Jianxing Song

Structural Characterization of the EphA4-Ephrin-B2 Complex Reveals New Features Enabling Eph-Ephrin Binding Promiscuity

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 Gln12 and Glu14 and ephrin-B2. Mutation of Gln12 and Glu14 does not cause significant structural changes in EphA4 or changes in its affinity for ephrin-A ligands. However, the EphA4 mutant has ∼10-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 Gln12 and Glu14 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.

A Small Molecule Agonist of EphA2 Receptor Tyrosine Kinase Inhibits Tumor Cell Migration In Vitro and Prostate Cancer Metastasis In Vivo

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.

ALS-Causing Mutations Significantly Perturb the Self-Assembly and Interaction with Nucleic Acid of the Intrinsically Disordered Prion-Like Domain of TDP-43.

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.

Structural characterization reveals that viperin is a radical S-adenosyl-l-methionine (SAM) enzyme

Viperin is an interferon-inducible protein inhibiting many DNA and RNA viruses. It contains an N-terminal transmembrane helix, a highly conserved C-terminus and a middle region carrying a CX3CX2C motif, characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. So far no structural characterization has been reported and reconstitution of the [4Fe-4S] cluster in viperin all failed. Here, by dissecting the 361-residue human viperin into 12 fragments, followed by extensive CD and NMR characterization, Viperin (45-361) was identified to be soluble and structured in buffers. Most importantly, we have successfully reconstituted the [4Fe-4S] cluster in Viperin (45-361), thus providing the first experimental evidence confirming that viperin is indeed a radical SAM enzyme. Furthermore, the C-terminus Viperin (214-361) which is insoluble in buffers but again can be solubilized in salt-free water appears to be only partially folded. Our results thus imply that the radical SAM enzyme activity may play a key role in the broad antiviral actions of viperin.

Crystal Structure and NMR Binding Reveal That Two Small Molecule Antagonists Target the High Affinity Ephrin-binding Channel of the EphA4 Receptor

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 β-sandwich architecture as shown previously for EphB2 and EphB4. The similarity with EphB receptors is high in the core β-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. We show that the two molecules bind to the EphA4 ligand-binding domain with Kd values of 20.4 and 26.4 μm, respectively. NMR heteronuclear single quantum coherence titrations revealed that upon binding, both molecules significantly perturb EphA4 residues Ile31-Met32 in the D-E loop, Gln43 in the E β-strand, and Ile131-Gly132 in the J-K loop. Molecular docking shows that they can occupy a cavity in the high affinity ephrin binding channel of EphA4 in a similar manner, by interacting mainly with the EphA4 residues in the E strand and D-E and J-K loops. However, many of the interactions observed in Eph receptor-ephrin complexes are absent, which is consistent with the small size of the two molecules and may account for their relatively weak binding affinity. Thus, our studies provide the first published structure of the ligand-binding domain of an EphA receptor of the A subclass. Furthermore, the results demonstrate that the high affinity ephrin binding channel of the Eph receptors is amenable to targeting with small molecule antagonists and suggest avenues for further optimization.

TDP-43 N terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssDNA

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 a double-edged sword indispensable for both physiology and proteinopathy, but its structure remains unknown due to aggregation. Here we revealed (i) the TDP-43 N terminus encodes a well-folded structure in equilibrium with its unfolded form; (ii) despite previous failure in detecting sequence homology to ubiquitin, the folded state assumes a novel ubiquitin-like fold; and (iii) this ubiquitin-like fold could bind ssDNA, thus representing the first capable of directly binding nucleic acid. Taken together, our results provide a molecular mechanism rationalizing the functional dichotomy of TDP-43 and further imply one therapeutic strategy for TDP-43–causing diseases. 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 long-range 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.

Identification, recombinant production and structural characterization of four silk proteins from the Asiatic honeybee Apis cerana.

Unlike silkworm and spider silks assembled from very large and repetitive fibrous proteins, the bee and ant silks were recently demonstrated to consist of four small and non-repetitive coiled-coil proteins. The design principle for this silk family remains largely unknown and so far no structural study is available on them in solution. The present study aimed to identify, express and characterize the Asiatic honeybee silk proteins using DLS, CD and NMR spectroscopy. Consequently, (1) four silk proteins are identified, with approximately 6, 10, 9 and 8% variations, respectively, from their European honeybee homologs. Strikingly, their recombinant forms can be produced in Escherichia coil with yields of 10-60 mg/l. (2) Despite containing approximately 65% coiled-coil sequences, four proteins have very low alpha-helix (9-27%) but unusually high random coil (45-56%) contents. Surprisingly, beta-sheet is also detected in four silk proteins (26-35%), implying the possible presence of beta-sheet in the bee and ant silks. (3) Four proteins lacking of the tight tertiary packing appear capable of interacting with each other weakly but this interaction triggers no significant formation of the tight tertiary packing. The study not only implies the promising potential to produce recombinant honeybee silk proteins for the development of various biomaterials; but also provides the first structural insight into the molecular mechanism underlying the formation of the coiled-coil silks.

The N- and C-termini of the human Nogo molecules are intrinsically unstructured: Bioinformatics, CD, NMR characterization, and functional implications

RTN4 or Nogo proteins are composed of three alternative splice forms, namely 1192‐residue Nogo‐A, 373‐residue Nogo‐B, and 199‐residue Nogo‐C. Nogo proteins have received intense attentions because they have been implicated in a variety of critical cellular processes including CNS neuronal regeneration, vascular remodeling, apoptosis, interaction with β‐amyloid protein converting enzyme, and generation/maintenance of the tubular network of the endoplasmic reticulum (ER). Despite their significantly‐different N‐terminal lengths, they share a conserved C‐terminal reticulon‐homology domain consisting of two transmembrane fragments, a 66‐residue extracellular loop Nogo‐66 and a 38‐residue C‐tail carrying ER retention motif. Nogo‐A owns the largest N‐terminus with 1016 residues while the Nogo‐B has an N‐terminus almost identical to the first 200 residues of Nogo‐A. So far, except for our previous determination of the Nogo‐66 solution structure, no structural characterization of the other Nogo regions has been reported. In the present study, we initiated a systematically investigation of structural properties of Nogo molecules by a combined use of bioinformatics, CD, and NMR spectroscopy. The results led to two striking findings: (1) in agreement with bioinformatics prediction, the N‐ and C‐termini of Nogo‐B were experimentally demonstrated to be intrinsically unstructured by CD, two‐dimensional 1H 15N NMR HSQC, hydrogen exchange, and 15N heteronuclear NOE characterization. (2) Further studies showed that the 1016‐residue N‐terminus of Nogo‐A was again highly disordered. Therefore, it appears that being intrinsically‐unstructured allows Nogo molecules to serve as double‐faceted functional players, with one set of functions involved in cellular signaling processes essential for CNS neuronal regeneration, vascular remodeling, apoptosis and so forth and with another in generating/maintaining membrane‐related structures. We propose that this mechanism may represent a general strategy to place the formation/maintenance of membrane‐related structures under the direct regulation of the cellular signaling. Proteins 2007.

A disalicylic acid-furanyl derivative inhibits ephrin binding to a subset of Eph receptors.

Eph receptor tyrosine kinases and ephrin ligands control many physiological and pathological processes, and molecules interfering with their interaction are useful probes to elucidate their complex biological functions. Moreover, targeting Eph receptors might enable new strategies to inhibit cancer progression and pathological angiogenesis as well as promote nerve regeneration. Because our previous work suggested the importance of the salicylic acid group in antagonistic small molecules targeting Eph receptors, we screened a series of salicylic acid derivatives to identify novel Eph receptor antagonists. This identified a disalicylic acid‐furanyl derivative that inhibits ephrin‐A5 binding to EphA4 with an IC50 of 3 μm in ELISAs. This compound, which appears to bind to the ephrin‐binding pocket of EphA4, also targets several other Eph receptors. Furthermore, it inhibits EphA2 and EphA4 tyrosine phosphorylation in cells stimulated with ephrin while not affecting phosphorylation of EphB2, which is not a target receptor. In endothelial cells, the disalicylic acid‐furanyl derivative inhibits EphA2 phosphorylation in response to TNFα and capillary‐like tube formation on Matrigel, two effects that depend on EphA2 interaction with endogenous ephrin‐A1. These findings suggest that salicylic acid derivatives could be used as starting points to design new small molecule antagonists of Eph receptors.

The catalysis of the SARS 3C‐like protease is under extensive regulation by its extra domain

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.