Cryo-EM analysis reveals human SID-1 transmembrane family member 1 dynamics underlying lipid hydrolytic activity.

Two mammalian homologs of systemic RNA interference defective protein 1 (SID-1) (SIDT1/2) are suggested to function as double-stranded RNA (dsRNA) transporters for extracellular dsRNA uptake or for release of incorporated dsRNA from lysosome to cytoplasm. SIDT1/2 is also suggested to be involved in cholesterol transport and lipid metabolism. Here, we determine the cryo-electron microscopy structures of human SIDT1, homodimer in a side-by-side arrangement, with two distinct conformations, the cholesterol-bound form and the unbound form. Our structures reveal that the membrane-spanning region of SIDT1 harbors conserved histidine and aspartate residues coordinating to putative zinc ion, in a structurally similar manner to alkaline ceramidases or adiponectin receptors that require zinc for ceramidase activity. We identify that SIDT1 has a ceramidase activity that is attenuated by cholesterol binding. Observations from two structures suggest that cholesterol molecules serve as allosteric regulator that binds the transmembrane region of SIDT1 and induces the conformation change and the reorientation of the catalytic residues. This study represents a contribution to the elucidation of the cholesterol-mediated mechanisms of lipid hydrolytic activity and RNA transport in the SID-1 family proteins.

In Silico Identification of Inhibitory Compounds for SARS-Cov-2 Papain-Like Protease.

Virtual screening with high-performance computers is a powerful and cost-effective technique in drug discovery. A chemical database is searched to find candidate compounds firmly bound to a target protein, judging from the binding poses and/or binding scores. The severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) infectious disease has spread worldwide for the last three years, causing severe slumps in economic and social activities. SARS-Cov-2 has two viral proteases: 3-chymotrypsin-like (3CL) and papain-like (PL) protease. While approved drugs have already been released for the 3CL protease, no approved agent is available for PL protease. In this work, we carried out in silico screening for the PL protease inhibitors, combining docking simulation and molecular mechanics calculation. Docking simulations were applied to 8,820 molecules in a chemical database of approved and investigational compounds. Based on the binding poses generated by the docking simulations, molecular mechanics calculations were performed to optimize the binding structures and to obtain the binding scores. Based on the binding scores, 57 compounds were selected for in vitro assay of the inhibitory activity. Five inhibitory compounds were identified from the in vitro measurement. The predicted binding structures of the identified five compounds were examined, and the significant interaction between the individual compound and the protease catalytic site was clarified. This work demonstrates that computational virtual screening by combining docking simulation with molecular mechanics calculation is effective for searching candidate compounds in drug discovery.

Bisabosqual A: A novel asparagine synthetase inhibitor suppressing the proliferation and migration of human non-small cell lung cancer A549 cells.

Asparagine synthetase (ASNS) is a crucial enzyme for the de novo biosynthesis of endogenous asparagine (Asn), and ASNS shows the positive relationship with the growth of several solid tumors. Most of ASNS inhibitors are analogs of transition-state in ASNS reaction, but their low cell permeability hinders their anticancer activity. Therefore, novel ASNS inhibitors with a new pharmacophore urgently need to be developed. In this study, we established and applied a system for in vitro screening of ASNS inhibitors, and found a promising unique bisabolane-type meroterpenoid molecule, bisabosqual A (Bis A), able to covalently modify K556 site of ASNS protein. Bis A targeted ASNS to suppress cell proliferation of human non-small cell lung cancer A549 cells and exhibited a synergistic effect with L-asparaginase (L-ASNase). Mechanistically, Bis A promoted oxidative stress and apoptosis, while inhibiting autophagy, cell migration and epithelial-mesenchymal transition (EMT), impeding cancer cell development. Moreover, Bis A induced negative feedback pathways containing the GCN2-eIF2α-ATF4, PI3K-AKT-mTORC1 and RAF-MEK-ERK axes, but combination treatment of Bis A and rapamycin/torin-1 overcame the potential drug resistance triggered by mTOR pathways. Our study demonstrates that ASNS inhibition is promising for cancer chemotherapy, and Bis A is a potential lead ASNS inhibitor for anticancer development.

A small molecule iCDM-34 identified by in silico screening suppresses HBV DNA through activation of aryl hydrocarbon receptor.

IFN-alpha have been reported to suppress hepatitis B virus (HBV) cccDNA via APOBEC3 cytidine deaminase activity through interferon signaling. To develop a novel anti-HBV drug for a functional cure, we performed in silico screening of the binding compounds fitting the steric structure of the IFN-alpha-binding pocket in IFNAR2. We identified 37 compounds and named them in silico cccDNA modulator (iCDM)-1-37. We found that iCDM-34, a new small molecule with a pyrazole moiety, showed anti-HCV and anti-HBV activities. We measured the anti-HBV activity of iCDM-34 dependent on or independent of entecavir (ETV). iCDM-34 suppressed HBV DNA, pgRNA, HBsAg, and HBeAg, and also clearly exhibited additive inhibitory effects on the suppression of HBV DNA with ETV. We confirmed metabolic stability of iCDM-34 was stable in human liver microsomal fraction. Furthermore, anti-HBV activity in human hepatocyte-chimeric mice revealed that iCDM-34 was not effective as a single reagent, but when combined with ETV, it suppressed HBV DNA compared to ETV alone. Phosphoproteome and Western blotting analysis showed that iCDM-34 did not activate IFN-signaling. The transcriptome analysis of interferon-stimulated genes revealed no increase in expression, whereas downstream factors of aryl hydrocarbon receptor (AhR) showed increased levels of the expression. CDK1/2 and phospho-SAMHD1 levels decreased under iCDM-34 treatment. In addition, AhR knockdown inhibited anti-HCV activity of iCDM-34 in HCV replicon cells. These results suggest that iCDM-34 decreases the phosphorylation of SAMHD1 through CDK1/2, and suppresses HCV replicon RNA, HBV DNA, and pgRNA formation.

Unsupervised deep learning for molecular dynamics simulations: a novel analysis of protein-ligand interactions in SARS-CoV-2 Mpro.

Molecular dynamics (MD) simulations, which are central to drug discovery, offer detailed insights into protein-ligand interactions. However, analyzing large MD datasets remains a challenge. Current machine-learning solutions are predominantly supervised and have data labelling and standardisation issues. In this study, we adopted an unsupervised deep-learning framework, previously benchmarked for rigid proteins, to study the more flexible SARS-CoV-2 main protease (Mpro). We ran MD simulations of Mpro with various ligands and refined the data by focusing on binding-site residues and time frames in stable protein conformations. The optimal descriptor chosen was the distance between the residues and the center of the binding pocket. Using this approach, a local dynamic ensemble was generated and fed into our neural network to compute Wasserstein distances across system pairs, revealing ligand-induced conformational differences in Mpro. Dimensionality reduction yielded an embedding map that correlated ligand-induced dynamics and binding affinity. Notably, the high-affinity compounds showed pronounced effects on the protein's conformations. We also identified the key residues that contributed to these differences. Our findings emphasize the potential of combining unsupervised deep learning with MD simulations to extract valuable information and accelerate drug discovery.

Wetting hysteresis induces effective unidirectional water transport through a fluctuating nanochannel.

We propose a water pump that actively transports water molecules through nanochannels. Spatially asymmetric noise fluctuations imposed on the channel radius cause unidirectional water flow without osmotic pressure, which can be attributed to hysteresis in the cyclic transition between the wetting/drying states. We show that the water transport depends on fluctuations, such as white, Brownian, and pink noises. Because of the high-frequency components in white noise, fast switching of open and closed states inhibits channel wetting. Conversely, pink and Brownian noises generate high-pass filtered net flow. Brownian fluctuation leads to a faster water transport rate, whereas pink noise has a higher capability to overcome pressure differences in the opposite direction. A trade-off relationship exists between the resonant frequency of the fluctuation and the flow amplification. The proposed pump can be considered as an analogy for the reversed Carnot cycle, which is the upper limit of the energy conversion efficiency.

Effect of temperature on the structure and drug-release behaviour of inclusion complex of ß-cyclodextrin with cyclophosphamide: a molecular dynamics study.

Cyclodextrins (CDs) are suitable drug carriers because of their doughnut-shaped cavities with hydrophilic outer and hydrophobic inner surfaces. Temperature-responsive CD-based drug carriers are expected to be one of the most promising candidates for drug delivery systems. In this study, we performed molecular dynamics simulations of the inclusion complex of ß-CD with cyclophosphamide (CP) at temperatures from 300 K to 400 K to investigate the temperature dependency of the release behaviour of CP and structural changes of ß-CD in an aqueous solution. We analysed the distance between the centres of mass of ß-CD and CP and the radius of gyration of ß-CD. The CP molecule was released from the ß-CD cavity at 400 K, whereas two different inclusion complexes, partially and completely, were observed at T < 400 K. ß-CD encapsulating a CP molecule had a more spherical shape and rigidity than ß-CD without a CP, and the rigidity of their inclusion complex decreased with increasing temperature. Our findings provide fundamental insights into the behaviours of the ß-CD/CP complex and drug release at the molecular level and can facilitate the development of new temperature-responsive drug delivery systems with CD nanocarriers triggered by localised temperature increases using focused ultrasound.

Differences in ligand-induced protein dynamics extracted from an unsupervised deep learning approach correlate with protein-ligand binding affinities.

Prediction of protein-ligand binding affinity is a major goal in drug discovery. Generally, free energy gap is calculated between two states (e.g., ligand binding and unbinding). The energy gap implicitly includes the effects of changes in protein dynamics induced by ligand binding. However, the relationship between protein dynamics and binding affinity remains unclear. Here, we propose a method that represents ligand-binding-induced protein behavioral change with a simple feature that can be used to predict protein-ligand affinity. From unbiased molecular simulation data, an unsupervised deep learning method measures the differences in protein dynamics at a ligand-binding site depending on the bound ligands. A dimension reduction method extracts a dynamic feature that strongly correlates to the binding affinities. Moreover, the residues that play important roles in protein-ligand interactions are specified based on their contribution to the differences. These results indicate the potential for binding dynamics-based drug discovery.

Molecular Dynamics Study of Conformational Changes of Tankyrase 2 Binding Subsites upon Ligand Binding.

The interactions between proteins and ligands are involved in various biological functions. While experimental structures provide key static structural information of ligand-unbound and ligand-bound proteins, dynamic information is often insufficient for understanding the detailed mechanism of protein-ligand binding. Here, we studied the conformational changes of the tankyrase 2 binding pocket upon ligand binding using molecular dynamics simulations of the ligand-unbound and ligand-bound proteins. The ligand-binding pocket has two subsites: the nicotinamide and adenosine subsite. Comparative analysis of these molecular dynamics trajectories revealed that the conformational change of the ligand-binding pocket was characterized by four distinct conformations of the ligand-binding pocket. Two of the four conformations were observed only in molecular dynamics simulations. We found that the pocket conformational change on ligand binding was based on the connection between the nicotinamide and adenosine subsites that are located adjacently in the pocket. From the analysis, we proposed the protein-ligand binding mechanism of tankyrase 2. Finally, we discussed the computational prediction of the ligand binding pose using the tankyrase 2 structures obtained from the molecular dynamics simulations.

Purification of Cytosolic Phospholipase A2α C2-domain after Expression in Soluble Form in Escherichia coli.

Previous expression/purification strategies for cytosolic phospholipase A2α C2-domain in Escherichia coli have relied on refolded protein recovered from inclusion bodies and sometimes containing C-terminal Cys139Ala and Cys141Ser substitutions to eliminate potential refolding complications induced by Cys residues. The protocol presented herein describes an effective method for the expression of cytosolic phospholipase A2α C2-domain in soluble form in E. coli and subsequent purification to homogeneity. This protocol, which utilizes a cleavable 6xHis-SUMO tag, has recently been used to gain insights into the structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase A2α ( Hirano et al., 2019 ).

Superior light-resistant dithieno[3,2-b:2',3'-d]arsole-based polymers exhibiting ultrastable amplified spontaneous emission.

Herein, by amplified spontaneous emission measurements, dithieno[3,2-b:2',3'-d]arsole (DTA)-bithiophene (DTA-BT) and DTA-ethynylbenzene (DTA-EB) polymer films exhibited considerable photostability under continuous pulsed pumping excitation for at least 15 h at the pumping energies, 28.9 and 20.7 µJ, respectively, showing an outstanding high light-resistance among the polymer-based gain media.

Mechanism of self/nonself-discrimination in Brassica self-incompatibility.

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

Drug binding dynamics of the dimeric SARS-CoV-2 main protease, determined by molecular dynamics simulation.

We performed molecular dynamics simulation of the dimeric SARS-CoV-2 (severe acute respiratory syndrome corona virus 2) main protease (Mpro) to examine the binding dynamics of small molecular ligands. Seven HIV inhibitors, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir, were used as the potential lead drugs to investigate access to the drug binding sites in Mpro. The frequently accessed sites on Mpro were classified based on contacts between the ligands and the protein, and the differences in site distributions of the encounter complex were observed among the ligands. All seven ligands showed binding to the active site at least twice in 28 simulations of 200 ns each. We further investigated the variations in the complex structure of the active site with the ligands, using microsecond order simulations. Results revealed a wide variation in the shapes of the binding sites and binding poses of the ligands. Additionally, the C-terminal region of the other chain often interacted with the ligands and the active site. Collectively, these findings indicate the importance of dynamic sampling of protein-ligand complexes and suggest the possibilities of further drug optimisations.

Polar recruitment of RLD by LAZY1-like protein during gravity signaling in root branch angle control.

In many plant species, roots maintain specific growth angles relative to the direction of gravity, known as gravitropic set point angles (GSAs). These contribute to the efficient acquisition of water and nutrients. AtLAZY1/LAZY1-LIKE (LZY) genes are involved in GSA control by regulating auxin flow toward the direction of gravity in Arabidopsis. Here, we demonstrate that RCC1-like domain (RLD) proteins, identified as LZY interactors, are essential regulators of polar auxin transport. We show that interaction of the CCL domain of LZY with the BRX domain of RLD is important for the recruitment of RLD from the cytoplasm to the plasma membrane by LZY. A structural analysis reveals the mode of the interaction as an intermolecular ß-sheet in addition to the structure of the BRX domain. Our results offer a molecular framework in which gravity signal first emerges as polarized LZY3 localization in gravity-sensing cells, followed by polar RLD1 localization and PIN3 relocalization to modulate auxin flow.

Structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase A2α.

Ca2+-stimulated translocation of cytosolic phospholipase A2α (cPLA2α) to the Golgi induces arachidonic acid production, the rate-limiting step in pro-inflammatory eicosanoid synthesis. Structural insights into the cPLA2α preference for phosphatidylcholine (PC)-enriched membranes have remained elusive. Here, we report the structure of the cPLA2α C2-domain (at 2.2 Å resolution), which contains bound 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and Ca2+ ions. Two Ca2+ are complexed at previously reported locations in the lipid-free C2-domain. One of these Ca2+ions, along with a third Ca2+, bridges the C2-domain to the DHPC phosphate group, which also interacts with Asn65. Tyr96 plays a key role in lipid headgroup recognition via cation-π interaction with the PC trimethylammonium group. Mutagenesis analyses confirm that Tyr96 and Asn65 function in PC binding selectivity by the C2-domain and in the regulation of cPLA2α activity. The DHPC-binding mode of the cPLA2α C2-domain, which differs from phosphatidylserine or phosphatidylinositol 4,5-bisphosphate binding by other C2-domains, expands and deepens knowledge of the lipid-binding mechanisms mediated by C2-domains.

Strigolactone perception and deactivation by a hydrolase receptor DWARF14.

The perception mechanism for the strigolactone (SL) class of plant hormones has been a subject of debate because their receptor, DWARF14 (D14), is an α/ß-hydrolase that can cleave SLs. Here we show via time-course analyses of SL binding and hydrolysis by Arabidopsis thaliana D14, that the level of uncleaved SL strongly correlates with the induction of the active signaling state. In addition, we show that an AtD14D218A catalytic mutant that lacks enzymatic activity is still able to complement the atd14 mutant phenotype in an SL-dependent manner. We conclude that the intact SL molecules trigger the D14 active signaling state, and we also describe that D14 deactivates bioactive SLs by the hydrolytic degradation after signal transmission. Together, these results reveal that D14 is a dual-functional receptor, responsible for both the perception and deactivation of bioactive SLs.

Influence of an Interfacial Effect on the Laser Performance of a Rhodamine 6G/Cellulose Acetate Waveguide on a Vinylidene Fluoride Copolymer Layer.

The fluorescent properties of dyes and fluorophores in condensed matter significantly affect the laser performance of organic dye lasers and fluorescent polymer lasers. Concentration quenching of fluorescence is commonly observed in condensed matter. Several approaches have been presented to suppress such quenching, such as the use of a dendrimer and the use of effective energy transfer in a guest-host system. The enhanced fluorescence of rhodamine 6G (R6G) dye on a vinylidene fluoride polymer is an alternative method for enhancing laser performance because of the roughness of the P(VDF-TrFE) surface and the interaction between polar ß-crystals of P(VDF-TrFE) and R6G dye. In this paper, a significant improvement in slope efficiency (SE) is demonstrated without a significant depression in the lasing threshold for distributed feedback (DFB) and distributed Bragg reflector (DBR) lasers fabricated using an R6G-dispersed cellulose acetate (CA) matrix spin-coated onto a copolymer of vinylidene fluoride and trifluoroethylene P(VDF-TrFE) thin film. SEs of 3.4 and 1.3% were measured for DBR and DFB laser devices with CA/R6G on a P(VDF-TrFE) thin film, respectively, whereas an SE of less than 1.0% was measured for both corresponding laser devices without a P(VDF-TrFE) thin film. From the aspect of simple fabrication procedures, repeatability in device fabrication and performance, stability of the device, time for device fabrication, the present approach is the most preferable way for industrial applications, requiring only the additional step of spin-coating of a P(VDF-TrFE) thin film.

The force-sensing device region of α-catenin is an intrinsically disordered segment in the absence of intramolecular stabilization of the autoinhibitory form.

Mechanotransduction by α-catenin facilitates the force-dependent development of adherens junctions (AJs) by recruiting vinculin to reinforce actin anchoring of AJs. The α-catenin mechanotransducing action is facilitated by its force-sensing device region that autoinhibits the vinculin-binding site 1 (VBS1). Here, we report the high-resolution structure of the force-sensing device region of α-catenin, which shows the autoinhibited form comprised of helix bundles E, F and G. The cryptic VBS1 is embedded into helix bundle E stabilized by direct interactions with the autoinhibitory region forming helix bundles F and G. Our molecular dissection study showed that helix bundles F and G are stable in solution in each isolated form, whereas helix bundle E that contains VBS1 is unstable and intrinsically disordered in solution in the isolated form. We successfully identified key residues mediating the autoinhibition and produced mutated α-catenins that display variable force sensitivity and autoinhibition. Using these mutants, we demonstrate both in vitro and in vivo that, in the absence of this stabilization, the helix bundle containing VBS1 would adopt an unfolded form, thus exposing VBS for vinculin binding. We provide evidence for importance of mechanotransduction with the intrinsic force sensitivity for vinculin recruitment to adherens junctions of epithelial cell sheets with mutated α-catenins.

Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM.

Adherens junctions (AJs) adaptively change their intensities in response to intercellular tension; therefore, they integrate tension generated by individual cells to drive multicellular dynamics, such as morphogenetic change in embryos. Under intercellular tension, α-catenin, which is a component protein of AJs, acts as a mechano-chemical transducer to recruit vinculin to promote actin remodeling. Although in vivo and in vitro studies have suggested that α-catenin-mediated mechanotransduction is a dynamic molecular process, which involves a conformational change of α-catenin under tension to expose a cryptic vinculin binding site, there are no suitable experimental methods to directly explore the process. Therefore, in this study, we developed a novel system by combining atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF). In this system, α-catenin molecules (residues 276-634; the mechano-sensitive M1-M3 domain), modified on coverslips, were stretched by AFM and their recruitment of Alexa-labeled full-length vinculin molecules, dissolved in solution, were observed simultaneously, in real time, using TIRF. We applied a physiologically possible range of tensions and extensions to α-catenin and directly observed its vinculin recruitment. Our new system could be used in the fields of mechanobiology and biophysics to explore functions of proteins under tension by coupling biomechanical and biochemical information.