Noboru Ishiyama

Structural and functional conservation of key domains in InsP3 and ryanodine receptors.

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca2+ channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP3R gating is initiated by InsP3 binding to the InsP3-binding core (IBC, residues 224–604 of InsP3R1) and it requires the suppressor domain (SD, residues 1–223 of InsP3R1). Here we present structures of the amino-terminal region (NT, residues 1–604) of rat InsP3R1 with (3.6 Å) and without (3.0 Å) InsP3 bound. The arrangement of the three NT domains, SD, IBC-β and IBC-α, identifies two discrete interfaces (α and β) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of InsP3R and of the ABC domains docked into RyR are remarkably similar. The importance of the α-interface for activation of InsP3R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations. Binding of InsP3 causes partial closure of the clam-like IBC, disrupting the β-interface and pulling the SD towards the IBC. This reorients an exposed SD loop (‘hotspot’ (HS) loop) that is essential for InsP3R activation. The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease. The HS loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A domain of RyR functionally replaced the SD in full-length InsP3R, and an InsP3R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP3 and blocked by ryanodine. Activation mechanisms are conserved between InsP3R and RyR. Allosteric modulation of two similar domain interfaces within an N-terminal subunit reorients the first domain (SD or A domain), allowing it, through interactions of the second domain of an adjacent subunit (IBC-β or B domain), to gate the pore.

p120-catenin binding masks an endocytic signal conserved in classical cadherins.

p120 regulates adhesive junction dynamics through binding to a dual-function motif in classical cadherins that alternately serves as a p120-binding interface and an endocytic signal.

Monomeric α-catenin links cadherin to the actin cytoskeleton.

The linkage of adherens junctions to the actin cytoskeleton is essential for cell adhesion. The contribution of the cadherin–catenin complex to the interaction between actin and the adherens junction remains an intensely investigated subject that centres on the function of α-catenin, which binds to cadherin through β-catenin and can bind F-actin directly or indirectly. Here, we delineate regions within Drosophila α-Catenin (α-Cat) that are important for adherens junction performance in static epithelia and dynamic morphogenetic processes. Moreover, we address whether persistent α-catenin-mediated physical linkage between cadherin and F-actin is crucial for cell adhesion and characterize the functions of α-catenin monomers and dimers at adherens junctions. Our data support the view that monomeric α-catenin acts as an essential physical linker between the cadherin–β-catenin complex and the actin cytoskeleton, whereas α-catenin dimers are cytoplasmic and form an equilibrium with monomeric junctional α-catenin.

Crystal structure of type I ryanodine receptor amino-terminal beta-trefoil domain reveals a disease-associated mutation "hot spot" loop.

Muscle contraction and relaxation is regulated by transient elevations of myoplasmic Ca2+. Ca2+ is released from stores in the lumen of the sarco(endo)plasmic reticulum (SER) to initiate formation of the Ca2+ transient by activation of a class of Ca2+ release channels referred to as ryanodine receptors (RyRs) and is pumped back into the SER lumen by Ca2+-ATPases (SERCAs) to terminate the Ca2+ transient. Mutations in the type 1 ryanodine receptor gene, RYR1, are associated with 2 skeletal muscle disorders, malignant hyperthermia (MH), and central core disease (CCD). The evaluation of proposed mechanisms by which RyR1 mutations cause MH and CCD is hindered by the lack of high-resolution structural information. Here, we report the crystal structure of the N-terminal 210 residues of RyR1 (RyRNTD) at 2.5 Å. The RyRNTD structure is similar to that of the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor (IP3Rsup), but lacks most of the long helix-turn-helix segment of the “arm” domain in IP3Rsup. The N-terminal β-trefoil fold, found in both RyR and IP3R, is likely to play a critical role in regulatory mechanisms in this channel family. A disease-associated mutation “hot spot” loop was identified between strands 8 and 9 in a highly basic region of RyR1. Biophysical studies showed that 3 MH-associated mutations (C36R, R164C, and R178C) do not adversely affect the global stability or fold of RyRNTD, supporting previously described mechanisms whereby mutations perturb protein–protein interactions.

An Autoinhibited Structure of α-catenin and Its Implications for Vinculin Recruitment to Adherens Junctions

Background: α-Catenin is an actin-binding protein that recruits vinculin to adherens junctions. Results: An elongated autoinhibited structure of α-catenin indicates structural and functional coupling of its vinculin- and actin-binding mechanisms. Conclusion: The anchoring strength of adherens junctions is dynamically regulated by α-catenin to match the actomyosin-generated tension. Significance: Multistate conformations of α-catenin facilitate the direct and vinculin-assisted linkages between the cadherin-catenin complex and the actin cytoskeleton. α-Catenin is an actin- and vinculin-binding protein that regulates cell-cell adhesion by interacting with cadherin adhesion receptors through β-catenin, but the mechanisms by which it anchors the cadherin-catenin complex to the actin cytoskeleton at adherens junctions remain unclear. Here we determined crystal structures of αE-catenin in the autoinhibited state and the actin-binding domain of αN-catenin. Together with the small-angle x-ray scattering analysis of full-length αN-catenin, we deduced an elongated multidomain assembly of monomeric α-catenin that structurally and functionally couples the vinculin- and actin-binding mechanisms. Cellular and biochemical studies of αE- and αN-catenins show that αE-catenin recruits vinculin to adherens junctions more effectively than αN-catenin, partly because of its higher affinity for actin filaments. We propose a molecular switch mechanism involving multistate conformational changes of α-catenin. This would be driven by actomyosin-generated tension to dynamically regulate the vinculin-assisted linkage between adherens junctions and the actin cytoskeleton.

Structural Studies of Inositol 1,4,5-Trisphosphate Receptor: COUPLING LIGAND BINDING TO CHANNEL GATING*

The three isoforms of the inositol 1,4,5-trisphosphate receptor (IP3R) exhibit distinct IP3 sensitivities and cooperativities in calcium (Ca2+) channel function. The determinants underlying this isoform-specific channel gating mechanism have been localized to the N-terminal suppressor region of IP3R. We determined the 1.9 Å crystal structure of the suppressor domain from type 3 IP3R (IP3R3SUP, amino acids 1–224) and revealed structural features contributing to isoform-specific functionality of IP3R by comparing it with our previously determined structure of the type 1 suppressor domain (IP3R1SUP). The molecular surface known to associate with the ligand binding domain (amino acids 224–604) showed marked differences between IP3R3SUP and IP3R1SUP. Our NMR and biochemical studies showed that three spatially clustered residues (Glu-20, Tyr-167, and Ser-217 in IP3R1 and Glu-19, Trp-168, and Ser-218 in IP3R3) within the N-terminal suppressor domains of IP3R1SUP and IP3R3SUP interact directly with their respective C-terminal fragments. Together with the accompanying paper (Yamazaki, H., Chan, J., Ikura, M., Michikawa, T., and Mikoshiba, K. (2010) J. Biol. Chem. 285, 36081–36091), we demonstrate that the single aromatic residue in this region (Tyr-167 in IP3R1 and Trp-168 in IP3R3) plays a critical role in the coupling between ligand binding and channel gating.

Structural Studies of FlaA1 from Helicobacter pylori Reveal the Mechanism for Inverting 4,6-Dehydratase Activity.

FlaA1 from the human pathogen Helicobacter pylori is an enzyme involved in saccharide biosynthesis that has been shown to be essential for pathogenicity. Here we present five crystal structures of FlaA1 in the presence of substrate, inhibitors, and bound cofactor, with resolutions ranging from 2.8 to 1.9 Å. These structures reveal that the enzyme is a novel member of the short-chain dehydrogenase/reductase superfamily. Additional electron microscopy studies show the enzyme to possess a hexameric doughnut-shaped quaternary structure. NMR analyses of “real time” enzyme-substrate reactions indicate that FlaA1 is a UDP-GlcNAc-inverting 4,6-dehydratase, suggesting that the enzyme catalyzes the first step in the biosynthetic pathway of a pseudaminic acid derivative, which is implicated in protein glycosylation. Guided by evidence from site-directed mutagenesis and computational simulations, a three-step reaction mechanism is proposed that involves Lys-133 functioning as both a catalytic acid and base.

Structural determination of the phosphorylation domain of the ryanodine receptor

The ryanodine receptor (RyR) is a large, homotetrameric sarcoplasmic reticulum membrane protein that is essential for Ca2+ cycling in both skeletal and cardiac muscle. Genetic mutations in RyR1 are associated with severe conditions including malignant hyperthermia (MH) and central core disease. One phosphorylation site (Ser 2843) has been identified in a segment of RyR1 flanked by two RyR motifs, which are found exclusively in all RyR isoforms as closely associated tandem (or paired) motifs, and are named after the protein itself. These motifs also contain six known MH mutations. In this study, we designed, expressed and purified the tandem RyR motifs, and show that this domain contains a putative binding site for the Ca2+/calmodulin‐dependent protein kinase β isoform. We present a 2.2 Å resolution crystal structure of the RyR domain revealing a two‐fold, symmetric, extended four‐helix bundle stabilized by a β sheet. Using mathematical modelling, we fit our crystal structure within a tetrameric electron microscopy (EM) structure of native RyR1, and propose that this domain is localized in the RyR clamp region, which is absent in its cousin protein inositol 1,4,5‐trisphosphate receptor.

Flagellin Glycosylation in Pseudomonas aeruginosa PAK Requires the O-antigen Biosynthesis Enzyme WbpO *□

Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-d-glucosamine to UDP-N-acetyl-d-glucuronic acid prior to the conversion to UDP-N-acetyl-d-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.

Structural insights into endoplasmic reticulum stored calcium regulation by inositol 1,4,5-trisphosphate and ryanodine receptors

The two major calcium (Ca²⁺) release channels on the sarco/endoplasmic reticulum (SR/ER) are inositol 1,4,5-trisphosphate and ryanodine receptors (IP3Rs and RyRs). They play versatile roles in essential cell signaling processes, and abnormalities of these channels are associated with a variety of diseases. Structural information on IP3Rs and RyRs determined using multiple techniques including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (EM), has significantly advanced our understanding of the mechanisms by which these Ca²⁺ release channels function under normal and pathophysiological circumstances. In this review, structural advances on the understanding of the mechanisms of IP3R and RyR function and dysfunction are summarized. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.