an initial attempt map out extra-domain residues critical dimerization, have systematically generated 15 point mutations, five deletions and one triple mutation...

Significance Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) inclusion is a histological hallmark of FTLD-TDP and amyotrophic lateral sclerosis. Its N terminus was just revealed as double-edged sword indispensable for both physiology proteinopathy, but its structure remains unknown due to aggregation. Here we ( i ) the TDP-43 encodes well-folded in equilibrium with unfolded form; ii despite previous failure detecting sequence homology ubiquitin, folded state assumes...

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 a unique manner: enhancement then dissolution. Currently, driving force is still under debate, while mechanism modulation remains completely undefined. Here, by NMR differential interference contrast (DIC) imaging, we characterized conformations,...

During tumor progression, EphA2 receptor can gain ligand-independent pro-oncogenic functions due to Akt activation and reduced ephrin-A ligand engagement. The effects be reversed by stimulation, which triggers the intrinsic suppressive signaling pathways of including inhibition PI3/Akt Ras/ERK pathways. These observations argue for development small molecule agonists as potential intervention agents. Through virtual screening cell-based assays, we report here identification characterization...

Previously we revealed that the extra domain of SARS 3CLpro mediated catalysis via different mechanisms. While R298A mutation completely abolished dimerization, thus resulting in inactive catalytic machinery, N214A inactivated enzyme by altering its dynamics without significantly perturbing structure. Here studied another mutant with S284-T285-I286 replaced Ala (STI/A) a 3.6-fold activity increase and slightly enhanced dimerization. We determined crystal structure, which still adopts dimeric...

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. NS2B-NS3pro essential for the proteolysis of polyprotein thereby replication. Thus represents an attractive target anti-Zika drug discovery/design. Here, we have characterized solution conformations catalytic parameters both linked unlinked complexes found that complex...

Abstract Adenosine triphosphate (ATP) provides energy for cellular processes but has recently been found to act also as a hydrotrope maintain protein homeostasis. ATP bivalently binds the disordered domain of FUS containing RG/RGG sequence motif and thereby affects liquid-liquid phase separation. Here, using NMR spectroscopy molecular docking studies, we report that specifically well-folded RRM at physiologically relevant concentrations with binding interface overlapping its physiological...

Dicer or Dicer-like (DCL) protein is a catalytic component involved in microRNA (miRNA) small interference RNA (siRNA) processing pathway, whose fragment structures have been partially solved. However, the structure and function of unique DUF283 domain within dicer largely unknown. Here we report first from Arabidopsis thaliana DCL4. The adopts an α-β-β-β-α topology resembles structural similarity to double-stranded RNA-binding domain. Notably, N-terminal α helix runs cross over C-terminal...

Abstract Mysteriously neurons maintain ATP concentrations of ~3 mM but whether modulates TDP-43 LLPS remains completely unexplored. Here we characterized the effect on PLD and seven mutants by DIC NMR. The results revealed: 1) induces subsequently dissolves specifically binding Arg saturated at 1:100. 2) modifies conformation-specific electrostatic property beyond just imposing screening effect. 3) Reversibility further exaggeration into aggregation appear to be controlled a delicate network...

SARS-CoV-2 nucleocapsid (N) protein with very low mutation rates is the only structural which not functions to package viral genomic RNA, but also manipulates host-cell machineries, thus representing a key target for drug development. Recent discovery of its liquid-liquid phase separation (LLPS) opens up new direction developing anti-SARS-CoV-2 strategies/drugs. However, so far high-resolution mechanism LLPS still remains unknown. Here by DIC and NMR characterization, we have demonstrated:...

The Eph receptor tyrosine kinases regulate a variety of physiological and pathological processes not only during development but also in adult organs, therefore they represent promising class drug targets. EphA4 plays important roles the inhibition regeneration injured axons, synaptic plasticity, platelet aggregation, likely certain types cancer. Here we report first crystal structure ligand-binding domain, which adopts same jellyroll beta-sandwich architecture as shown previously for EphB2...

Despite utilizing the same chymotrypsin fold to host catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C in acquiring an extra helical domain evolution. Previously, was demonstrated regulate catalysis of SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts dimeric structure almost identical that...

Significance FAT10, a ubiquitin-like modifier, is an oncogene that interacts with mitotic arrest-deficient 2 (MAD2) and confers cellular malignancy. Here we identified the MAD2-binding residues of FAT10 determined first solution structure, to our knowledge, domain. Importantly, demonstrated proof-of-mechanism for novel specific drug-targeting strategy entails inhibition pathological activity therapeutic target but not its reported physiological function, thus minimizing undesirable side...

526-residue FUS functions to self-assemble into reversible droplets/hydrogels, which could be further solidified pathological fibrils. is intrinsically prone 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 required for manifesting cytotoxicity but the underlying mechanism remains unknown. Here, we characterized solution conformations five differentially...

Primordial germ cell (PGC) specification occurs early in development. PGC specifiers have been identified Drosophila, mouse, and human but remained elusive most animals. Here we identify the RNA-binding protein Dnd as a critical specifier medaka fish (Oryzias latipes). depletion specifically abolished PGCs, its overexpression boosted PGCs. We established single-cell culture procedure enabling lineage tracing vitro. show that individual blastomeres from cleavage embryos at 32- 64-cell stages...