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

Inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca(2+) 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. InsP(3)R gating is initiated by InsP(3) binding to the InsP(3)-binding core (IBC, residues 224-604 of InsP(3)R1) and it requires the suppressor domain (SD, residues 1-223 of InsP(3)R1). Here we present structures of the amino-terminal region (NT, residues 1-604) of rat InsP(3)R1 with (3.6 Å) and without (3.0 Å) InsP(3) 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 InsP(3)R and of the ABC domains docked into RyR are remarkably similar. The importance of the α-interface for activation of InsP(3)R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations. Binding of InsP(3) 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 InsP(3)R 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 InsP(3)R, and an InsP(3)R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP(3) and blocked by ryanodine. Activation mechanisms are conserved between InsP(3)R 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

Force-dependent allostery of the α-catenin actinbinding domain controls adherens junction dynamics and functions

α-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which α-catenin engages F-actin under tension remained elusive. Here we show that the α1-helix of the α-catenin actin-binding domain (αcat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. αcat-ABD containing an α1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length α-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that α1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of αcat-ABD-H1 homodimer suggest that α-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of αcat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis

多重ダイアコプティックスの説明例

　先に著者の一人或いは両者で単純化且つ双対化したダイアコプティックス及びコダイアコプティックスなるものを山大紀要第4巻第2号等に発表したが更に複雑な回路網ではこの両者を交互にほどこすことによって一層便利な手段を提供することが解ったので，これを”多重ダイアコプティックス”と称することにした。この方法で最も有効なのは”二重ダイアコプティックス”で，ダイアコプティックス→コダイアコプティックス又はコダイアコプティックス→ダイアコプテイィックスの組合せである。本文では前者について説明例をあげてその計算過程を明かにした

Structural studies of the O antigen biosynthetic enzymes from Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that can cause a chronic lung infection in individuals with cystic fibrosis. The O antigen of lipopolysaccharide is a major virulence factor for Gram-negative bacteria and plays a critical role in bacterium-host interactions. The inhibition of O antigen biosynthesis is known to significantly reduce the virulence of P. aeruginosa. The O antigen biosynthetic enzymes are considered as promising novel targets to develop new antimicrobials to treat a P. aeruginosa infection.WbpP and WbpM are two UDP-GlcNAc-modifying enzymes that are involved in the initial steps of the O-antigen biosynthesis in P. aeruginosa serotype 06. WbpP is a UDP-GlcNAc 4-epimerase that preferentially catalyzes the conversion of acetylated substrates over non-acetylated substrates, such as UDP-Glc. In contrast, WbpM is a membrane protein with a catalytic domain that is capable of UDP-GlcNAc 4,6-dehydratase activity. The structural and biological studies were pursued to gain insight into the mechanism of action for WbpP and WbpM. Specifically for WbpP, the crystal structures of this enzyme in complex with cofactor and either UDP-Glc or UDP-GalNAc were determined at 2.5 and 2.1 A, respectively. These represent the first structural studies of a genuine UDP-GlcNAc 4-epimerase. For WbpM, crystallographic studies proved difficult. Hence, the crystal structures of FlaA1, a close soluble relative from Helicobacter pylori, were determined in the presence of substrate, inhibitors and bound cofactor, with resolutions ranging from 2.8-1.9 A. These represent the first structural studies of a 4,6-dehydratase that can catalyze a UDP-hexose. Third, the crystal structures of the Y141M FlaA1 mutant were determined in oxidized and reduced forms at 2.2 and 2.1 A, respectively, to study the structure of an unusual T-M-K triad found in WbpM. Finally, a homology model of the catalytic domain of WbpM was built based on the structures of FlaA1 wild type and mutant to unveil its unique structural features.In conclusion, the work described in this thesis gives insight into the structures and reaction mechanisms of WbpP and WbpM and provide a basis for future studies aimed at understanding the O antigen biosynthesis of P. aeruginosa

双対ダイアコプテイクス

　最近G. Kronによって紹介された“ダイアコブテイクス”が複雑な電気回路網の解析に極めて有効であることが認められている[Kdk, Kic, Kmp及びKpi]。更に特殊な条件を持つ回路網では一層簡単化された方法も紹介されている[Wsd及びTse]。それらの方法を要約すれば次のような段階に分けることができる。　(i) 　与えられた回路網を数個の部分回路網に分割する。　(ii) 各部分回路網を各個独立に解き等価部分回路網を作る。　(iii)　等価部分回路網を組合せて与えられた回路網の等価回路網を作る。　(iv) 等価回路網の解析によって与えられた回路網の問題を解く。　(i)の段階は切断という操作で行われる。この切断には開放操作と短絡操作の二者を考えることができるが未だ後者については紹介されていない，更に(ii)の段階では等価部分回路網が作られるのであるが，これには端子型と閉路型の二者を考えることができ未だ後者の利用については紹介されていない。(iii)の操作も(i),(ii)の段階に従って導かれる。　(iv)では等価回路網の未知数選たくに点電位と閉路電流の二者を考えることができるが夫夫上の段階に従って便利なるものを使用することが望ましい。　　従つて著者はまずKronの方法を単純化し，更に種々の電源の存在する一般回路網に拡張した場合のダイアコプテイクスを紹介し，上述の工夫のもとに“双対ダイアコプテイクス”を新しく提案しようとするものである。その優劣については全く与えられた回路網がいずれの型に属するかによって定まる

Backbone resonance assignments of the F-actin binding domain of mouse αN-catenin.

α-Catenin is a filamentous actin (F-actin) binding protein that links the classical cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). Its C-terminal F-actin binding domain is required for regulating the dynamic interaction between AJs and the actin cytoskeleton during tissue development. Thus, obtaining the molecular details of this interaction is a crucial step towards understanding how α-catenin plays critical roles in biological processes, such as morphogenesis, cell polarity, wound healing and tissue maintenance. Here we report the backbone atom (H, N, C, C and C') resonance assignments of the C-terminal F-actin binding domain of αN-catenin

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

AbstractThe 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

Exploring the evolution and function of Canoe's intrinsically disordered region in linking cell-cell junctions to the cytoskeleton during embryonic morphogenesis.

One central question for cell and developmental biologists is defining how epithelial cells can change shape and move during embryonic development without tearing tissues apart. This requires robust yet dynamic connections of cells to one another, via the cell-cell adherens junction, and of junctions to the actin and myosin cytoskeleton, which generates force. The last decade revealed that these connections involve a multivalent network of proteins, rather than a simple linear pathway. We focus on Drosophila Canoe, homolog of mammalian Afadin, as a model for defining the underlying mechanisms. Canoe and Afadin are complex, multidomain proteins that share multiple domains with defined and undefined binding partners. Both also share a long carboxy-terminal intrinsically disordered region (IDR), whose function is less well defined. IDRs are found in many proteins assembled into large multiprotein complexes. We have combined bioinformatic analysis and the use of a series of canoe mutants with early stop codons to explore the evolution and function of the IDR. Our bioinformatic analysis reveals that the IDRs of Canoe and Afadin differ dramatically in sequence and sequence properties. When we looked over shorter evolutionary time scales, we identified multiple conserved motifs. Some of these are predicted by AlphaFold to be alpha-helical, and two correspond to known protein interaction sites for alpha-catenin and F-actin. We next identified the lesions in a series of eighteen canoe mutants, which have early stop codons across the entire protein coding sequence. Analysis of their phenotypes are consistent with the idea that the IDR, including the conserved motifs in the IDR, are critical for protein function. These data provide the foundation for further analysis of IDR function