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...

The Eph receptor tyrosine kinases regulate a variety of physiological and pathological processes not only during development but also in adult organs, and therefore they represent a promising class of drug targets. The EphA4 receptor plays important roles in the inhibition of the regeneration of injured axons, synaptic plasticity, platelet aggregation, and likely in certain types of cancer. Here we report the first crystal structure of the EphA4 ligand-binding domain, which adopts the same jellyroll ␤-sandwich architecture as shown previously for EphB2 and EphB4. The similarity with EphB receptors is high in the core ␤-stranded regions, whereas large variations exist in the loops, particularly the D-E and J-K loops, which form the high affinity ephrin binding channel. We also used isothermal titration calorimetry, NMR spectroscopy, and computational docking to characterize the binding to EphA4 of two small molecules, 4-and 5-(2,5 dimethyl-pyrrol-1-yl)-2-hydroxybenzoic acid which antagonize ephrin-induced effects in EphA4-expressing cells. The erythropoietin-producing hepatocellular (Eph) 3 carcinoma receptors constitute the largest family of receptor tyrosine kinases, with 16 individual receptors throughout the animal kingdom, which are activated by nine ephrins (1-6). Eph receptors and their ligands are both anchored onto the plasma membrane and are subdivided into two subclasses (A and B) based on their sequence conservation and binding preferences. Usually, EphA receptors (EphA1-A10) interact with glycosylphosphatidylinositol-anchored ephrin-A ligands (ephrin-A1-A6), whereas EphB receptors (EphB1-B6) interact with transmembrane ephrin-B ligands (ephrin-B1-B3) that have a short cytoplasmic portion carrying both Src homology domain 2 and PDZ domain-binding motifs (7,8).The Eph receptors have a modular structure, consisting of a unique N-terminal ephrin-binding domain followed by a cysteine-rich linker and two fibronectin type III repeats in the extracellular region. The intracellular region is composed of a conserved tyrosine kinase domain, a C-terminal sterile ␣-domain, and a PDZ-binding motif. The N-terminal 180-residue globular domain of the Eph receptors has been shown to be sufficient for high affinity ephrin binding (9 -11). EphA subclass receptors remarkably differ from EphB receptors because they lack a 4-residue insert in the H-I loop of the ligand-binding domain. Previously, the structures of the EphB2 and EphB4 ligand-binding domains have been determined in both the free state and in complex with ephrins or peptide antagonists (10,11,(12)(13)(14)(15). These studies have shown that the ligand-binding domains of EphB2 and EphB4 adopt the same jellyroll ␤-sandwich architecture composed of 11 antiparallel ␤-strands connected by loops of various lengths. In particular, the D-E and * This work was supported by National Medical Research Council of Singapore Grant R-154-000-382-213 (to J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must the...

During tumor progression, EphA2 receptor can gain ligand-independent pro-oncogenic functions due to Akt activation and reduced ephrin-A ligand engagement. The effects can be reversed by ligand stimulation, which triggers the intrinsic tumor suppressive signaling pathways of EphA2 including inhibition of PI3/Akt and Ras/ERK pathways. These observations argue for development of small molecule agonists for EphA2 as potential tumor intervention agents. Through virtual screening and cell-based assays, we report here the identification and characterization of doxazosin as a novel small molecule agonist for EphA2 and EphA4, but not for other Eph receptors tested. NMR studies revealed extensive contacts of doxazosin with EphA2/A4, recapitulating both hydrophobic and electrostatic interactions recently found in the EphA2/ephrin-A1 complex. Clinically used as an α1-adrenoreceptor antagonist (Cardura®) for treating hypertension and benign prostate hyperplasia, doxazosin activated EphA2 independent of α1-adrenoreceptor. Similar to ephrin-A1, doxazosin inhibited Akt and ERK kinase activities in an EphA2-dependent manner. Treatment with doxazosin triggered EphA2 receptor internalization, and suppressed haptotactic and chemotactic migration of prostate cancer, breast cancer, and glioma cells. Moreover, in an orthotopic xenograft model, doxazosin reduced distal metastasis of human prostate cancer cells and prolonged survival in recipient mice. To our knowledge, doxazosin is the first small molecule agonist of a receptor tyrosine kinase that is capable of inhibiting malignant behaviors in vitro and in vivo.

EphA and EphB receptors preferentially bind ephrin-A and ephrin-B ligands, respectively, but EphA4 is exceptional for its ability to bind all ephrins. Here, we report the crystal structure of the EphA4 ligand-binding domain in complex with ephrin-B2, which represents the first structure of an EphA-ephrin-B interclass complex. A loose fit of the ephrin-B2 G-H loop in the EphA4 ligand-binding channel is consistent with a relatively weak binding affinity. Additional surface contacts also exist between EphA4 residues Gln 12 and Glu 14 and ephrin-B2. Mutation of Gln 12 and Glu 14 does not cause significant structural changes in EphA4 or changes in its affinity for ephrin-A ligands. However, the EphA4 mutant has ϳ10-fold reduced affinity for ephrin-B ligands, indicating that the surface contacts are critical for interclass but not intraclass ephrin binding. Thus, EphA4 uses different strategies to bind ephrin-A or ephrin-B ligands and achieve binding promiscuity. NMR characterization also suggests that the contacts of Gln 12 and Glu 14 with ephrin-B2 induce dynamic changes throughout the whole EphA4 ligand-binding domain. Our findings shed light on the distinctive features that enable the remarkable ligand binding promiscuity of EphA4 and suggest that diverse strategies are needed to effectively disrupt different Eph-ephrin complexes.The Eph receptors represent the largest family of tyrosine kinases, with 16 members divided into two classes, EphA and EphB. This subdivision is based on sequence conservation and binding preferences for their ligands, the ephrins, which are also divided into A and B classes. There are 10 EphA and 6 EphB receptors in mammals and chick, which can bind to six glycosylphosphatidylinositol-anchored ephrin-A ligands or three transmembrane ephrin-B ligands to mediate an extremely wide spectrum of biological responses through signals that are generated by both receptor and ligand activation (1, 2).All of the Eph receptors share the same modular structure, which comprises a juxtamembrane region, a tyrosine kinase domain, a C-terminal sterile ␣-motif domain, and a PDZbinding motif in the intracellular region. In the extracellular portion, there are an N-terminal ligand-binding domain, a cysteine-rich region, and two fibronectin type III repeats. The ephrin-binding domain is responsible for ligand recognition and is composed of 11 antiparallel ␤-strands organized in a jellyroll ␤-sandwich architecture, which is conserved among EphA and EphB receptors (3-8). The ectodomain of the ephrins is also conserved and consists of an eight-stranded ␤-barrel with a Greek key topology, including several large and highly conserved functional loops, such as the G-H and C-D loops (4,5,8,9), which are very flexible in solution (10).The formation of a complex between an Eph receptor and an ephrin is centered around the insertion of the solventexposed ephrin G-H loop into the Eph receptor hydrophobic channel formed by the convex sheet of four ␤-strands together with the D-E, J-K, and G-H loops. These interactions are...

Dicer or Dicer-like (DCL) protein is a catalytic component involved in microRNA (miRNA) or small interference RNA (siRNA) processing pathway, whose fragment structures have been partially solved. However, the structure and function of the unique DUF283 domain within dicer is largely unknown. Here we report the first structure of the DUF283 domain from the Arabidopsis thaliana DCL4. The DUF283 domain adopts an a-b-b-b-a topology and resembles the structural similarity to the doublestranded RNA-binding domain. Notably, the N-terminal a helix of DUF283 runs cross over the C-terminal a helix orthogonally, therefore, N-and C-termini of DUF283 are in close proximity. Biochemical analysis shows that the DUF283 domain of DCL4 displays weak dsRNA binding affinity and specifically binds to double-stranded RNA-binding domain 1 (dsRBD1) of Arabidopsis DRB4, whereas the DUF283 domain of DCL1 specifically binds to dsRBD2 of Arabidopsis HYL1. These data suggest a potential functional role of the Arabidopsis DUF283 domain in target selection in small RNA processing.

Many proteins expressed in Escherichia coli cells form inclusion bodies that are neither refoldable nor soluble in buffers. Very surprisingly, we recently discovered that all 11 buffer-insoluble protein fragments/domains we have, with a great diversity of cellular function, location, and molecular size, could be easily solubilized in salt-free water. The circular dichroism (CD) and NMR characterization led to classification of these proteins into three groups: group 1, with no secondary structure by CD and with narrowly-dispersed but sharp (1)H-(15)N heteronuclear single quantum correlation (HSQC) peaks; group 2, with secondary structure by CD but with HSQC peaks broadened and, consequently, only a small set of peaks detectable; and group 3, with secondary structure by CD and also well-separated HSQC peaks. Intriguingly, we failed to find any protein with a tight tertiary packing. Therefore, we propose that buffer-insoluble proteins may lack intrinsic ability to reach or/and to maintain a well-packed conformation, and thus are trapped in partially-folded states with many hydrophobic side chains exposed to the bulk solvent. As such, a very low ionic strength is sufficient to screen out intrinsic repulsive interactions and, consequently, allow the hydrophobic clustering/aggregation to occur. Marvelously enough, it appears that in pure water, proteins have the potential to manifest their full spectrum of structural states by utilizing intrinsic repulsive interactions to suppress the attractive hydrophobic clustering. Our discovery not only gives a novel insight into the properties of insoluble proteins, but also sheds the first light that we know of on previously unknown regimes associated with proteins.

Additional to involvement in diverse physiological and pathological processes such as axon regeneration, synaptic plasticity, and cancers, EphA4 receptor has been recently identified as the only amyotrophic lateral sclerosis (ALS) modifier. Previously, we found that two small molecules bind the same EphA4 channel at almost equivalent affinities but mysteriously trigger opposite signaling outputs: one activated but another inhibited. Here, we determined the solution structure of the 181-residue EphA4 LBD, which represents the first for 16 Eph receptors. Further NMR dynamic studies deciphered that the agonistic and antagonistic effects of two small molecules are dynamically driven, which are achieved by oppositely modulating EphA4 dynamics. Consequently, in design of drugs to target EphA4, the dynamic requirement also needs to be satisfied in addition to the classic criteria. For example, to increase the survival of ALS patients by inhibiting EphA4, the drugs must enhance, or at least not suppress, the EphA4 dynamics.

T46I is the second mutation on the hVAPB MSP domain which was recently identified from non-Brazilian kindred to cause a familial amyotrophic lateral sclerosis (ALS). Here using CD, NMR and molecular dynamics (MD) simulations, we characterized the structure, stability, dynamics and binding capacity of the T46I-MSP domain. The results reveal: 1) unlike P56S which we previously showed to completely eliminate the native MSP structure, T46I leads to no significant disruption of the native secondary and tertiary structures, as evidenced from its far-UV CD spectrum, as well as Cα and Cβ NMR chemical shifts. 2) Nevertheless, T46I does result in a reduced thermodynamic stability and loss of the cooperative urea-unfolding transition. As such, the T46I-MSP domain is more prone to aggregation than WT at high protein concentrations and temperatures in vitro, which may become more severe in the crowded cellular environments. 3) T46I only causes a 3-fold affinity reduction to the Nir2 peptide, but a significant elimination of its binding to EphA4. 4) EphA4 and Nir2 peptide appear to have overlapped binding interfaces on the MSP domain, which strongly implies that two signaling networks may have a functional interplay in vivo. 5) As explored by both H/D exchange and MD simulations, the MSP domain is very dynamic, with most loop residues and many residues on secondary structures highly fluctuated or/and exposed to bulk solvent. Although T46I does not alter overall dynamics, it does trigger increased dynamics of several local regions of the MSP domain which are implicated in binding to EphA4 and Nir2 peptide. Our study provides the structural and dynamic understanding of the T46I-causing ALS; and strongly highlights the possibility that the interplay of two signaling networks mediated by the FFAT-containing proteins and Eph receptors may play a key role in ALS pathogenesis.