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, by circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopy, we have successfully determined conformations, dynamics, and self-associations of the full-length prion-like domains of the wild type and three ALS-causing mutants (A315E, Q331K, and M337V) in both aqueous solutions and membrane environments. The study decodes the following: (1) The TDP-43 prion-like domain is intrinsically disordered only with some nascent secondary structures in aqueous solutions, but owns the capacity to assemble into dynamic oligomers rich in β-sheet structures. By contrast, despite having highly similar conformations, three mutants gained the ability to form amyloid oligomers. The wild type and three mutants all formed amyloid fibrils after incubation as imaged by electron microscopy. (2) The interaction with nucleic acid enhances the self-assembly for the wild type but triggers quick aggregation for three mutants. (3) A membrane-interacting subdomain has been identified over residues Met311-Gln343 indispensable for TDP-43 neurotoxicity, which transforms into a well-folded Ω-loop-helix structure in membrane environments. Furthermore, despite having very similar membrane-embedded conformations, three mutants will undergo further self-association in the membrane environment. Our study implies that the TDP-43 prion-like domain appears to have an energy landscape, which allows the assembly of the wild-type sequence into dynamic oligomers only under very limited condition sets, and ALS-causing point mutations are sufficient to remodel it to more favor the amyloid formation or irreversible aggregation, thus supporting the emerging view that the pathologic aggregation may occur via the exaggeration of functionally important assemblies. Furthermore, the coupled capacity of TDP-43 in aggregation and membrane interaction may critically account for its high neurotoxicity, and therefore its decoupling may represent a promising therapeutic strategy to treat TDP-43 causing neurodegenerative diseases.

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 is under extensive regulation by the unique extra domain. Despite intense studies, two puzzles still remain: (i) how the dimer-monomer switch is controlled and (ii) why dimerization is absolutely required for catalysis. Here we report the monomeric crystal structure of the SARS-CoV 3CLpro mutant R298A at a resolution of 1.75 Å. Detailed analysis reveals that Arg298 serves as a key component for maintaining dimerization, and consequently, its mutation will trigger a cooperative switch from a dimer to a monomer. The monomeric enzyme is irreversibly inactivated because its catalytic machinery is frozen in the collapsed state, characteristic of the formation of a short 3 10 -helix from an active-site loop. Remarkably, dimerization appears to be coupled to catalysis in 3CLpro through the use of overlapped residues for two networks, one for dimerization and another for the catalysis.

The 3C‐like protease of the severe acute respiratory syndrome (SARS) coronavirus has a C‐terminal extra domain in addition to the chymotrypsin‐fold adopted by piconavirus 3C proteases hosting the complete catalytic machinery. Previously we identified the extra domain to be involved in enzyme dimerization which has been considered essential for the catalytic activity. In an initial attempt to map out the extra‐domain residues critical for dimerization, we have systematically generated 15 point mutations, five deletions and one triple mutation and subsequently characterized them by enzymatic assay, dynamic light scattering, CD and NMR spectroscopy. The results led to identification of four regions critical for enzyme dimerization. Interestingly, Asn214Ala mutant with a significant tendency to form a monomer still retained ≈ 30% activity, indicating that the relationship between the activity and dimerization might be very complex. Very surprisingly, two regions (one over Ser284–Thr285–Ile286 and another around Phe291) were discovered on which Ala‐mutations significantly increased the enzymatic activities. Based on this, a super‐active triple‐mutant STI/A with a 3.7‐fold activity enhancement was thus engineered by mutating residues Ser284, Thr285 and Ile286 to Ala. The dynamic light scattering, CD and NMR characterizations indicate that the wild‐type (WT) and STI/A mutant share similar structural and dimerization properties, thus implying that in addition to dimerization, the extra domain might have other mechanisms to regulate the catalytic machinery. We rationalized these results based on the enzyme structure and consequently observed an interesting picture: the majority of the dimerization‐critical residues plus Ser284–Thr285–Ile286 and Phe291 are clustered together to form a nano‐scale channel passing through the central region of the enzyme. We therefore speculate that this channel might play a role in relaying regulatory effects from the extra domain to the catalytic machinery.

Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) is the principal component of ubiquitinated inclusions characteristic of most forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia-frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), as well as an increasing spectrum of other neurodegenerative diseases. Previous structural and functional studies on TDP-43 have been mostly focused on its recognized domains. Very recently, however, its extreme N terminus was identified to be a double-edged sword indispensable for both physiology and proteinopathy, but thus far its structure remains unknown due to the severe aggregation. Here as facilitated by our previous discovery that protein aggregation can be significantly minimized by reducing salt concentrations, by circular dichroism and NMR spectroscopy we revealed that the TDP-43 N terminus encodes a well-folded structure in concentration-dependent equilibrium with its unfolded form. Despite previous failure in detecting any sequence homology to ubiquitin, the folded state was determined to adopt a novel ubiquitin-like fold by the CS-Rosetta program with NMR chemical shifts and 78 unambiguous longrange nuclear Overhauser effect (NOE) constraints. Remarkably, this ubiquitin-like fold could bind ssDNA, and the binding shifted the conformational equilibrium toward reducing the unfolded population. To the best of our knowledge, the TDP-43 N terminus represents the first ubiquitin-like fold capable of directly binding nucleic acid. Our results provide a molecular mechanism rationalizing the functional dichotomy of TDP-43 and might also shed light on the formation and dynamics of cellular ribonucleoprotein granules, which have been recently linked to ALS pathogenesis. As a consequence, one therapeutic strategy for TDP-43-causing diseases might be to stabilize its ubiquitin-like fold by ssDNA or designed molecules. 1, 2). Since then, numerous studies have confirmed that TDP43 protein is mechanistically linked to neurodegeneration (3, 4). TDP43 is a 414-residue protein that has been previously recognized to be composed of a nuclear localization signal (NLS), two RNA recognition motifs (RRM1 and RRM2) hosting a nuclear export signal (NES), and C-terminal glycine-rich prion-like domain (Fig. 1A). The NLS and NES regulate the shuttling of TDP-43 between the nucleus and the cytoplasm (5), whereas the RRM1 and RRM2 are responsible for binding to nucleic acids including single-or double-stranded DNA/RNA (5-8). The prion-like domain mediates protein-protein interactions between TDP-43 and other hnRNP members (9), which also hosts most known ALS-associated TDP-43 mutations.TDP43 is an aggregation-prone protein (1-4, 10-14), and its abnormal aggregation has been found in ∼97% ALS and ∼45% frontotemporal dementia (FTD) patients. Additionally, TDP-43 immunoreactive inclusions have also been observed in an increasing spectrum of other neurodegenerative disorders, which include ALS/parkinsonism-dementia complex of Guam, Alzhei...

BgK is a K؉ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 ؎ 1.0 Å. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K ؉ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the threedimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.Functional properties of proteins are frequently associated with a small number of important residues. For example, enzyme activities depend on a few residues that are essential for catalysis. Also, protein-protein recognition processes have been predicted (1) and recently demonstrated (2) to be energetically driven by a small proportion of the residues forming the contacting areas in protein-protein complexes, as identified by x-ray studies (3, 4). Among the proteins whose major functions require protein-protein interactions are animal toxins, which bind to various molecular targets, such as receptors or ion channels, using a small number of binding residues (5-8). As has been shown for enzymes (9), toxins with different architectures are capable of exerting similar functions (10). However, in contrast to enzymes, the molecular basis associated with the conservation of the function in structurally unrelated toxins remains unknown. In this paper, we show that two families of animal toxins with different folding patterns but a comparable capacity to bind to potassium channels include similar functional diads, composed of a critical lysine and an aromatic amino acid separated from each other by 6.6 Ϯ 1.0 Å. MATERIALS AND METHODS Synthesis of Toxin and Mutants-The amino acid sequence of BgK 1 was proposed a few years ago (11). However, chemical synthesis attempts, based on these data, systematically failed. The proposed amino acid sequence was therefore questioned, re-examined, and ultimately corrected.2 The revised amino acid sequence of BgK from Bunodosoma granulifera is: VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTC-ELC. BgK and each alanine-substituted analog were synthesized by solid phase synthesis using an Applied Biosystems model 431A peptide synthesizer, starting from 0.1 mmol of Rink-resin (4-(2Ј,4Ј-dimethoxyphenylhydroxymethylphenoxy resin; 0.48 mmol/g). A 10-fold excess (1 mmol) of Fmoc (N-(9-fluorenyl)methoxycarbonyl)-protected amino acid was used and coupled in N-methylpyrrolidone in the presence of N,NЈ-dicyclohexylcarbodiimide/1-hydroxybenzotriazole. The following side chain protections wer...