Construction of 2,2-Dimethyloxepane Frameworks from Ene-Diols Catalyzed by Metal Catalyst or Brønsted Acid via 7-Endo-Trig Cyclization.

The synthesis of 2,2-dimethyloxepane frameworks based on the 7-endo-trig cyclization of ene-diol using a catalytic amount of metal catalysts (Au, Ag) or Brønsted acid (TfOH) has been developed. Also, the spiro-type dioxabicyclic products were also derived from the diene-diols. For the condition using metal catalysts, the cyclization selectively reacted between the 1,1,3-trisubstituted alkenes and alcohols to form the 2,2-dimethyloxepane frameworks. On the other hand, the TfOH reacted with not only the 1,1,2-trisubstituted alkene, but also the 1-substituted and 1,2-disubstituted alkenes providing the corresponding cyclic ethers, which is quite different from the conditions of the metal catalysts.

Fluorescent-Oxaboroles: Synthesis and Optical Property by Sugar Recognition.

The optical property of fluorescent unit-conjugated aliphatic oxaboroles has been investigated. The oxaboroles provide good fluorescence quantum yields and selective recognition toward D-ribose and D-ribose containing molecules. The molecular recognition induced significant fluorescence quenching. The property of the boroles showed the possibility of the boron-based nicotinamide adenine dinucleotide (NAD) sensor probe.

Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodisks Using Native Lipids.

We successfully reconstituted single Natronomonas pharaonis halorhodopsin (NpHR) trimers into a nanodisk (ND) using the native archaeal lipid (NL) and an artificial lipid having a zwitterionic headgroup, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Incorporation of single trimeric NpHR into NDs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, and visible circular dichroism spectroscopy. The Cl- binding affinity of NpHR in NDs using NL (NL-ND NpHR) or POPC (POPC-ND NpHR) was examined by absorption spectroscopy, showing that the Cl--releasing affinities (Kd,N↔O) of these ND-reconstituted NpHRs are more than 10 times higher than that obtained from native NpHR membrane fragments (MFs) harvested from a NpHR-overexpressing archaeal strain (MF NpHR). The photoreaction kinetics of these ND-reconstituted NpHRs revealed that the Cl- uptake was faster than that of MF NpHR. These differences in the Cl--releasing and uptake properties of ND-reconstituted NpHRs and MF NpHR may arise from suppression of protein conformational changes associated with Cl- release from the trimeric NpHR caused by ND reconstitution, conformational perturbation in the trimeric state, and loss of the trimer-trimer interactions. On the other hand, POPC-ND NpHR demonstrated accelerated Cl- uptake compared to NL-ND NpHR, suggesting that the negative charge on the archaeal membrane surface regulates the photocycle of NpHR. Although NL-ND NpHR and MF NpHR are embedded in the same lipid, the lower Cl--binding affinity at the initial state (Kd,initial) and faster recovering from the NpHR' state to the original state of the photoreaction cycle were observed for NL-ND NpHR, probably because of insufficient interactions with a chromophore in the native membrane, bacterioruberin in reconstituted NDs. Our results indicate that specific interactions of NpHR with surrounding lipids and bacterioruberin, structural flexibility of the membrane, and interactions between trimeric NpHRs may be necessary for efficient Cl- pumping.

Structural basis of Atg8 activation by a homodimeric E1, Atg7.

E1 enzymes activate ubiquitin-like proteins and transfer them to cognate E2 enzymes. Atg7, a noncanonical E1, activates two ubiquitin-like proteins, Atg8 and Atg12, and plays a crucial role in autophagy. Here, we report crystal structures of full-length Atg7 and its C-terminal domain bound to Atg8 and MgATP, as well as a solution structure of Atg8 bound to the extreme C-terminal domain (ECTD) of Atg7. The unique N-terminal domain (NTD) of Atg7 is responsible for Atg3 (E2) binding, whereas its C-terminal domain is comprised of a homodimeric adenylation domain (AD) and ECTD. The structural and biochemical data demonstrate that Atg8 is initially recognized by the C-terminal tail of ECTD and is then transferred to an AD, where the Atg8 C terminus is attacked by the catalytic cysteine to form a thioester bond. Atg8 is then transferred via a trans mechanism to the Atg3 bound to the NTD of the opposite protomer within a dimer.

Activation of disulfide bond cleavage triggered by hydrophobization and lipophilization of functionalized dihydroasparagusic acid.

Concisely synthesized and functionalized dihydroasparagusic acid (DHAA) derivatives were used to show that the introduction of a hydrophobic functional group dramatically reduced air oxidation activity at the dithiol moieties and dominantly activated the cleavage of S-S bonds in proteins, presumably due to the hydrophobization and lipophilization. Notably, the reaction sites of water-reactive dithiol moieties behaved similarly to hydrophobic and lipophilic functional groups, which suggests impersonation of the reaction site.

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site.

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.

CO2-Selective Absorbents in Air: Reverse Lipid Bilayer Structure Forming Neutral Carbamic Acid in Water without Hydration.

Emission gas and air contain not only CO2 but also plentiful moisture, making it difficult to achieve selective CO2 absorption without hydration. To generate absorbed CO2 (wet CO2) under heating, the need for external energy to release the absorbed water has been among the most serious problems in the fields of carbon dioxide capture and storage (CCS) and direct air capture (DAC). We found that the introduction of the hydrophobic phenyl group into alkylamines of CO2 absorbents improved the absorption selectivity between CO2 and water. Furthermore, ortho-, meta-, and para-xylylenediamines (OXDA, MXDA, PXDA, respectively) absorbed only CO2 in air without any hydration. Notably, MXDA·CO2 was formed as an anhydrous carbamic acid even in water, presumably because it was covered with hydrophobic phenyl groups, which induces a reverse lipid bilayer structure. Dry CO2 was obtained from heating MXDA·CO2 at 103-120 °C, which was revealed to involve chemically the Grignard reaction to form the resulting carboxylic acids in high yields.

Biophysical characterization of drug-resistant mutants of fibroblast growth factor receptor 1.

Over-expression and aberrant activation of tyrosine kinases occur frequently in human cancers. Various tyrosine kinase inhibitors (TKIs) are under clinical use, but acquisition of resistance to these drugs is a major problem. Here, we studied the interaction between two drug-resistant mutants of fibroblast growth factor receptor 1 (FGFR1), N546K and V561M, and four ATP-competitive inhibitors, ponatinib, dovitinib, PD173074 and BGJ-398. Among these protein-drug systems, the only marked reduction in affinity was that of PD173074 for the V561M mutant. We also examined the interaction of these FGFR1 variants to AMP-PNP, a nonhydrolyzable analogue of ATP, and showed that N546K showed increased affinity for the ATP analogue as compared with the wild type. These findings will help to clarify the mechanism of drug resistance in mutant tyrosine kinases.

Nuclear magnetic resonance analysis of the conformational state of cancer mutant of fibroblast growth factor receptor 1 tyrosine kinase domain.

Tyrosine kinases are key enzymes that play critical roles in growth signaling, the abnormal activation of which is associated with various human cancers. Activation of tyrosine kinases is mediated by tyrosine phosphorylation in the activation-loop, which transforms the catalytic domain to the active state conformation. Cancer mutations are supposed to transform the conformation of the catalytic domain into the active-form independent of the phosphorylation state of the activation-loop. Here, we report structural and biophysical analyses of cancer mutations of the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1). Based on the nuclear magnetic resonance analyses, phosphorylation of the activation-loop exhibited cooperative structural transition in the activation-loop, C-helix and P-loop regions, whereas cancer mutations induced structural transformation at either one or two of these regions.

Functional interfaces between TICAM-2/TRAM and TICAM-1/TRIF in TLR4 signaling.

Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS), produces pro-inflammatory cytokines and type I interferons, and associates with a trigger of endotoxin shock. TLR4 is interacted with a TIR domain-containing adaptor molecule-2 (TICAM-2)/TRAM [TRIF (TIR domain-containing adaptor-inducing interferon-ß)-related adaptor molecule] via its Toll-interleukin-1 receptor homology (TIR) domain. TICAM-2 acts as a scaffold protein and activates TIR domain-containing adaptor molecule-1 (TICAM-1)/TRIF. According to the structural analysis by NMR, TICAM-2 interacts with TICAM-1 by the acidic amino acids motif, E87/D88/D89. The TIR domain of TICAM-2 couples with the dimer of TIR domain of TLR4 beneath the membrane, and TICAM-2 itself also forms dimer and constitutes a binding site with TICAM-1. Endosomal localization of TICAM-2 is essential for TLR4-mediated type I interferon-inducing signal from the endosome. N-terminal myristoylation allows TICAM-2 to anchor to the endosomal membrane. Additionally, we have identified two acidic amino acids, D91/E92, as a functional motif that cooperatively determines endosomal localization of TICAM-2. This structural information of TICAM-2 suggests that the specific structure is indispensable for the endosomal localization and type I interferon production of TICAM-2. Taken together with the knowledge on cytoplasmic sensors for LPS, TICAM-2/TICAM-1 may conform to a signal network on TLR4 to facilitate induction of cytokine disorders.

Identification of a Regulatory Acidic Motif as the Determinant of Membrane Localization of TICAM-2.

TLR4 triggers LPS signaling through the adaptors Toll/IL-1R domain-containing adaptor molecule (TICAM)-2 (also called TRAM) and TICAM-1 (also called TRIF), together with Toll/IL-1R domain-containing adaptor protein (TIRAP) and MyD88. The MyD88 pathway mediates early phase responses to LPS on the plasma membrane, whereas the TICAM pathway mediates late-phase responses, which induce the production of type I IFN and activation of inflammasomes. TICAM-2 bridges TLR4 and TICAM-1 for LPS signaling in the endosome. Recently, we identified an acidic motif, E87/D88/D89 in TICAM-2, that provides the interaction surfaces between TICAM-2 and TICAM-1. In the present study, we found additional D91/E92 in TICAM-2, conserved across species, that is crucial for TICAM-1 activation. The D91A/E92A mutant protein was distributed largely to the cytosol, despite myristoylation, suggesting its importance for assistance of membrane localization of TICAM-2. An ectopically expressed D91A/E92A mutant per se failed to activate TICAM-1, unlike its wild-type counterpart that forms self-aggregation, but it still retained the ability to pass LPS-mediated IFN regulatory factor (IRF)3 activation. In a TICAM-2 knockout human cell line expressing TLR4/MD-2 with or without CD14, overexpression of the D91A/E92A mutant did not activate IRF3, but upon LPS stimulation, it induced sufficient TLR4-mediated IRF3 activation with high coefficient colocalization. Hence, the D91/E92 motif guides TICAM-2 membrane localization and self-activation for signaling. Our results suggest the presence of two distinct steps underlying endosomal LPS signaling on TICAM-2 for TICAM-1 activation: TICAM-2 assembling in TLR4 and/or TICAM-2 self-activation. D91A/E92A of TICAM-2 selectively associates the TLR4-dependent TICAM-2 assembling, but not cytosolic TICAM-2 self-aggregation, to activate TICAM-1.

Substrate Specific Silver(I)-Catalyzed Cycloisomerization of Diene Involving Alkyl Rearrangements: Syntheses of 1,2,5,6-Tetrahydrocuminic Acid, p-Menth-3-en-7-ol, and p-Menth-3-en-7-al.

The novel cationic Ag(I)-catalyzed cycloisomerization, which is associated with alkyl rearrangements, from dimethyl 2-allyl-2-prenylmalonate (1) to dimethyl 4-isopropylcyclohex-3-ene-1,1-dicarboxylate (2) has been developed. Derivatization from the diester 2 into the diol 3 and its X-ray crystallographic analysis determined the structure. The mechanisms of the novel reaction were investigated by isotopic experiments, which supported the unusual alkyl shifts. In addition, the product 2 was used for the total syntheses of three natural products, 1,2,5,6-tetrahydrocuminic acid (12), p-menth-3-en-7-ol (13), and p-menth-3-en-7-al (14) in short steps.

The Thermotolerant Yeast Kluyveromyces marxianus Is a Useful Organism for Structural and Biochemical Studies of Autophagy.

Autophagy is a conserved degradation process in which autophagosomes are generated by cooperative actions of multiple autophagy-related (Atg) proteins. Previous studies using the model yeast Saccharomyces cerevisiae have provided various insights into the molecular basis of autophagy; however, because of the modest stability of several Atg proteins, structural and biochemical studies have been limited to a subset of Atg proteins, preventing us from understanding how multiple Atg proteins function cooperatively in autophagosome formation. With the goal of expanding the scope of autophagy research, we sought to identify a novel organism with stable Atg proteins that would be advantageous for in vitro analyses. Thus, we focused on a newly isolated thermotolerant yeast strain, Kluyveromyces marxianus DMKU3-1042, to utilize as a novel system elucidating autophagy. We developed experimental methods to monitor autophagy in K. marxianus cells, identified the complete set of K. marxianus Atg homologs, and confirmed that each Atg homolog is engaged in autophagosome formation. Biochemical and bioinformatic analyses revealed that recombinant K. marxianus Atg proteins have superior thermostability and solubility as compared with S. cerevisiae Atg proteins, probably due to the shorter primary sequences of KmAtg proteins. Furthermore, bioinformatic analyses showed that more than half of K. marxianus open reading frames are relatively short in length. These features make K. marxianus proteins broadly applicable as tools for structural and biochemical studies, not only in the autophagy field but also in other fields.

Investigation of the redox-dependent modulation of structure and dynamics in human cytochrome c.

Redox-dependent changes in the structure and dynamics of human cytochrome c (Cyt c) were investigated by solution NMR. We found significant structural changes in several regions, including residues 23-28 (loop 3), which were further corroborated by chemical shift differences between the reduced and oxidized states of Cyt c. These differences are essential for discriminating redox states in Cyt c by cytochrome c oxidase (CcO) during electron transfer reactions. Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments identified that the region around His33 undergoes conformational exchanges on the µs-ms timescale, indicating significant redox-dependent structural changes. Because His33 is not part of the interaction site for CcO, our data suggest that the dynamic properties of the region, which is far from the interaction site for CcO, contribute to conformational changes during electron transfer to CcO.

Structural analysis of the mechanism of phosphorylation of a critical autoregulatory tyrosine residue in FGFR1 kinase domain.

Receptor and nonreceptor tyrosine kinases are enzymes that play important roles in regulating signal transduction pathways in a variety of normal cellular process and in many pathological conditions. Ordered phosphorylation is required for receptor tyrosine kinase (RTK) activation, a process mediated by transient dimer formation of the kinase domains. This process is triggered by the tyrosine phosphorylation in the activation loop. Here, we report structural and biochemical analyses of the tyrosine kinase domain interaction of fibroblast growth factor receptor 1 (FGFR1) required for the initial phosphorylation step. On the basis of nuclear magnetic resonance (NMR) analysis and covalent cross-linking experiments, we propose a parallel symmetric dimer model where specific contacts are formed between the N-lobes and C-lobes, respectively, in the FGFR1 kinase domains. Moreover, assignment of the contact sites between two FGFR1 kinase domains are supported by a trans-phosphorylation assay and by mutational analyses. The present report shows the molecular mechanism underlying the control of trans-phosphorylation of a critical auto-regulatory site in FGF receptors' catalytic domain.

Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses.

A DExD/H protein, RIG-I, is critical in innate antiviral responses by sensing viral RNA. Here we show that RIG-I recognizes two distinct viral RNA patterns: double-stranded (ds) and 5'ppp single-stranded (ss) RNA. The binding of RIG-I with dsRNA or 5'ppp ssRNA in the presence of ATP produces a common structure, as suggested by protease digestion. Further analyses demonstrated that the C-terminal domain of RIG-I (CTD) recognizes these RNA patterns and CTD coincides with the autorepression domain. Structural analysis of CTD by NMR spectroscopy in conjunction with mutagenesis revealed that the basic surface of CTD with a characteristic cleft interacts with RIG-I ligands. Our results suggest that the bipartite structure of CTD regulates RIG-I on encountering viral RNA patterns.

Structures and interface mapping of the TIR domain-containing adaptor molecules involved in interferon signaling.

Homotypic and heterotypic interactions between Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors (TLRs) and downstream adaptors are essential to evoke innate immune responses. However, such oligomerization properties present intrinsic difficulties in structural studies of TIR domains. Here, using BB-loop mutations that disrupt homotypic interactions, we determined the structures of the monomeric TIR domain-containing adaptor molecule (TICAM)-1 and TICAM-2 TIR domains. Docking of the monomeric structures, together with yeast two hybrid-based mutagenesis assays, reveals that the homotypic interaction between TICAM-2 TIR is indispensable to present a scaffold for recruiting the monomeric moiety of the TICAM-1 TIR dimer. This result proposes a unique idea that oligomerization of upstream TIR domains is crucial for binding of downstream TIR domains. Furthermore, the bivalent nature of each TIR domain dimer can generate a large signaling complex under the activated TLRs, which would recruit downstream signaling molecules efficiently. This model is consistent with previous reports that BB-loop mutants completely abrogate downstream signaling.

Energyless CO2 Absorption, Generation, and Fixation Using Atmospheric CO2.

From an economic and ecological perspective, the efficient utilization of atmospheric CO2 as a carbon resource should be a much more important goal than reducing CO2 emissions. However, no strategy to harvest CO2 using atmospheric CO2 at room temperature currently exists, which is presumably due to the extremely low concentration of CO2 in ambient air (approximately 400 ppm=0.04 vol%). We discovered that monoethanolamine (MEA) and its derivatives efficiently absorbed atmospheric CO2 without requiring an energy source. We also found that the absorbed CO2 could be easily liberated with acid. Furthermore, a novel CO2 generator enabled us to synthesize a high value-added material (i.e., 2-oxazolidinone derivatives based on the metal catalyzed CO2-fixation at room temperature) from atmospheric CO2.

Structure of the Atg12-Atg5 conjugate reveals a platform for stimulating Atg8-PE conjugation.

Atg12 is conjugated to Atg5 through enzymatic reactions similar to ubiquitination. The Atg12-Atg5 conjugate functions as an E3-like enzyme to promote lipidation of Atg8, whereas lipidated Atg8 has essential roles in both autophagosome formation and selective cargo recognition during autophagy. However, the molecular role of Atg12 modification in these processes has remained elusive. Here, we report the crystal structure of the Atg12-Atg5 conjugate. In addition to the isopeptide linkage, Atg12 forms hydrophobic and hydrophilic interactions with Atg5, thereby fixing its position on Atg5. Structural comparison with unmodified Atg5 and mutational analyses showed that Atg12 modification neither induces a conformational change in Atg5 nor creates a functionally important architecture. Rather, Atg12 functions as a binding module for Atg3, the E2 enzyme for Atg8, thus endowing Atg5 with the ability to interact with Atg3 to facilitate Atg8 lipidation.

Air-stable cationic gold(I) catalyst featuring a Z-type ligand: promoting enyne cyclizations.

An air-stable cationic Au(I) complex featuring a Z-type ligand (boron atom) as a σ-acceptor was developed for elucidating the effect of B on catalytic reactions. An enyne cyclization in the presence of either [Au→B](+) or [Au](+) showed that [Au→B](+) promotes the reactivity, which enabled the effective construction of not only five- and six-membered rings, but also seven-membered rings.