ALS mutations in the TIA-1 prion-like domain trigger highly condensed pathogenic structures.

T cell intracellular antigen-1 (TIA-1) plays a central role in stress granule (SG) formation by self-assembly via the prion-like domain (PLD). In the TIA-1 PLD, amino acid mutations associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Welander distal myopathy (WDM), have been identified. However, how these mutations affect PLD self-assembly properties has remained elusive. In this study, we uncovered the implicit pathogenic structures caused by the mutations. NMR analysis indicated that the dynamic structures of the PLD are synergistically determined by the physicochemical properties of amino acids in units of five residues. Molecular dynamics simulations and three-dimensional electron crystallography, together with biochemical assays, revealed that the WDM mutation E384K attenuated the sticky properties, whereas the ALS mutations P362L and A381T enhanced the self-assembly by inducing ß-sheet interactions and highly condensed assembly, respectively. These results suggest that the P362L and A381T mutations increase the likelihood of irreversible amyloid fibrillization after phase-separated droplet formation, and this process may lead to pathogenicity.

Volatile Organic Compound Detection by Graphene Field-Effect Transistors Functionalized with Fly Olfactory Receptor Mimetic Peptides.

An olfactory receptor mimetic peptide-modified graphene field-effect transistor (gFET) is a promising solution to overcome the principal challenge of low specificity graphene-based sensors for volatile organic compound (VOC) sensing. Herein, peptides mimicking a fruit fly olfactory receptor, OR19a, were designed by a high-throughput analysis method that combines a peptide array and gas chromatography for the sensitive and selective gFET detection of the signature citrus VOC, limonene. The peptide probe was bifunctionalized via linkage of a graphene-binding peptide to facilitate one-step self-assembly on the sensor surface. The limonene-specific peptide probe successfully achieved highly sensitive and selective detection of limonene by gFET, with a detection range of 8-1000 pM, while achieving facile sensor functionalization. Taken together, our target-specific peptide selection and functionalization strategy of a gFET sensor demonstrates advancement of a precise VOC detection system.

Importin α2 association with chromatin: Direct DNA binding via a novel DNA-binding domain.

The nuclear transport of proteins is important for facilitating appropriate nuclear functions. The importin α family proteins play key roles in nuclear transport as transport receptors for copious nuclear proteins. Additionally, these proteins possess other functions, including chromatin association and gene regulation. However, these nontransport functions of importin α are not yet fully understood, especially their molecular-level mechanisms and consequences for functioning with chromatin. Here, we report the novel molecular characteristics of importin α binding to diverse DNA sequences in chromatin. We newly identified and characterized a DNA-binding domain-the Nucleic Acid Associating Trolley pole domain (NAAT domain)-in the N-terminal region of importin α within the conventional importin ß binding (IBB) domain that is necessary for nuclear transport of cargo proteins. Furthermore, we found that the DNA binding of importin α synergistically coupled the recruitment of its cargo protein to DNA. This is the first study to delineate the interaction between importin α and chromatin DNA via the NAAT domain, indicating the bifunctionality of the importin α N-terminal region for nuclear transport and chromatin association.

Genetic loss of importin α4 causes abnormal sperm morphology and impacts on male fertility in mouse.

Importin α proteins play a central role in the transport of cargo from the cytoplasm to the nucleus. In this study, we observed that male knock-out mice for importin α4, which is encoded by the Kpna4 gene (Kpna4-/- ), were subfertile and yielded smaller litter sizes than those of wild-type (WT) males. In contrast, mice lacking the closely related importin α3 (Kpna3-/- ) were fertile. In vitro fertilization and sperm motility assays demonstrated that sperm from Kpna4-/- mice had significantly reduced quality and motility. In addition, acrosome reaction was also impaired in Kpna4-/- mice. Transmission electron microscopy revealed striking defects, including abnormal head morphology and multiple axoneme structures in the flagella of Kpna4-/- mice. A five-fold increase in the frequency of abnormalities in Kpna4-/- mice compared to WT mice indicates the functional importance of importin α4 in normal sperm development. Moreover, Nesprin-2, which is a component of the linker of nucleus and cytoskeleton complex, was expressed at lower levels in sperm from Kpna4-/- mice and was localized with abnormal axonemes, suggesting incorrect formation of the nuclear membrane-cytoskeleton structure during spermiogenesis. Proteomics analysis of Kpna4-/- testis showed significantly altered expression of proteins related to sperm formation, which provided evidence that genetic loss of importin α4 perturbed chromatin status. Collectively, these findings indicate that importin α4 is critical for establishing normal sperm morphology in mice, providing new insights into male germ cell development by highlighting the requirement of importin α4 for normal fertility.

Conformational Space Sampled by Domain Reorientation of Linear Diubiquitin Reflected in Its Binding Mode for Target Proteins.

Linear polyubiquitin chains regulate diverse signaling proteins, in which the chains adopt various conformations to recognize different target proteins. Thus, the structural plasticity of the chains plays an important role in controlling the binding events. Herein, paramagnetic NMR spectroscopy is employed to explore the conformational space sampled by linear diubiquitin, a minimal unit of linear polyubiquitin, in its free state. Rigorous analysis of the data suggests that, regarding the relative positions of the ubiquitin units, particular regions of conformational space are preferentially sampled by the molecule. By combining these results with further data collected for charge-reversal derivatives of linear diubiquitin, structural insights into the factors underlying the binding events of linear diubiquitin are obtained.

Tensin2 is important for podocyte-glomerular basement membrane interaction and integrity of the glomerular filtration barrier.

Tensin2 (Tns2), an integrin-linked protein, is enriched in podocytes within the glomerulus. Previous studies have revealed that Tns2-deficient mice exhibit defects of the glomerular basement membrane (GBM) soon after birth in a strain-dependent manner. However, the mechanisms for the onset of defects caused by Tns2 deficiency remains unidentified. Here, we aimed to determine the role of Tns2 using newborn Tns2-deficient mice and murine primary podocytes. Ultrastructural analysis revealed that developing glomeruli during postnatal nephrogenesis exhibited abnormal GBM processing due to ectopic laminin-α2 accumulation followed by GBM thickening. In addition, analysis of primary podocytes revealed that Tns2 deficiency led to impaired podocyte-GBM interaction and massive expression of laminin-α2 in podocytes. Our study suggests that weakened podocyte-GBM interaction due to Tns2 deficiency causes increased mechanical stress on podocytes by continuous daily filtration after birth, resulting in stressed podocytes ectopically producing laminin-α2, which interrupts GBM processing. We conclude that Tns2 plays important roles in the podocyte-GBM interaction and maintenance of the glomerular filtration barrier.

Design and synthesis of ß-strand-fixed peptides inhibiting aggregation of amyloid ß-protein.

Aggregation of 42-residue amyloid ß-protein (Aß42) can be prevented by ß-sheet breaker peptides (BSBps) homologous to LVFFA residues, which are included in a ß-sheet region of Aß42 aggregates. To enhance the affinity of BSBps to the Aß42 aggregates, we designed and synthesized ß-strand-fixed peptides (BSFps) whose side chains were cross-linked by ring closing metathesis. Conformation analysis verified that the designed peptides could be fixed in ß-strand conformation. Among the synthesized pentapeptides, 1 and 12, whose side chains of 2nd and 4th residues were cross-linked, significantly inhibited the aggregation of Aß42. This suggested that ß-strand-fixation of BSBps could enhance their inhibitory activity against the Aß42 aggregation. However, pentapeptides 1 and 12 had little effect on morphology of Aß42 aggregates (fibrils) and neurotoxicity of Aß42 against SH-SY5Y cells.

Mechanistic analyses of the suppression of amyloid ß42 aggregation by apomorphine.

(R)-Apomorphine (1) has the potential to reduce the accumulation of amyloid ß-protein (Aß42), a causative agent of Alzheimer's disease (AD). Although the inhibition of Aß42 aggregation by 1 is ascribable to the antioxidative effect of its phenol moiety, its inhibitory mechanism at the molecular level remains to be fully elucidated. LC-MS and UV analyses revealed that 1 is autoxidized during incubation to produce an unstable o-quinone form (2), which formed a Michael adduct with Lys 16 and 28 of Aß42. A further autoxidized form of 1 (3) with o-quinone and phenanthrene moieties suppressed Aß42 aggregation comparable to 1, whereas treating 1 with a reductant, tris(2-carboxyethyl)phosphine diminished its inhibitory activity. 1H-15N SOFAST-HMQC NMR studies suggested that 1 interacts with Arg5, His13,14, Gln15, and Lys16 of the Aß42 monomer. These regions form intermolecular ß-sheets in Aß42 aggregates. Since 3 did not perturb the chemical shift of monomeric Aß42, we performed aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aß42 to investigate whether 3 associates with Aß42 oligomers. Compounds 1 and 3 delayed the onset of the oligomer-driven nucleation phase. Despite their cytotoxicity, they did not exacerbate Aß42-mediated neurotoxicity in SH-SY5Y neuroblastoma cells. These results demonstrate that extension of the conjugated system in 1 by autoxidation can promote its planarity, which is required for intercalation into the ß-sheet of Aß42 nuclei, thereby suppressing further aggregation.

UCP3 is associated with Hax-1 in mitochondria in the presence of calcium ion.

Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two-hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca(2+). The hydrophilic sequences within loop 2, and the matrix-localized hydrophilic domain of mouse UCP3, were necessary for binding to Hax-1 at the C-terminal domain, adjacent to the mitochondrial inner membrane. Interestingly, interaction of these proteins occurred in a calcium-dependent manner. Moreover, the NMR spectrum of the C-terminal domain of Hax-1 was dramatically changed by removal of Ca(2+), suggesting that the C-terminal domain of Hax-1 underwent a Ca(2+)-induced conformational change. In the Ca(2+)-free state, the C-terminal Hax-1 tended to unfold, suggesting that Ca(2+) binding may induce protein folding of the Hax-1 C-terminus. These results suggested that the UCP3-Hax-1 complex may regulate mitochondrial functional changes caused by mitochondrial Ca(2+).

Structural insights into mechanisms for inhibiting amyloid ß42 aggregation by non-catechol-type flavonoids.

The prevention of 42-mer amyloid ß-protein (Aß42) aggregation is promising for the treatment of Alzheimer's disease. We previously described the site-specific inhibitory mechanism for Aß42 aggregation by a catechol-type flavonoid, (+)-taxifolin, targeting Lys16,28 after its autoxidation. In contrast, non-catechol-type flavonoids (morin, datiscetin, and kaempferol) inhibited Aß42 aggregation without targeting Lys16,28 with almost similar potencies to that of (+)-taxifolin. We herein provided structural insights into their mechanisms for inhibiting Aß42 aggregation. Physicochemical analyses revealed that their inhibition did not require autoxidation. The (1)H-(15)N SOFAST-HMQC NMR of Aß42 in the presence of morin and datiscetin revealed the significant perturbation of chemical shifts of His13,14 and Gln15, which were close to the intermolecular ß-sheet region, Gln15-Ala21. His13,14 also played a role in radical formation at Tyr10, thereby inducing the oxidation of Met35, which has been implicated in Aß42 aggregation. These results suggest the direct interaction of morin and datiscetin with the Aß42 monomer. Although only kaempferol was oxidatively-degraded during incubation, its degradation products as well as kaempferol itself suppressed Aß42 aggregation. However, neither kaempferol nor its decomposed products perturbed the chemical shifts of the Aß42 monomer. Aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aß42 demonstrated that kaempferol and its degradation products inhibited the elongation rather than nucleation phase, implying that they interacted with small aggregates of Aß42, but not with the monomer. In contrast, morin and datiscetin inhibited both phases. The position and number of hydroxyl groups on the B-ring of non-catechol-type flavonoids could be important for their inhibitory potencies and mechanisms against Aß42 aggregation.

Synthesis and characterization of the amyloid ß40 dimer model with a linker at position 30 adjacent to the intermolecular ß-sheet region.

Amyloid fibrils in senile plaque mainly consist of the 40-mer and 42-mer amyloid ß-proteins (Aß40 and Aß42). Although Aß42 plays more important role in the pathogenesis of Alzheimer's disease (AD), Aß40 could be involved in the progression of AD pathology because of its large amount. Recent studies revealed that variable sizes of Aß oligomers contributed to the neuronal death and cognitive dysfunction. However, how large oligomeric species are responsible for AD pathogenesis remains unclear. We previously proposed a toxic dimer model of Aß with turn structure at positions 22 and 23 using solid-state NMR and systematic proline replacement. Based on this model, we herein show the synthesis and biological activities of an E22P-Aß40 dimer at position 30, which was connected to l,l-2,6-diaminopimeric acid. The E22P-Aß40 dimer formed stable 6∼8-mer oligomers without amyloid fibrils, but was not neurotoxic on human neuroblastoma cells. On the other hand, E22P-Aß40 generated high molecular-weight oligomers into fibrils, and showed the neurotoxicity. These results suggest that such kind of Aß40 dimer with a parallel ß-sheet might not be pathological.

Site-specific inhibitory mechanism for amyloid ß42 aggregation by catechol-type flavonoids targeting the Lys residues.

The aggregation of the 42-residue amyloid ß-protein (Aß42) is involved in the pathogenesis of Alzheimer disease (AD). Numerous flavonoids exhibit inhibitory activity against Aß42 aggregation, but their mechanism remains unclear in the molecular level. Here we propose the site-specific inhibitory mechanism of (+)-taxifolin, a catechol-type flavonoid, whose 3',4'-dihydroxyl groups of the B-ring plays a critical role. Addition of sodium periodate, an oxidant, strengthened suppression of Aß42 aggregation by (+)-taxifolin, whereas no inhibition was observed under anaerobic conditions, suggesting the inhibition to be associated with the oxidation to form o-quinone. Because formation of the Aß42-taxifolin adduct was suggested by mass spectrometry, Aß42 mutants substituted at Arg(5), Lys(16), and/or Lys(28) with norleucine (Nle) were prepared to identify the residues involved in the conjugate formation. (+)-Taxifolin did not suppress the aggregation of Aß42 mutants at Lys(16) and/or Lys(28) except for the mutant at Arg(5). In addition, the aggregation of Aß42 was inhibited by other catechol-type flavonoids, whereas that of K16Nle-Aß42 was not. In contrast, some non-catechol-type flavonoids suppressed the aggregation of K16Nle-Aß42 as well as Aß42. Furthermore, interaction of (+)-taxifolin with the ß-sheet region in Aß42 was not observed using solid-state NMR unlike curcumin of the non-catechol-type. These results demonstrate that catechol-type flavonoids could specifically suppress Aß42 aggregation by targeting Lys residues. Although the anti-AD activity of flavonoids has been ascribed to their antioxidative activity, the mechanism that the o-quinone reacts with Lys residues of Aß42 might be more intrinsic. The Lys residues could be targets for Alzheimer disease therapy.

Identification of a new interaction mode between the Src homology 2 domain of C-terminal Src kinase (Csk) and Csk-binding protein/phosphoprotein associated with glycosphingolipid microdomains.

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the ßB/ßC loop of the SH2 domain.

Interaction modes of human orexin 2 receptor with selective and nonselective antagonists studied by NMR spectroscopy.

Orexin neuropeptides have many physiological roles in the sleep-wake cycle, feeding behavior, reward demands, and stress responses by activating cognitive receptors, the orexin receptors (OX1R and OX2R), distributed in the brain. There are only subtle differences between OX1R and OX2R in the orthosteric site, which has hindered the rational development of subtype-selective antagonists. In this study, we utilized solution-state NMR to capture the structural plasticity of OX2R labeled with 13CH3-ε-methionine in complex with antagonists. Mutations in the orthosteric site allosterically affected the intracellular tip of TM6. Ligand exchange experiments with the subtype-selective EMPA and the nonselective suvorexant identified three methionine residues that were substantially perturbed. The NMR spectra suggested that the suvorexant-bound state exhibited more structural plasticity than the EMPA-bound state, which has not been foreseen from the close similarity of their crystal structures, providing insights into dynamic features to be considered in understanding the ligand recognition mode.

Analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria.

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid-selective unlabeling method. Because we found that the zinc ion-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

Structure-activity relationship for (+)-taxifolin isolated from silymarin as an inhibitor of amyloid ß aggregation.

Silymarin, the seed extract of Silybium marianum, has preventive effects against Alzheimer's disease-like pathogenesis in vivo. We isolated (+)-taxifolin (4) from silymarin as an inhibitor of aggregation of the 42-residue amyloid ß-protein. Structure-activity relationship studies revealed the 3',4'-dihydroxyl groups to be critical to the anti-aggregative ability, whereas the 7-hydroxyl group and the stereochemistry at positions 2 and 3 were not important.

NMR analysis of Lys63-linked polyubiquitin recognition by the tandem ubiquitin-interacting motifs of Rap80.

Ubiquitin is a post-translational modifier that is involved in cellular functions through its covalent attachment to target proteins. Ubiquitin can also be conjugated to itself at seven lysine residues and at its amino terminus to form eight linkage-specific polyubiquitin chains for individual cellular processes. The Lys63-linked polyubiquitin chain is recognized by tandem ubiquitin-interacting motifs (tUIMs) of Rap80 for the regulation of DNA repair. To understand the recognition mechanism between the Lys63-linked diubiquitin (K63-Ub(2)) and the tUIMs in solution, we determined the solution structure of the K63-Ub(2):tUIMs complex by using NOE restraints and RDC data derived from NMR spectroscopy. The structure showed that the tUIMs adopts a nearly straight and single continuous α-helix, and the two ubiquitin units of the K63-Ub(2) separately bind to each UIM motif. The interfaces are formed between Ile44-centered patches of the two ubiquitin units and the hydrophobic residues of the tUIMs. We also showed that the linker region between the two UIM motifs possesses a random-coil conformation in the free state, but undergoes the coil-to-helix transition upon complex formation, which simultaneously fixes the relative position of ubiquitin subunits. These data suggest that the relative position of ubiquitin subunits in the K63-Ub(2):tUIMs complex is essential for linkage-specific binding of Rap80 tUIMs.

Solid-state NMR analysis of the ß-strand orientation of the protofibrils of amyloid ß-protein.

Alzheimer's disease (AD) is caused by abnormal deposition (fibrillation) of a 42-residue amyloid ß-protein (Aß42) in the brain. During the process of fibrillation, the Aß42 takes the form of protofibrils with strong neurotoxicity, and is thus believed to play a crucial role in the pathogenesis of AD. To elucidate the supramolecular structure of the Aß42 protofibrils, the intermolecular proximity of the Ala-21 residues in the Aß42 protofibrils was analyzed by (13)C-(13)C rotational resonance experiments in the solid state. Unlike the Aß42 fibrils, an intermolecular (13)C-(13)C correlation was not found in the Aß42 protofibrils. This result suggests that the ß-strands of the Aß42 protofibrils are not in an in-register parallel orientation. Aß42 monomers would assemble to form protofibrils with the ß-strand conformation, then transform into fibrils by forming intermolecular parallel ß-sheets.

19F chemical library and 19F-NMR for a weakly bound complex structure.

Fragment-based drug discovery (FBDD), which involves small compounds <300 Da, has been recognized as one of the most powerful tools for drug discovery. In FBDD, the affinity of hit compounds tends to be low, and the analysis of protein-compound interactions becomes difficult. In an effort to overcome such difficulty, we developed a 19F-NMR screening method optimizing a 19F chemical library focusing on highly soluble monomeric molecules. Our method was successfully applied to four proteins, including protein kinases and a membrane protein. For FKBP12, hit compounds were carefully validated by protein thermal shift analysis, 1H-15N HSQC NMR spectroscopy, and isothermal titration calorimetry to determine dissociation constants and model complex structures. It should be noted that the 1H and 19F saturation transfer difference experiments were crucial to obtaining highly precise model structures. The combination of 19F-NMR analysis and the optimized 19F chemical library enables the modeling of the complex structure made up of a weak binder and its target protein.

Solid-state NMR analysis of interaction sites of curcumin and 42-residue amyloid ß-protein fibrils.

Aggregation of 42-residue amyloid ß-protein (Aß42) plays a pivotal role in the etiology of Alzheimer's disease (AD). Curcumin, the yellow pigment in the rhizome of turmeric, attracts considerable attention as a food component potentially preventing the pathogenesis of AD. This is because curcumin not only inhibits the aggregation of Aß42 but also binds to its aggregates (fibrils), resulting in disaggregation. However, the mechanism of interaction between curcumin and the Aß42 fibrils remains unclear. In this study, we analyzed the binding mode of curcumin to the Aß42 fibrils by solid-state NMR using dipolar-assisted rotational resonance (DARR). To improve the quality of 2D spectra, 2D DARR data were processed with the covariance NMR method, which enabled us to detect weak cross peaks between carbons of curcumin and those of the Aß42 fibrils. The observed (13)C-(13)C cross peaks indicated that curcumin interacts with the 12th and 17-21st residues included in the ß-sheet structure in the Aß42 fibrils. Interestingly, aromatic carbons adjacent to the methoxy and/or hydroxy groups of curcumin showed clear cross peaks with the Aß42 fibrils. This suggested that these functional groups of curcumin play an important role in its interaction with the Aß42 fibrils.