Toyoyuki Ose

Efficient Leukocyte Ig-like Receptor Signaling and Crystal Structure of Disulfide-linked HLA-G Dimer

HLA-G is a nonclassical major histocompatibility complex class I (MHCI) molecule, which is expressed in trophoblasts and confers immunological tolerance in the maternal-fetal interface by binding to leukocyte Ig-like receptors (LILRs, also called as LIR/ILT/CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Interestingly, MHCI dimer formations have been involved in pathogenesis and T cell activation. The structure and receptor binding characteristics of MHCI dimers have never been evaluated. Here we performed binding studies showing that the HLA-G dimer exhibited higher overall affinity to LILRB1/2 than the monomer by significant avidity effects. Furthermore, the cell reporter assay demonstrated that the dimer formation remarkably enhanced the LILRB1-mediated signaling at the cellular level. We further determined the crystal structure of the wild-type dimer of HLA-G with the intermolecular Cys42-Cys42 disulfide bond. This dimer structure showed the oblique configuration to expose two LILR/CD8-binding sites upward from the membrane easily accessible for receptors, providing plausible 1:2 (HLA-G dimer:receptors) complex models. These results indicated that the HLA-G dimer conferred increased avidity in a proper structural orientation to induce efficient LILR signaling, resulting in the dominant immunosuppressive effects. Moreover, structural and functional implications for other MHCI dimers observed in activated T cells and the pathogenic allele, HLA-B27, are discussed.

Structure of the measles virus hemagglutinin bound to its cellular receptor SLAM

Measles virus, a major cause of childhood morbidity and mortality worldwide, predominantly infects immune cells using signaling lymphocyte activation molecule (SLAM) as a cellular receptor. Here we present crystal structures of measles virus hemagglutinin (MV-H), the receptor-binding glycoprotein, in complex with SLAM. The MV-H head domain binds to a β-sheet of the membrane-distal ectodomain of SLAM using the side of its β-propeller fold. This is distinct from attachment proteins of other paramyxoviruses that bind receptors using the top of their β-propeller. The structure provides templates for antiviral drug design, an explanation for the effectiveness of the measles virus vaccine, and a model of the homophilic SLAM-SLAM interaction involved in immune modulations. Notably, the crystal structures obtained show two forms of the MV-H–SLAM tetrameric assembly (dimer of dimers), which may have implications for the mechanism of fusion triggering.

Insight into a natural Diels-Alder reaction from the structure of macrophomate synthase.

The Diels–Alder reaction, which forms a six-membered ring from an alkene (dienophile) and a 1,3-diene, is synthetically very useful for construction of cyclic products with high regio- and stereoselectivity under mild conditions. It has been applied to the synthesis of complex pharmaceutical and biologically active compounds. Although evidence on natural Diels–Alderases has been accumulated in the biosynthesis of secondary metabolites, there has been no report on the structural details of the natural Diels–Alderases. The function and catalytic mechanism of the natural Diels–Alderase are of great interest owing to the diversity of molecular skeletons in natural Diels–Alder adducts. Here we present the 1.70 Å resolution crystal structure of the natural Diels–Alderase, fungal macrophomate synthase (MPS), in complex with pyruvate. The active site of the enzyme is large and hydrophobic, contributing amino acid residues that can hydrogen-bond to the substrate 2-pyrone. These data provide information on the catalytic mechanism of MPS, and suggest that the reaction proceeds via a large-scale structural reorganization of the product.

Tom20 recognizes mitochondrial presequences through dynamic equilibrium among multiple bound states.

Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N‐terminal presequences of mitochondrial‐precursor proteins contain a diverse consensus motif (ϕχχϕϕ, ϕ is hydrophobic and χ is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20–presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR 15N relaxation analyses suggested motion on a sub‐millisecond timescale at the Tom20–presequence interface. We suggest that the dynamic, multiple‐mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.

Structural analysis for glycolipid recognition by the C-type lectins Mincle and MCL.

Significance Here we report the crystal structures of human C-type lectin receptors Mincle (macrophage inducible C-type “calcium-dependent” lectin; CLEC4E) and MCL (macrophage C-type lectin; CLEC4D), both of which are receptors for mycobacterial glycolipid adjuvant cord factor (also called trehalose-6,6′-dimycolate; TDM). Our structural and functional studies clearly reveal the simultaneous recognition of sugar and lipid moieties by Mincle and MCL, distinct from other C-type lectin receptors. Because better adjuvants are desired for enhancing vaccination effects of medical treatments for infectious diseases, cancer, and so forth, these structures provide a framework for the rational design of more effective adjuvants than TDM. Mincle [macrophage inducible Ca2+-dependent (C-type) lectin; CLEC4E] and MCL (macrophage C-type lectin; CLEC4D) are receptors for the cord factor TDM (trehalose-6,6′-dimycolate), a unique glycolipid of mycobacterial cell-surface components, and activate immune cells to confer adjuvant activity. Although it is known that receptor–TDM interactions require both sugar and lipid moieties of TDM, the mechanisms of glycolipid recognition by Mincle and MCL remain unclear. We here report the crystal structures of Mincle, MCL, and the Mincle–citric acid complex. The structures revealed that these receptors are capable of interacting with sugar in a Ca2+-dependent manner, as observed in other C-type lectins. However, Mincle and MCL uniquely possess shallow hydrophobic regions found adjacent to their putative sugar binding sites, which reasonably locate for recognition of fatty acid moieties of glycolipids. Functional studies using mutant receptors as well as glycolipid ligands support this deduced binding mode. These results give insight into the molecular mechanism of glycolipid recognition through C-type lectin receptors, which may provide clues to rational design for effective adjuvants.

Structural basis for mRNA recognition by elongation factor SelB.

In bacteria, incorporation of selenocysteine, the 21st amino acid, into proteins requires elongation factor SelB, which has the unusual property of binding to both transfer RNA (tRNA) and mRNA. SelB binds to an mRNA hairpin formed by the selenocysteine insertion sequence (SECIS) with extremely high specificity, the molecular basis of which has been unknown. We have determined the crystal structure of the mRNA-binding domain of SelB in complex with SECIS RNA at a resolution of 2.3 Å. This is the first example of a complex between an RNA and a winged-helix (WH) domain, a motif found in many DNA-binding proteins and recently discovered in RNA-binding proteins. Notably, RNA binding does not induce a major conformational change in the WH motif. The structure reveals a new mode of RNA recognition with a geometry that allows the complex to wrap around the small ribosomal subunit.

Crystal Structure of 1-Aminocyclopropane-1-carboxylate Deaminase from Hansenula saturnus

The pyridoxal 5′-phosphate (PLP)-dependent enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) catalyzes a reaction that involves a ring opening of cyclopropanoid amino acid, yielding α-ketobutyrate and ammonia. Unlike other PLP-dependent enzymes, this enzyme has no α-hydrogen atom in the substrate. Thus, a unique mechanism for the bond cleavage is expected. The crystal structure of ACCD fromHansenula saturnus has been determined at 2.0 Å resolution by the multiple wavelength anomalous diffraction method using mercury atoms as anomalous scatterers. The model was built on the electron density map, which was obtained by the density averaging of multiple crystal forms. The final model was refined to an R-factor of 22.5% and an R free-factor of 26.8%. The ACCD folds into two domains, each of which has an open twisted α/β structure similar to the β-subunit of tryptophan synthase. However, in ACCD, unlike in other members of the β family of PLP-dependent enzymes, PLP is buried deep in the molecule. The structure provides the first view of the catalytic center of the cyclopropane ring opening.

Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis

Roquin is an RNA-binding protein that prevents autoimmunity and inflammation via repression of bound target mRNAs such as inducible costimulator (Icos). When Roquin is absent or mutated (Roquinsan), Icos is overexpressed in T cells. Here we show that Roquin enhances Dicer-mediated processing of pre-miR-146a. Roquin also directly binds Argonaute2, a central component of the RNA-induced silencing complex, and miR-146a, a microRNA that targets Icos mRNA. In the absence of functional Roquin, miR-146a accumulates in T cells. Its accumulation is not due to increased transcription or processing, rather due to enhanced stability of mature miR-146a. This is associated with decreased 3′ end uridylation of the miRNA. Crystallographic studies reveal that Roquin contains a unique HEPN domain and identify the structural basis of the ‘san’ mutation and Roquin’s ability to bind multiple RNAs. Roquin emerges as a protein that can bind Ago2, miRNAs and target mRNAs, to control homeostasis of both RNA species.

Structural basis of the 3′-end recognition of a leading strand in stalled replication forks by PriA

In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3′ end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N‐terminal domain of PriA possesses a binding pocket for the 3′‐terminal nucleotide residue of DNA. The interaction with the deoxyribose 3′‐OH is essential for the 3′‐terminal recognition. In contrast, the direct interaction with 3′‐end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3′ end. Thus, the N‐terminal domain of PriA recognizes the 3′‐end base in a base‐non‐selective manner, in addition to the deoxyribose and 5′‐side phosphodiester group, of the 3′‐terminal nucleotide to acquire both sufficient affinity and non‐selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double‐stranded DNA.

The structure of alanyl-tRNA synthetase with editing domain

Alanyl-tRNA synthetase (AlaRS) catalyzes synthesis of Ala-tRNAAla and hydrolysis of mis-acylated Ser- and Gly-tRNAAla at 2 different catalytic sites. Here, we describe the monomer structures of C-terminal truncated archaeal AlaRS, with both activation and editing domains in the apo form, in complex with an Ala-AMP analog, and in a high-resolution lysine-methylated form. The structures show docking of the editing domain to the activation domain opposite from the predicted tRNA-binding surface. Thus, the editing site is positioned >35 Å from the activation site, prompting us to model 2 different tRNA complexes: one binding tRNA at the activation site, and the other binding tRNA at the editing site. Interestingly, a gel-shift assay also implies the presence of 2 types of tRNA complex with different mobility. These results suggest that tRNA translocation via a canonical CCA flipping is unlikely to occur in AlaRS. The structure also demonstrated the binding of zinc in the editing site, in which the specific coordination of zinc would be facilitated by a conserved GGQ motif, implying that the editing mechanism may not be the same as in ThrRS. As Asn-194 in eubacterial AlaRS important for Ser misactivation is replaced by Thr-213 in archaeal AlaRS, a different Ser accommodation mechanism is proposed.