Hideaki Unno

Biological and immunological characteristics of hepatitis E virus-like particles based on the crystal structure

Hepatitis E virus (HEV) is a causative agent of acute hepatitis. The crystal structure of HEV-like particles (HEV-LP) consisting of capsid protein was determined at 3.5-Å resolution. The capsid protein exhibited a quite different folding at the protruding and middle domains from the members of the families of Caliciviridae and Tombusviridae, while the shell domain shared the common folding. Tyr-288 at the 5-fold axis plays key roles in the assembly of HEV-LP, and aromatic amino acid residues are well conserved among the structurally related viruses. Mutational analyses indicated that the protruding domain is involved in the binding to the cells susceptive to HEV infection and has some neutralization epitopes. These structural and biological findings are important for understanding the molecular mechanisms of assembly and entry of HEV and also provide clues in the development of preventive and prophylactic measures for hepatitis E.

Functional, structural, and spectroscopic characterization of a glutathione-ligated [2Fe-2S] cluster in poplar glutaredoxin C1

When expressed in Escherichia coli, cytosolic poplar glutaredoxin C1 (CGYC active site) exists as a dimeric iron–sulfur-containing holoprotein or as a monomeric apoprotein in solution. Analytical and spectroscopic studies of wild-type protein and site-directed variants and structural characterization of the holoprotein by using x-ray crystallography indicate that the holoprotein contains a subunit-bridging [2Fe–2S] cluster that is ligated by the catalytic cysteines of two glutaredoxins and the cysteines of two glutathiones. Mutagenesis data on a variety of poplar glutaredoxins suggest that the incorporation of an iron–sulfur cluster could be a general feature of plant glutaredoxins possessing a glycine adjacent to the catalytic cysteine. In light of these results, the possible involvement of plant glutaredoxins in oxidative stress sensing or iron–sulfur biosynthesis is discussed with respect to their intracellular localization.

Atomic Structure of Plant Glutamine Synthetase: A KEY ENZYME FOR PLANT PRODUCTIVITY

Plants provide nourishment for animals and other heterotrophs as the sole primary producer in the food chain. Glutamine synthetase (GS), one of the essential enzymes for plant autotrophy catalyzes the incorporation of ammonia into glutamate to generate glutamine with concomitant hydrolysis of ATP, and plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Elucidation of the atomic structure of higher plant GS is important to understand its detailed reaction mechanism and to obtain further insight into plant productivity and agronomical utility. Here we report the first crystal structures of maize (Zea mays L.) GS. The structure reveals a unique decameric structure that differs significantly from the bacterial GS structure. Higher plants have several isoenzymes of GS differing in heat stability and catalytic properties for efficient responses to variation in the environment and nutrition. A key residue responsible for the heat stability was found to be Ile-161 in GS1a. The three structures in complex with substrate analogues, including phosphinothricin, a widely used herbicide, lead us to propose a mechanism for the transfer of phosphate from ATP to glutamate and to interpret the inhibitory action of phosphinothricin as a guide for the development of new potential herbicides.

Structural and Mutational Studies of Anthocyanin Malonyltransferases Establish the Features of BAHD Enzyme Catalysis

The BAHD family is a class of acyl-CoA-dependent acyltransferases that are involved in plant secondary metabolism and show a diverse range of specificities for acyl acceptors. Anthocyanin acyltransferases make up an important class of the BAHD family and catalyze the acylation of anthocyanins that are responsible for most of the red-to-blue colors of flowers. Here, we describe crystallographic and mutational studies of three similar anthocyanin malonyltransferases from red chrysanthemum petals: anthocyanidin 3-O-glucoside-6″-O-malonyltransferase (Dm3MaT1), anthocyanidin 3-O-glucoside-3″, 6″-O-dimalonyltransferase (Dm3MaT2), and a homolog (Dm3MaT3). Mutational analyses revealed that seven amino acid residues in the N- and C-terminal regions are important for the differential acyl-acceptor specificity between Dm3MaT1 and Dm3MaT2. Crystallographic studies of Dm3MaT3 provided the first structure of a BAHD member, complexed with acyl-CoA, showing the detailed interactions between the enzyme and acyl-CoA molecules. The structure, combined with the results of mutational analyses, allowed us to identify the acyl-acceptor binding site of anthocyanin malonyltransferases, which is structurally different from the corresponding portion of vinorine synthase, another BAHD member, thus permitting the diversity of the acyl-acceptor specificity of BAHD family to be understood.

The Role and Structure of the Carboxyl-terminal Domain of the Human Voltage-gated Proton Channel Hv1

The voltage-gated proton channel Hv1 has a voltage sensor domain but lacks a pore domain. Although the C-terminal domain of Hv1 is known to be responsible for dimeric architecture of the channel, its role and structure are not known. We report that the full-length Hv1 is mainly localized in intracellular compartment membranes rather than the plasma membrane. Truncation of either the N or C terminus alone or both together revealed that the N-terminal deletion did not alter localization, but deletion of the C terminus either alone or together with the N terminus resulted in expression throughout the cell. These results indicate that the C terminus is essential for Hv1 localization but not the N terminus. In the 2.0 Å structure of the C-terminal domain, the two monomers form a dimer via a parallel α-helical coiled-coil, in which one chloride ion binds with the Nη atom of Arg264. A pH-dependent structural change of the protein has been observed, but it remains a dimer irrespective of pH value.

New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

Structural basis for substrate recognition and hydrolysis by mouse carnosinase CN2.

l-Carnosine is a bioactive dipeptide (β-alanyl-l-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of l-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn2+, and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn2+ at a resolution of 1.7Å and that with Mn2+ at 2.3Å. CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn2+ and Zn2+ complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes.

C-type Lectin-like Carbohydrate Recognition of the Hemolytic Lectin CEL-III Containing Ricin-type β-Trefoil Folds

CEL-III is a Ca2+-dependent hemolytic lectin, isolated from the marine invertebrate Cucumaria echinata. The three-dimensional structure of CEL-III/GalNAc and CEL-III/methyl α-galactoside complexes was solved by x-ray crystallographic analysis. In these complexes, five carbohydrate molecules were found to be bound to two carbohydrate-binding domains (domains 1 and 2) located in the N-terminal 2/3 portion of the polypeptide and that contained β-trefoil folds similar to ricin B-chain. The 3-OH and 4-OH of bound carbohydrate molecules were coordinated with Ca2+ located at the subdomains 1α, 1γ, 2α, 2β, and 2γ, simultaneously forming hydrogen bond networks with nearby amino acid side chains, which is similar to carbohydrate binding in C-type lectins. The binding of carbohydrates was further stabilized by aromatic amino acid residues, such as tyrosine and tryptophan, through a stacking interaction with the hydrophobic face of carbohydrates. The importance of amino acid residues in the carbohydrate-binding sites was confirmed by the mutational analyses. The orientation of bound GalNAc and methyl α-galactoside was similar to the galactose moiety of lactose bound to the carbohydrate-binding site of the ricin B-chain, although the ricin B-chain does not require Ca2+ ions for carbohydrate binding. The binding of the carbohydrates induced local structural changes in carbohydrate-binding sites in subdomains 2α and 2β. Binding of GalNAc also induced a slight change in the main chain structure of domain 3, which could be related to the conformational change upon binding of specific carbohydrates to induce oligomerization of the protein.

Hemolytic Lectin CEL-III Heptamerizes via a Large Structural Transition from α-Helices to a β-Barrel during the Transmembrane Pore Formation Process

Background: The hemolytic lectin CEL-III self-oligomerizes upon binding to cell surface carbohydrates to form transmembrane pores. Results: The CEL-III oligomer crystal structure revealed a membrane-inserted pore structure. Conclusion: CEL-III forms a heptamer containing a long β-barrel as the pore structure, resulting from large conformational changes. Significance: The heptameric structure of CEL-III provides insights into the action mechanisms of pore-forming proteins. CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer.

Covalent modification of reduced flavin mononucleotide in type-2 isopentenyl diphosphate isomerase by active-site-directed inhibitors

Evidence for an unusual catalysis of protonation/deprotonation by a reduced flavin mononucleotide cofactor is presented for type-2 isopentenyl diphosphate isomerase (IDI-2), which catalyzes isomerization of the two fundamental building blocks of isoprenoid biosynthesis, isopentenyl diphosphate and dimethylallyl diphosphate. The covalent adducts formed between irreversible mechanism-based inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor were investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of IDI-2 binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5, which had been proposed previously. In addition, the high-resolution crystal structures of IDI-2-substrate complexes and the kinetic studies of new mutants confirmed that only the flavin cofactor can catalyze protonation of the substrates and suggest that N5 of flavin is most likely to be involved in proton transfer. These data provide support for a mechanism where the reduced flavin cofactor acts as a general acid/base catalyst and helps stabilize the carbocationic intermediate formed by protonation.