I23-Long wavelength MX
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Tai-Ying
Chu
,
Céline
Zheng-Gérard
,
Kuan-Yeh
Huang
,
Yu-Chi
Chang
,
Ying-Wen
Chen
,
Kuan-Yu
I
,
Yu-Ling
Lo
,
Nien-Yi
Chiang
,
Hsin-Yi
Chen
,
Martin
Stacey
,
Siamon
Gordon
,
Wen-Yi
Tseng
,
Chiao-Yin
Sun
,
Yen-Mu
Wu
,
Yi-Shin
Pan
,
Chien-Hao
Huang
,
Chun-Yen
Lin
,
Tse-Ching
Chen
,
Kamel
El Omari
,
Marilina
Antonelou
,
Scott R.
Henderson
,
Alan
Salama
,
Elena
Seiradake
,
Hsi-Hsien
Lin
Open Access
Abstract: Neutrophils play essential anti-microbial and inflammatory roles in host defense, however, their activities require tight regulation as dysfunction often leads to detrimental inflammatory and autoimmune diseases. Here we show that the adhesion molecule GPR97 allosterically activates CD177-associated membrane proteinase 3 (mPR3), and in conjugation with several protein interaction partners leads to neutrophil activation in humans. Crystallographic and deletion analysis of the GPR97 extracellular region identified two independent mPR3-binding domains. Mechanistically, the efficient binding and activation of mPR3 by GPR97 requires the macromolecular CD177/GPR97/PAR2/CD16b complex and induces the activation of PAR2, a G protein-coupled receptor known for its function in inflammation. Triggering PAR2 by the upstream complex leads to strong inflammatory activation, prompting anti-microbial activities and endothelial dysfunction. The role of the complex in pathologic inflammation is underscored by the finding that both GPR97 and mPR3 are upregulated on the surface of disease-associated neutrophils. In summary, we identify a PAR2 activation mechanism that directs neutrophil activation, and thus inflammation. The PR3/CD177/GPR97/PAR2/CD16b protein complex, therefore, represents a potential therapeutic target for neutrophil-mediated inflammatory diseases.
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Oct 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Onno
Akkermans
,
Céline
Delloye-Bourgeois
,
Claudia
Peregrina
,
Maria
Carrasquero-Ordaz
,
Maria
Kokolaki
,
Miguel
Berbeira-Santana
,
Matthieu
Chavent
,
Florie
Reynaud
,
Ritu
Raj
,
Jon
Agirre
,
Metin
Aksu
,
Eleanor S.
White
,
Edward
Lowe
,
Dounia
Ben Amar
,
Sofia
Zaballa
,
Jiandong
Huo
,
Irene
Pakos
,
Patrick T. N.
Mccubbin
,
Davide
Comoletti
,
Raymond J.
Owens
,
Carol V.
Robinson
,
Valérie
Castellani
,
Daniel
Del Toro
,
Elena
Seiradake
Diamond Proposal Number(s):
[18069]
Open Access
Abstract: Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.
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Oct 2022
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I03-Macromolecular Crystallography
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Daniel
Del Toro
,
Maria A.
Carrasquero-Ordaz
,
Amy
Chu
,
Tobias
Ruff
,
Meriam
Shahin
,
Verity A.
Jackson
,
Matthieu
Chavent
,
Miguel
Berbeira-Santana
,
Goenuel
Seyit-Bremer
,
Sara
Brignani
,
Rainer
Kaufmann
,
Edward
Lowe
,
Rüdiger
Klein
,
Elena
Seiradake
Diamond Proposal Number(s):
[12346, 1838]
Open Access
Abstract: Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.
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Jan 2020
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346, 1838]
Open Access
Abstract: Teneurins are ancient cell–cell adhesion receptors that are vital for brain development and synapse organisation. They originated in early metazoan evolution through a horizontal gene transfer event when a bacterial YD-repeat toxin fused to a eukaryotic receptor. We present X-ray crystallography and cryo-EM structures of two Teneurins, revealing a ~200 kDa extracellular super-fold in which eight sub-domains form an intricate structure centred on a spiralling YD-repeat shell. An alternatively spliced loop, which is implicated in homophilic Teneurin interaction and specificity, is exposed and thus poised for interaction. The N-terminal side of the shell is ‘plugged’ via a fibronectin-plug domain combination, which defines a new class of YD proteins. Unexpectedly, we find that these proteins are widespread amongst modern bacteria, suggesting early metazoan receptor evolution from a distinct class of proteins, which today includes both bacterial proteins and eukaryotic Teneurins.
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Mar 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Verity A.
Jackson
,
Shahid
Mehmood
,
Matthieu
Chavent
,
Pietro
Roversi
,
Maria
Carrasquero
,
Daniel
Del Toro
,
Goenuel
Seyit-Bremer
,
Fanomezana M.
Ranaivoson
,
Davide
Comoletti
,
Mark S. P.
Sansom
,
Carol V.
Robinson
,
Rüdiger
Klein
,
Elena
Seiradake
Diamond Proposal Number(s):
[9306, 8423, 1747]
Open Access
Abstract: Latrophilin adhesion-GPCRs (Lphn1–3 or ADGRL1–3) and Unc5 cell guidance receptors (Unc5A–D) interact with FLRT proteins (FLRT1–3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger ‘super-complex’ (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes.
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Apr 2016
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Latrophilins, receptors for spider venom α-latrotoxin, are adhesion type G-protein-coupled receptors with emerging functions in synapse development. The N-terminal region binds the endogenous cell adhesion molecule FLRT, a major regulator of cortical and synapse development. We present crystallographic data for the mouse Latrophilin3 lectin and olfactomedin-like (Olf) domains, thereby revealing the Olf β-propeller fold and conserved calcium-binding site. We locate the FLRT-Latrophilin binding surfaces by a combination of sequence conservation analysis, point mutagenesis, and surface plasmon resonance experiments. In stripe assays, we show that wild-type Latrophilin3 and its high-affinity interactor FLRT2, but not the binding-impaired mutants we generated, promote HeLa cell adhesion. In contrast, cortical neurons expressing endogenous FLRTs are repelled by wild-type Latrophilin3 and not by the binding-impaired mutant. Taken together, we present molecular level insights into Latrophilin structure, its FLRT-binding mechanism, and a role for Latrophilin and FLRT that goes beyond a simply adhesive interaction.
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Apr 2015
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Elena
Seiradake
,
Daniel
Del toro
,
Daniel
Nagel
,
Florian
Cop
,
Ricarda
Härtl
,
Tobias
Ruff
,
Gönül
Seyit-Bremer
,
Karl
Harlos
,
Ellen clare
Border
,
Amparo
Acker-Palmer
,
E. Yvonne
Jones
,
Rüdiger
Klein
Diamond Proposal Number(s):
[8423]
Open Access
Abstract: FLRTs are broadly expressed proteins with the unique property of acting as homophilic cell adhesion molecules and as heterophilic repulsive ligands of Unc5/Netrin receptors. How these functions direct cell behavior and the molecular mechanisms involved remain largely unclear. Here we use X-ray crystallography to reveal the distinct structural bases for FLRT-mediated cell adhesion and repulsion in neurons. We apply this knowledge to elucidate FLRT functions during cortical development. We show that FLRTs regulate both the radial migration of pyramidal neurons, as well as their tangential spread. Mechanistically, radial migration is controlled by repulsive FLRT2-Unc5D interactions, while spatial organization in the tangential axis involves adhesive FLRT-FLRT interactions. Further, we show that the fundamental mechanisms of FLRT adhesion and repulsion are conserved between neurons and vascular endothelial cells. Our results reveal FLRTs as powerful guidance factors with structurally encoded repulsive and adhesive surfaces.
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Oct 2014
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Abstract: Functional outcomes of ephrin binding to Eph receptors (Ephs) range from cell repulsion to adhesion. Here we used cell collapse and stripe assays, showing contrasting effects of human ephrinA5 binding to EphA2 and EphA4. Despite equivalent ligand binding affinities, EphA4 triggered greater cell collapse, whereas EphA2-expressing cells adhered better to ephrinA5-coated surfaces. Chimeric receptors showed that the ectodomain is a major determinant of cell response. We report crystal structures of EphA4 ectodomain alone and in complexes with ephrinB3 and ephrinA5. These revealed closed clusters with a dimeric or circular arrangement in the crystal lattice, contrasting with extended arrays previously observed for EphA2 ectodomain. Localization microscopy showed that ligand-stimulated EphA4 induces smaller clusters than does EphA2. Mutant Ephs link these characteristics to interactions observed in the crystal lattices, suggesting a mechanism by which distinctive ectodomain surfaces determine clustering, and thereby signaling, properties.
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Jun 2013
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Brain wiring depends on cells making highly localized and selective connections through surface protein-protein interactions, including those between NetrinGs and NetrinG ligands (NGLs). The NetrinGs are members of the structurally uncharacterized netrin family. We present a comprehensive crystallographic analysis comprising NetrinG1-NGL1 and NetrinG2-NGL2 complexes, unliganded NetrinG2 and NGL3. Cognate NetrinG-NGL interactions depend on three specificity-conferring NetrinG loops, clasped tightly by matching NGL surfaces. We engineered these NGL surfaces to implant custom-made affinities for NetrinG1 and NetrinG2. In a cellular patterning assay, we demonstrate that NetrinG-binding selectivity can direct the sorting of a mixed population of NGLs into discrete cell surface subdomains. These results provide a molecular model for selectivity-based patterning in a neuronal recognition system, dysregulation of which is associated with severe neuropsychological disorders.
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Dec 2011
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