I03-Macromolecular Crystallography
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Kunimichi
Suzuki
,
Jonathan
Elegheert
,
Inseon
Song
,
Hiroyuki
Sasakura
,
Oleg
Senkov
,
Keiko
Matsuda
,
Wataru
Kakegawa
,
Veronica T.
Chang
,
Maura
Ferrer-ferrer
,
Eriko
Miura
,
Rahul
Kaushik
,
Masashi
Ikeno
,
Yuki
Morioka
,
Yuka
Takeuchi
,
Tatsuya
Shimada
,
Shintaro
Otsuka
,
Stoyan
Stoyanov
,
Masahiko
Watanabe
,
Kosei
Takeuchi
,
Alexander
Dityatev
,
A. Radu
Aricescu
,
Michisuke
Yuzaki
Open Access
Abstract: Neuronal synapses undergo structural and functional changes throughout life, which are essential for nervous system physiology. However, these changes may also perturb the excitatory–inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we designed and characterized CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1. CPTX can interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors and induced the formation of excitatory synapses both in vitro and in vivo. CPTX restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimer’s disease, and spinal cord injury, respectively. Thus, CPTX represents a prototype for structure-guided biologics that can efficiently repair or remodel neuronal circuits.
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Aug 2020
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I04-Macromolecular Crystallography
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Wataru
Kakegawa
,
Nikolaos
Mitakidis
,
Eriko
Miura
,
Manabu
Abe
,
Keiko
Matsuda
,
Yukari h.
Takeo
,
Kazuhisa
Kohda
,
Junko
Motohashi
,
Akiyo
Takahashi
,
Soichi
Nagao
,
Shin-ichi
Muramatsu
,
Masahiko
Watanabe
,
Kenji
Sakimura
,
Alexandru
Aricescu
,
Michisuke
Yuzaki
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Kakegawa et al. demonstrate that C1ql1 provided by climbing fibers (CFs) serves as a new antegrade signal to determine and maintain a single-winner CF throughout development and adulthood by binding to its postsynaptic receptor Bai3 in cerebellar Purkinje cells.
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Jan 2015
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I02-Macromolecular Crystallography
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Abstract: Two members of the paramyxovirus family, Nipah virus (NiV) and Hendra virus (HeV), are recent additions to a growing number of agents of emergent diseases which use bats as a natural host. Identification of ephrin-B2 and ephrin-B3 as cellular receptors for these viruses has enabled the development of immunotherapeutic reagents which prevent virus attachment and subsequent fusion. Here we present the structural analysis of the protein and carbohydrate components of the unbound viral attachment glycoprotein of NiV glycoprotein (NiV-G) at a 2.2-Å resolution. Comparison with its ephrin-B2-bound form reveals that conformational changes within the envelope glycoprotein are required to achieve viral attachment. Structural differences are particularly pronounced in the 579-590 loop, a major component of the ephrin binding surface. In addition, the 236-245 loop is rather disordered in the unbound structure. We extend our structural characterization of NiV-G with mass spectrometric analysis of the carbohydrate moieties. We demonstrate that NiV-G is largely devoid of the oligomannose-type glycans that in viruses such as human immunodeficiency virus type 1 and Ebola virus influence viral tropism and the host immune response. Nevertheless, we find putative ligands for the endothelial cell lectin, LSECtin. Finally, by mapping structural conservation and glycosylation site positions from other members of the paramyxovirus family, we suggest the molecular surface involved in oligomerization. These results suggest possible pathways of virus-host interaction and strategies for the optimization of recombinant vaccines.
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Nov 2008
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[8423]
Open Access
Abstract: Type-A ?-aminobutyric acid receptors (GABAARs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in GABAAR signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a GABAAR, the human ?3 homopentamer, at 3?Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows an unexpected structural role for a conserved N-linked glycan. The receptor was crystallized bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of GABAAR modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitized state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission.
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Aug 2014
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Krios I-Titan Krios I at Diamond
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Simonas
Masiulis
,
Rooma
Desai
,
Tomasz
Uchanski
,
Itziar
Serna Martin
,
Duncan
Laverty
,
Dimple
Karia
,
Tomas
Malinauskas
,
Jasenko
Zivanov
,
Els
Pardon
,
Abhay
Kotecha
,
Jan
Steyaert
,
Keith W.
Miller
,
A. Radu
Aricescu
Abstract: Type-A γ-aminobutyric (GABAA) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABAA receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1β3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABAA receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABAA receptor modulators.
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Jan 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Veronica T
Chang
,
Ricardo A
Fernandes
,
Kristina A
Ganzinger
,
Steven F
Lee
,
Christian
Siebold
,
James
Mccoll
,
Peter
Jönsson
,
Matthieu
Palayret
,
Karl
Harlos
,
Charlotte H
Coles
,
Edith
Jones
,
Yuan
Lui
,
Elizabeth
Huang
,
Robert J C
Gilbert
,
David
Klenerman
,
A Radu
Aricescu
,
Simon J
Davis
Abstract: It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.
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Mar 2016
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Tomasz
Uchański
,
Simonas
Masiulis
,
Baptiste
Fischer
,
Valentina
Kalichuk
,
Uriel
López-sánchez
,
Eleftherios
Zarkadas
,
Miriam
Weckener
,
Andrija
Sente
,
Philip
Ward
,
Alexandre
Wohlkonig
,
Thomas
Zogg
,
Han
Remaut
,
James
Naismith
,
Hugues
Nury
,
Wim
Vranken
,
A. Radu
Aricescu
,
Els
Pardon
,
Jan
Steyaert
Abstract: Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water–air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.
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Jan 2021
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Abstract: Wnt morphogens control embryonic development and homeostasis in adult tissues. In vertebrates the N-terminal WIF domain (WIF-1(WD)) of Wnt inhibitory factor 1 (WIF-1) binds Wnt ligands. Our crystal structure of WIF-1(WD) reveals a previously unidentified binding site for phospholipid; two acyl chains extend deep into the domain, and the head group is exposed to the surface. Biophysical and cellular assays indicate that there is a WIF-1(WD) Wnt-binding surface proximal to the lipid head group but also implicate the five epidermal growth factor (EGF)-like domains (EGFs I-V) in Wnt binding. The six-domain WIF-1 crystal structure shows that EGFs I-V are wrapped back, interfacing with WIF-1(WD) at EGF III. EGFs II-V contain a heparan sulfate proteoglycan (HSPG)-binding site, consistent with conserved positively charged residues on EGF IV. This combination of HSPG- and Wnt-binding properties suggests a modular model for the localization of WIF-1 and for signal inhibition within morphogen gradients.
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Dec 2011
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Abstract: Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) regulate numerous cell surface signaling events, with typically opposite effects on cell function. CSPGs inhibit nerve regeneration through receptor protein tyrosine phosphatase sigma (RPTPσ). Here we report that RPTPσ acts bimodally in sensory neuron extension, mediating CSPG inhibition and HSPG growth promotion. Crystallographic analyses of a shared HSPG-CSPG binding site reveal a conformational plasticity that can accommodate diverse glycosaminoglycans with comparable affinities. Heparan sulfate and analogs induced RPTPσ ectodomain oligomerization in solution, which was inhibited by chondroitin sulfate. RPTPσ and HSPGs colocalize in puncta on sensory neurons in culture, whereas CSPGs occupy the extracellular matrix. These results lead to a model where proteoglycans can exert opposing effects on neuronal extension by competing to control the oligomerization of a common receptor.
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Dec 2011
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Jonathan
Elegheert
,
Vedrana
Cvetkovska
,
Amber J.
Clayton
,
Christina
Heroven
,
Kristel M.
Vennekens
,
Samuel N.
Smukowski
,
Michael C.
Regan
,
Wanyi
Jia
,
Alexandra C.
Smith
,
Hiro
Furukawa
,
Jeffrey N.
Savas
,
Joris
De Wit
,
Jo
Begbie
,
Ann Marie
Craig
,
Alexandru
Aricescu
Diamond Proposal Number(s):
[8423]
Open Access
Abstract: Neuroligin-neurexin (NL-NRX) complexes are fundamental synaptic organizers in the central nervous system. An accurate spatial and temporal control of NL-NRX signaling is crucial to balance excitatory and inhibitory neurotransmission, and perturbations are linked with neurodevelopmental and psychiatric disorders. MDGA proteins bind NLs and control their function and interaction with NRXs via unknown mechanisms. Here, we report crystal structures of MDGA1, the NL1-MDGA1 complex, and a spliced NL1 isoform. Two large, multi-domain MDGA molecules fold into rigid triangular structures, cradling a dimeric NL to prevent NRX binding. Structural analyses guided the discovery of a broad, splicing-modulated interaction network between MDGA and NL family members and helped rationalize the impact of autism-linked mutations. We demonstrate that expression levels largely determine whether MDGAs act selectively or suppress the synapse organizing function of multiple NLs. These results illustrate a potentially brain-wide regulatory mechanism for NL-NRX signaling modulation.
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Aug 2017
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