Search

Prostaglandins (PGs), lipid mediators, exert various effects in vivo through receptor-mediated signal transduction, and the elucidation of the molecular mechanisms of signal transduction by structural biology of PG receptors is expected to contribute to drug discovery. We have elucidated the molecular mechanisms of ligand binding mode, signal transduction mechanism, and G protein selectivity through structural analysis of PG receptors. In this paper, we would like to introduce the structure and function of GPCRs revealed through structural biology of PG receptors. [ABSTRACT FROM AUTHOR]

Adhesion is an essential ability for bacteria to survive and occupy a niche in the environment. Filamentous adhesive machinery called “pilus” or “fimbria” plays an important role in colonization, biofilm formation, and infection of many bacteria. Newly characterized type V pilus is common in the class Bacteroidia bacteria including a major oral pathogen Porphyromonas gingivalis. The monomer form and polymerized form of type V pilus subunits were revealed by X-ray crystallography and cryo-EM analysis, which led to the proposal of a unique model of type V pilus assembly mechanism involving protease-mediated strand exchange. These insights broaden our knowledge of infection mechanisms and microbiota development of class Bacteroidia bacteria. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase in the mitochondrial respiratory chain mostly exists as an independent monomer, or as a monomer associated with the complexes I and III in the supercomplex, although its molecular mechanism has been elucidated based on the crystal structure of its dimeric form. Here, we report detailed comparison of the activity and crystal structure between the monomer and the dimer, and the possibility that the monomer is an activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. [ABSTRACT FROM AUTHOR]

When we have food intake, pH of the stomach reaches around 1. This highly acidic environment is indispensable for the digestion. Gastric proton pump, H+,K+-ATPase is a membrane protein responsible for the gastric acid secretion. Its crystal structures now reveal how this proton pump extrudes H+ even into the pH 1 environment of the stomach. [ABSTRACT FROM AUTHOR]

Vibrio cholerae, an etiological agent of cholera, is attracted by various amino acids and taurine, a component of bile. A chemoreceptor homolog (Mlp37) was found to mediate taxis to both taurine and amino acids. Direct binding of taurine and amino acids to the periplasmic fragment of Mlp37 was observed by isothermal titration calorimetry. Ligand-occupied structures of the Mlp37 fragment revealed that the ligand-binding pocket has an opening, large enough to accommodate a larger side chain, and contains several common residues that can interact with taurine and different amino acids without substantial displacements. This structural information allowed us to visualize ligand binding to Mlp37 with fluorescently labeled serine. [ABSTRACT FROM AUTHOR]

P-glycoprotein (P-gp) is an ATP-binding cassette (ABC) transporter that transports various substrates from the cells. So far, crystal structures of several transporters structurally similar to P-gp have been reported. However, the multidrug transport mechanism of P-gp remains unclear, because their resolutions are low or the transporters have different functions from P-gp. Recently, we determined the crystal structures of the structural and functional homolog of P-gp, CmABCB1, from Cyanidioschyzon merolae at 2.4 Å resolution. Based on the structural comparison between CmABCB1 and the other ABC transporters, we explain structural features conserved in the true P-gp homologs, but unconserved in the other structurally similar ones. [ABSTRACT FROM AUTHOR]

Oxygenic photosynthesis synthesizes sugars from water and carbon dioxide using light energy from the sun, thereby converts light energy into chemical energy and provides oxygen for aerobic life on the earth. The light-harvesting, electron transfer, and water-splitting reactions of photosynthesis are catalyzed by two large membrane-protein complexes photosystem II (PSII) and photosystem I (PSI). Through high-resolution crystal structural analysis by synchrotron X-rays as well as femtosecond X-ray free electron lasers, the mechanisms of these reactions have become understandable at the atomic level. Here we review the recent progresses in analyzing the structures of PSII and PSI as well as their functional implications. [ABSTRACT FROM AUTHOR]

The Tm-1 gene of tomato encodes a 754-aa protein that binds tomato mosaic virus (ToMV) replication proteins and inhibits viral RNA replication. We determined a crystal structure of complex of an N-terminal fragment of Tm-1 and helicase domain of ToMV (ToMVHel). The residues in ToMV-Hel that are changed in resistance-breaking mutants are directly involved in the interaction. The positively selected region of Tm-1 formed the binding surface with ToMV-Hel. Thus, the antagonistic coevolution between a resistance protein and a viral protein has occurred at the interaction interface on both sides. [ABSTRACT FROM AUTHOR]

In purple photosynthetic bacteria, the light-harvesting core antenna (LH1) and the reaction center (RC) form a supramolecular complex (LH1-RC). The crystal structure of the LH1-RC complex from Thermochromatium tepidum was determined at a resolution of 3.0 Å. The RC is completely surrounded by the LH1 ring which is composed of 16 αβ-heterodimers. The atomic structure enables us to meet further challenge to elucidating the mechanism of color tuning, excited energy transfer and ubiquinone shuttling in the bacterial photo-system. [ABSTRACT FROM AUTHOR]

Membrane transporters transport their substrates across the membrane, thereby contributing to the maintenance of the cellular environments. One of the largest superfamily of the membrane transporters is Major Facilitator Superfamily (MFS), and many of MFS transporters are involved in the cellular uptake of various compounds utilizing the proton motive force across the membrane. Although several crystal structures of MFS transporters have been reported so far, their active transport mechanism has still remained elusive. Proton-dependent oligopeptide transporter, POT, is a member of MFS, and is involved in the uptake of oligopeptides as well as peptide-like drugs. In this article, we explain the active transporter mechanism by POT based on our recent results of the structural and computational analyses. [ABSTRACT FROM AUTHOR]

Channelrhodopsin (ChR) is a light-gated cation channel derived from algae. Since the inward fl ow of cations triggers the neuron firing, neurons expressing ChRs can be optically controlled even within freely moving mammals. Although ChR has been broadly applied to neuroscience research, little is known about its molecular mechanisms. We determined the crystal structure of chimeric ChR at 2.3 Å resolution and revealed its molecular architecture. The integration of structural and electrophysiological analyses provided insight into the molecular basis for the channel function of ChR, and paved the way for the principled design of ChR variants with novel properties. [ABSTRACT FROM AUTHOR]

Calcium (Ca2+) release from the sarco/endoplasmic reticulum stores generates vital regulatory signals in eukaryotes. The two major groups of Ca2+ release channels include the ubiquitously expressed inositol 1,4,5-trisphosphate receptors (IP3Rs) and the ryanodine receptors (RyRs). This review focuses on recently reported structural findings on the N- terminal regions of IP3Rs and RyRs and their arrangement into full-length tetrameric channels, revealing remarkable mechanistic homologies in Ca2+-release function. Additionally, we discuss genomic analyses identifying this class of Ca2+ release channels in unicellular organisms and the implications of these data in understanding the molecular evolution of these fundamental receptors in nature. [ABSTRACT FROM AUTHOR]

Actin assembly activates ATP hydrolysis, which provides structural cues for lament turnover. Polymerized actin supports cellular signaling, intracellular trafficking, and cytokinesis. We present the cryo-electron microscopic structure of F-actin in the presence of phosphate, with the visualization of some α-helical backbones and large side chains. A complete atomic model based on the cryo-EM identified intermolecular interactions, some of which were mediated by magnesium or phosphate ions. A critical role for bending of the proline-rich loop (residues 108-112) in activating ATPase was revealed. Crystal structures of G-actin mutants, which trap the catalytic site in two intermediate states, were solved. These structures combined with cryo-EM data allows us to propose a molecular mechanism for actin assembly and ATPase activation, critical for filament dynamics. [ABSTRACT FROM AUTHOR]

Half titanocene complexes bearing pyridinethiolate ligands, Cp*TiCl2(SPy) (1, Cp*=η5-pentamethylcyclopentadienyl, SPy=2-pyridinethiolate) and Cp* TiCl (3-R-SPy)2 ⋅ H (3-R-SPy) (3-R-SPy = 3-R-2-pyridinethiolate: R = H (2a) , Me3Si (2b)), were synthesized by the reaction of Cp*TiCl3 and one or two equivalents of K(3-R-SPy). The structure of 2a was revealed by X-ray crystallography. The the reaction of Cp*TiCl3 and one or two equivalents of K(3-R-SPy). The structure of 2a was revealed by X-ray crystallography. The 1-MMAO (MMAO = modified methylaluminoxane) system showed moderate activity for ethylene polymerization to give linear polyethylene with high molecular weight and relatively narrow molecular weight distribution. The 2a- and 2b-MMAO systems showed lower activity than that of 1-MMAO. The 1-MMAO system also polymerized styrene to produce highly syndiotactic polystyrene, while the 2a- and 2b-MMAO systems were almost inactive for styrene polymerization. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD), including nonalcoholic fatty liver (NAFL) and a more advanced condition with inflammation/fibrosis, nonalcoholic steatohepatitis (NASH), is emerging as one of the most prevalent chronic diseases associated with the worldwide expansion of the obese population; however, there are currently only symptomatic therapy but no cure. Among multiple candidate drugs that have been developed and tried in clinical trials against NAFLD/NASH, peroxisome proliferator-activated receptor (PPAR) dual/pan agonists continue to be the most expected ones. This review summarizes the current condition of several PPAR agonists that were and are in clinical trials against NAFLD/NASH. In addition, we recently expanded structural information about PPARα/δ/γ-ligand interactions by X-ray crystallography and executed comparative functional analyses of PPARα/δ/γ activation by those ligands; based on those knowledge, we propose the reevaluation or repositioning of currently approved PPAR agonists, saroglitazar, bezafibrate, and pemafibrate, for the treatment of NAFLD/NASH.

Peroxisome proliferator-activated receptors (PPARs) are transcription factors that are activated by endogenous fatty acids and synthetic compounds as ligands. We have been developing new phenylpropanoic acid derivatives based on structure-activity relationship studies that could reduce the side effects of existing clinical drugs. As a result, we have obtained many partial agonists that exhibit a moderate transcriptional activity while maintaining high specificity towards the receptors. However, because most of them are poorly soluble, protein-ligand interaction information has not yet been obtained by X-ray crystallography, which is essential for structure-activity relationship studies. In this paper, we report our ongoing crystallization experiments, which are aimed to develope a crystallization method for PPAR LBDs in solid-phase hydrogels that enables high-throughput protein-ligand complex crystal structure determination, using poorly soluble ligands.

Prostanoids [prostaglandins (PGs) and thromboxanes (TXs)] are a series of bioactive lipid metabolites that function in an autacoid manner via activation of cognate G protein-coupled receptors (GPCRs). The nine subtypes of prostanoid receptors (DP1, DP2, EP1, EP2, EP3, EP4, FP, IP, TP) are involved in a wide range of functions, including inflammation, immune response, reproduction, and homeostasis of the intestinal mucosa and cardiovascular system. Among the prostanoid receptors, the structure of antagonist-bound DP2, which belongs to the chemoattractant receptor family, was previously determined. However, the mechanisms of prostanoid recognition and receptor activation remained elusive. To address this issue, we determined the crystal structures of antagonist-bound EP4 and PGE 2 -bound EP3. The EP3-PGE 2 complex exhibits an active-like conformation, including outward movement of the cytoplasmic end of transmembrane (TM) 6 relative to the cytoplasmic end of TM6 of the EP4 complex. The carboxyl moiety of PGE 2 is recognized through three hydrogen bonds formed by highly conserved residues: Y114 2.65 , T206 Extracelluar loop 2 (ECL2) , and R333 7.40 (superscripts denote Ballesteros-Weinstein numbering). In addition, the ω-chain of PGE 2 orients toward TM6, which appears to contribute to receptor activation. The structure reveals important insights into the activation mechanism of prostanoid receptors and provides a molecular basis for the binding modes of endogenous ligands. These findings should facilitate the development of subtype-selective and non-PG-like ligands.

Translesion DNA synthesis (TLS) is an emergency system activated to inhibit cell death caused by DNA damage-induced replication arrest. Thus, TLS enables cancer cells to acquire resistance to alkylate anticancer drugs. REV7 functions as the hub protein that interacts with both the inserter DNA polymerase REV1 and the extender DNA polymerase REV3 in TLS. REV7-mediated protein-protein interactions (PPIs) are essential for the activation of TLS, and are therefore attractive targets for anticancer drug development. To clarify the REV7-REV3 and REV7-REV1 PPIs, we determined the structures of REV7-REV3 and REV7-REV3-REV1 complexes. In the structures of REV7-REV3 and REV7-REV3-REV1 complexes, REV7 wraps around the REV3 fragment, and the REV1-binding interface is distinct from the REV3-binding site of REV7. We also identified a novel REV7 binding protein, transcription factor II-I (TFII-I), which is required for TLS. Of note, TFII-I binds the REV7-REV3-REV1 complex, suggesting that REV7-TFII-I PPIs are independent of other REV7-mediated PPIs. Furthermore, we found a small-molecule compound that inhibits TLS by targeting the REV7-REV3 PPIs. Lastly, we determined the structure of REV7 in complex with chromosome alignment maintaining phosphoprotein (CAMP), a known kinetochore-microtubule attachment protein. The overall structure of the REV7-CAMP complex is similar to that of the REV7-REV3 complex, but the REV7-CAMP PPIs are markedly different from the REV7-REV3 PPIs. These findings improve our understanding of multifunctional hub proteins, and are helpful for designing small-molecule compounds for novel anticancer drug development.

Detailed structural characterization of protein biopharmaceuticals is a critical step in research and development; however, this step is often hampered by the structural complexities associated with glycosylation. Most protein biopharmaceuticals are modified with structurally heterogeneous and dynamic oligosaccharides which govern the physicochemical properties, functionality, pharmacokinetics, and potential pathogenicity of these glycoproteins. Considering this, we have developed a structural biological approach to describe the dynamic three-dimensional structures and interactions of glycoproteins as biopharmaceuticals. We developed an NMR technique assisted by metabolic stable-isotope labeling that can provide useful atomic-level probes for detecting and characterizing structural perturbations of glycoproteins caused by alterations in solution conditions and production protocols, as well as by mutagenesis. We have applied this method in conjunction with X-ray crystallography to investigate the structural impacts of varying glycoforms of the Fc region of immunoglobulin G (IgG), thereby elucidating the functional roles of the Fc glycans. In particular, we have successfully elucidated the structural mechanisms by which defucosylation of the IgG-Fc region increases its affinity for Fcγ receptor IIIa, leading to an improvement in ameliorating antibody-dependent cell-mediated cytotoxicity. In addition, we applied our stable-isotope-assisted NMR method to analyzing biomolecular interactions in serum environments, which are characterized by molecular crowding and promiscuous intermolecular interactions. An integrative structural biological approach combining NMR spectroscopy, X-ray crystallography, neutron scattering, atomic force microscopy, and molecular dynamics simulation will provide new research tools that will enable the visualization of dynamic structures and interactions of glycoproteins of pharmaceutical interest, thereby providing valuable insights for the development of biopharmaceuticals.