Photosystem II （PSII） is a multi-subunit protein complex embedded in the thylakoid membrane and splits water molecules, producing electrons, protons and molecular oxygen in photosynthesis. We started to purify and crystallize PSII in 1990, which led to the first crystal structure analysis at a resolution of 3.7 Å in 2003 using SPring-8. The resolution of the structure was raised to 1.9 Å in 2011, which was considered a breakthrough in the filed of photosynthesis because, on the basis of this atomic resolution structure, it became possible to discuss the molecular mechanism of the water-splitting reaction. We are continuing our studies to obtain much more precise information on the oxygen-evolving complex （OEC） at the S1-state of the Kok cycle and to analyze the intermediate state structures using femtosecond X-ray pulses from XFEL of SACLA.
In situ synchrotron powder diffraction measurement of gas adsorption and crystal structure analysis for porous coordination polymers（PCPs）were performed. From the obtained accurate crystal structure in both atomic and charge density levels, not only the position and orientation of adsorbed gas molecules but also the interaction between the adsorbed gas molecule and host framework were found. The information enables us to understand the mechanism of gas adsorption phenomena and functions of PCPs. It will give us the guiding principles for the novel functional materials design.
Three-dimensional protein structures bring us a deeper insight into the biological function. The main aim of my group is the X-ray structure determination of the biological macromolecular assemblies including membrane protein complexes in order to elucidate the molecular mechanism of the highly organized biological processes at atomic level. Here, I report two examples; One is structural studies of photosynthetic membrane protein complex and related redox enzymes, and the other is crystal structure analyses of dynein motor.
Electron 3D crystallography is a useful method for structure analysis from tiny and thin crystals of membrane proteins and protein complexes, which often yield crystals too small or too thin for even the synchrotron X-ray beam and X-ray free electron laser. More importantly, it can visualize the charged states of amino-acid residues and metals, as the diffraction pattern formed by elastically scattered electrons is directly related to the distribution of Coulomb potential. Here we introduce the development of this technique and structure determination with charges, and discuss further applications including a suitable treatment of electron scattering factors of charged atoms.
Pheganomycins（PGMs）derived from Streptomyces cirratus are one of the peptide antibiotics and synthesized under the cooperation of a novel peptide ligase, PGM1, and ribosome, in contrast of typical peptide antibiotics. In the biosynthesis, PGM1, which is a peptide ligase with broad substrate permissivity, can catalyze the linkage between several types of nonproteinogenic amino acid including a 2-guanidino group and some ribosomal peptides to generate N-terminal capping peptide. Herein, I present structural studies for the recognition mechanisms of N-terminal capping substrates and C-terminal ribosomal peptides.
Cyclic-AMP is one of the most important second messengers，regulating many crucial cellular events in both prokaryotes and eukaryotes. Precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase（PAC）from the photosynthetic cyanobacterium Oscillatoria acuminata is a small homodimer eminently suitable for this task，requiring only a simple flavin as chromophore. Here we describe its structure using X-ray crystallography and crystal microspectrophotometry. Site-directed mutants show signal transduction over 30 Ångstroms across the protein with minimal conformational rearrangement. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase，showing a rapid and stable response to light over many hours and activation cycles.
Fertilization, the crucial step in sexual reproduction, requires the fusion of haploid sperm and egg to create a genetically distinct offspring. Despite many studies on mammalian fertilization, the molecular mechanisms underlining the membrane fusion remain largely unknown. At present, IZUMO1 on the sperm surface and JUNO on the egg surface are known as the only protein pair essential for fertilization. In this review, we focus on the crystal structures of human IZUMO1, JUNO and IZUMO1-JUNO complex. These structures reveal the molecular mechanism of mammalian gamete recognition, and provide information for development of non-hormonal contraceptive agents.
The dystrophin glycoprotein complex, which connects the cell membrane to the basement membrane, is essential for a variety of biological events, including maintenance of muscle integrity. An O-mannose-type GalNAc-β3-GlcNAc-β4-Man（-6-phosphate）（core M3） structure of α-dystroglycan （αDG）, a subunit of the complex that is anchored to the cell membrane, interacts directly with laminin in the basement membrane. Hypo-glycosylation of αDG is linked to some types of inherited muscular dystrophy; consistent with this relationship, many disease-related mutations have been detected in genes involved in O-mannosyl glycan synthesis. Defects in protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 （POMGnT1）, a glycosyltransferase that participates in the formation of GlcNAc-β2-Man glycan, are causally related to muscle-eye-brain disease （MEB）, a congenital muscular dystrophy, although the role of POMGnT1 in post-phosphoryl modification of core M3 glycan remains elusive. We found that N-terminal domain of POMGnT1 （called stem domain） recognizes the β-linked GlcNAc of O-mannosyl glycan, an enzymatic product of POMGnT1. This interaction may recruit POMGnT1 to a specific site of α-DG to promote GlcNAc-β2-Man （core M1） clustering and also may recruit other enzymes that interact with POMGnT1, e.g., FKTN which is required for ribitol-phosphate modification of the core M3 glycan that is the first step of post-phosphoryl modification of core M3 glycan. These findings explain how POMGnT1 attaches GlcNAc-β to clustered O-mannose sites and influences post-phosphoryl modification of core M3. Our study provides important insight into how disease-associated mutations cause inherited muscular dystrophy pathogenesis.
The ring-shaped cohesin complex entraps chromosomes and regulates chromosome biology. The cohesin core consists of Smc1, Smc3, Scc1 and Scc3. Scc2-Scc4 complex is known as a cohesin loader and loads cohesin onto chromosomes. Mutations of Scc2 cause human developmental diseases termed cohesinopathy. Here we determined the crystal structure of Chaetomium thermophilum（Ct）Scc2 and its interaction with cohesin. The structure consists mostly of HEAT repeats and it forms a hook-shaped structure that similar with Scc3 and Pds5. Many cohesinopathy mutations in Scc2 diminish the Scc2- Scc1 interaction. Our study defines a functionally important interaction between cohesin and Scc2.