B21-High Throughput SAXS
I03-Macromolecular Crystallography
Krios IV-Titan Krios IV at Diamond
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Ross A.
Robinson
,
Samuel C.
Griffiths
,
Lieke L.
Van De Haar
,
Tomas
Malinauskas
,
Eljo Y.
Van Battum
,
Pavol
Zelina
,
Rebekka A.
Schwab
,
Dimple
Karia
,
Lina
Malinauskaite
,
Sara
Brignani
,
Marleen H.
Van Den Munkhof
,
Özge
Düdükcü
,
Anna A.
De Ruiter
,
Dianne M.a.
Van Den Heuvel
,
Benjamin
Bishop
,
Jonathan
Elegheert
,
A. Radu
Aricescu
,
R. Jeroen
Pasterkamp
,
Christian
Siebold
Diamond Proposal Number(s):
[19946, 20223]
Open Access
Abstract: During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a “trimer-of-trimers” super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.
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Mar 2021
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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
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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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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-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Abstract: Type A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of inhibitory neurotransmission in the human brain. Endogenous neurosteroids interact with GABAARs to regulate acute and chronic anxiety and are potent sedative, analgesic, anticonvulsant and anesthetic agents. Their mode of binding and mechanism of receptor potentiation, however, remain unknown. Here we report crystal structures of a chimeric GABAAR construct in apo and pregnanolone-bound states. The neurosteroid-binding site is mechanically coupled to the helices lining the ion channel pore and modulates the desensitization-gate conformation. We demonstrate that the equivalent site is responsible for physiological, heteromeric GABAAR potentiation and explain the contrasting modulatory properties of 3a versus 3b neurosteroid epimers. These results illustrate how peripheral lipid ligands can regulate the desensitization gate of GABAARs, a process of broad relevance to pentameric ligand-gated ion channels.
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Oct 2017
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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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Jonathan
Elegheert
,
W.
Kakegawa
,
J.
Clay
,
N. F.
Shanks
,
E.
Behiels
,
K.
Matsuda
,
K.
Kohda
,
E.
Miura
,
M.
Rossmann
,
Nikolaos
Mitakidis
,
J.
Motohashi
,
Veronica T.
Chang
,
Christian
Siebold
,
Ingo H.
Greger
,
Terunaga
Nakagawa
,
M.
Yuzaki
,
A. Radu
Aricescu
Diamond Proposal Number(s):
[8423, 10627]
Abstract: Ionotropic glutamate receptor (iGluR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGluR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGluR δ2 (GluD2) and presynaptic β-neurexin 1 (β-NRX1) via Cbln1, a C1q-like synaptic organizer. We show how Cbln1 hexamers “anchor” GluD2 amino-terminal domain dimers to monomeric β-NRX1. This arrangement promotes synaptogenesis and is essential for d-serine–dependent GluD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber–Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGluR function.
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Jul 2016
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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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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]
Abstract: Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as a crucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesion G-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions.
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Jan 2015
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8423]
Open Access
Abstract: Receptor protein tyrosine phosphatase sigma (RPTPσ) regulates neuronal extension and acts as a presynaptic nexus for multiple protein and proteoglycan interactions during synaptogenesis. Unknown mechanisms govern the shift in RPTPσ function, from outgrowth promotion to synaptic organization. Here, we report crystallographic, electron microscopic and small-angle X-ray scattering analyses, which reveal sufficient inter-domain flexibility in the RPTPσ extracellular region for interaction with both cis (same cell) and trans (opposite cell) ligands. Crystal structures of RPTPσ bound to its postsynaptic ligand TrkC detail an interaction surface partially overlapping the glycosaminoglycan-binding site. Accordingly, heparan sulphate and heparin oligomers compete with TrkC for RPTPσ binding in vitro and disrupt TrkC-dependent synaptic differentiation in neuronal co-culture assays. We propose that transient RPTPσ ectodomain emergence from the presynaptic proteoglycan layer allows capture by TrkC to form a trans-synaptic complex, the consequent reduction in RPTPσ flexibility potentiating interactions with additional ligands to orchestrate excitatory synapse formation.
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Nov 2014
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