Publications by authors named "Elena Seiradake"

28 Publications

  • Page 1 of 1

Structural Basis of Teneurin-Latrophilin Interaction in Repulsive Guidance of Migrating Neurons.

Cell 2020 01 9;180(2):323-339.e19. Epub 2020 Jan 9.

Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978801PMC
January 2020

Homozygous Missense Variants in NTNG2, Encoding a Presynaptic Netrin-G2 Adhesion Protein, Lead to a Distinct Neurodevelopmental Disorder.

Am J Hum Genet 2019 11 24;105(5):1048-1056. Epub 2019 Oct 24.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

NTNG2 encodes netrin-G2, a membrane-anchored protein implicated in the molecular organization of neuronal circuitry and synaptic organization and diversification in vertebrates. In this study, through a combination of exome sequencing and autozygosity mapping, we have identified 16 individuals (from seven unrelated families) with ultra-rare homozygous missense variants in NTNG2; these individuals present with shared features of a neurodevelopmental disorder consisting of global developmental delay, severe to profound intellectual disability, muscle weakness and abnormal tone, autistic features, behavioral abnormalities, and variable dysmorphisms. The variants disrupt highly conserved residues across the protein. Functional experiments, including in silico analysis of the protein structure, in vitro assessment of cell surface expression, and in vitro knockdown, revealed potential mechanisms of pathogenicity of the variants, including loss of protein function and decreased neurite outgrowth. Our data indicate that appropriate expression of NTNG2 plays an important role in neurotypical development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2019.09.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849109PMC
November 2019

Teneurin Structures Are Composed of Ancient Bacterial Protein Domains.

Front Neurosci 2019 13;13:183. Epub 2019 Mar 13.

Department of Biochemistry, University of Oxford, Oxford, United Kingdom.

Pioneering bioinformatic analysis using sequence data revealed that teneurins evolved from bacterial tyrosine-aspartate (YD)-repeat protein precursors. Here, we discuss how structures of the C-terminal domain of teneurins, determined using -ray crystallography and electron microscopy, support the earlier findings on the proteins' ancestry. This chapter describes the structure of the teneurin scaffold with reference to a large family of teneurin-like proteins that are widespread in modern prokaryotes. The central scaffold of modern eukaryotic teneurins is decorated by additional domains typically found in bacteria, which are re-purposed in eukaryotes to generate highly multifunctional receptors. We discuss how alternative splicing contributed to further diversifying teneurin structure and thereby function. This chapter traces the evolution of teneurins from a structural point of view and presents the state-of-the-art of how teneurin function is encoded by its specific structural features.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnins.2019.00183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425310PMC
March 2019

DIPping into the Fly Visual System.

Neuron 2018 12;100(6):1270-1272

Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

The nervous system relies on a combinatorial code of protein interactions that determine wiring specificity. Two complementary studies by Cosmanescu et al. (2018) and Xu et al. (2018) present the most comprehensive biophysical and structural analysis of the cell-recognition proteins DIP and Dpr to date, and the discovery of key functions of DIP-α and Dpr6/10 in the wiring of the Drosophila visual system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2018.11.044DOI Listing
December 2018

Interactions of the EphA2 Kinase Domain with PIPs in Membranes: Implications for Receptor Function.

Structure 2018 07 7;26(7):1025-1034.e2. Epub 2018 Jun 7.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

EphA2 is a member of the receptor tyrosine kinase family. Interactions of the cytoplasmic region of EphA2 with the cell membrane are functionally important and yet remain incompletely characterized. Molecular dynamics simulations combined with biochemical studies reveal the interactions of the transmembrane, juxtamembrane (JM), and kinase domains with the membrane. We describe how the kinase domain is oriented relative to the membrane and how the JM region can modulate this interaction. We highlight the role of phosphatidylinositol phosphates (PIPs) in mediating the interaction of the kinase domain with the membrane and, conversely, how positively charged patches at the kinase surface and in the JM region induce the formation of nanoclusters of PIP molecules in the membrane. Integration of these results with those from previous studies enable computational reconstitution of a near complete EphA2 receptor within a membrane, suggesting a role for receptor-lipid interactions in modulation of EphA2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2018.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039763PMC
July 2018

Structures of Teneurin adhesion receptors reveal an ancient fold for cell-cell interaction.

Nat Commun 2018 03 14;9(1):1079. Epub 2018 Mar 14.

Department of Biochemistry, Oxford University, OX1 3QU, Oxford, UK.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-03460-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5851990PMC
March 2018

Robo1 Forms a Compact Dimer-of-Dimers Assembly.

Structure 2018 02 4;26(2):320-328.e4. Epub 2018 Jan 4.

European Molecular Biology Laboratory, Grenoble Outstation, 71 avenue des Martyrs, 38042 Grenoble, France. Electronic address:

Roundabout (Robo) receptors provide an essential repulsive cue in neuronal development following Slit ligand binding. This important signaling pathway can also be hijacked in numerous cancers, making Slit-Robo an attractive therapeutic target. However, little is known about how Slit binding mediates Robo activation. Here we present the crystal structure of Robo1 Ig1-4 and Robo1 Ig5, together with a negative stain electron microscopy reconstruction of the Robo1 ectodomain. These results show how the Robo1 ectodomain is arranged as compact dimers, mainly mediated by the central Ig domains, which can further interact in a "back-to-back" fashion to generate a tetrameric assembly. We also observed no change in Robo1 oligomerization upon interaction with the dimeric Slit2-N ligand using fluorescent imaging. Taken together with previous studies we propose that Slit2-N binding results in a conformational change of Robo1 to trigger cell signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2017.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807052PMC
February 2018

Understanding the Structural Basis of Adhesion GPCR Functions.

Handb Exp Pharmacol 2016 ;234:67-82

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

Unlike conventional G-protein-coupled receptors (GPCRs), adhesion GPCRs (aGPCRs) have large extracellular regions that are autoproteolytically cleaved from their membrane-embedded seven-pass transmembrane helices. Autoproteolysis occurs within the conserved GPCR-Autoproteolysis INducing (GAIN) domain that is juxtaposed to the transmembrane domain and cleaves the last beta strand of the GAIN domain. The other domains of the extracellular region are variable and specific to each aGPCR and are likely involved in adhering to various ligands. Emerging evidence suggest that extracellular regions may modulate receptor function and that ligand binding to the extracellular regions may induce receptor activation via multiple mechanisms. Here, we summarize current knowledge about the structural understanding for the extracellular regions of aGPCRs and discuss their possible functional roles that emerge from the available structural information.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-3-319-41523-9_4DOI Listing
June 2017

Structural Perspectives on Axon Guidance.

Annu Rev Cell Dev Biol 2016 10 24;32:577-608. Epub 2016 Aug 24.

Max Planck Institute of Neurobiology, 82152 Munich-Martinsried, Germany; email:

Axon guidance relies on a combinatorial code of receptor and ligand interactions that direct adhesive/attractive and repulsive cellular responses. Recent structural data have revealed many of the molecular mechanisms that govern these interactions and enabled the design of sophisticated mutant tools to dissect their biological functions. Here, we discuss the structure/function relationships of four major classes of guidance cues (ephrins, semaphorins, slits, netrins) and examples of morphogens (Wnt, Shh) and of cell adhesion molecules (FLRT). These cell signaling systems rely on specific modes of receptor-ligand binding that are determined by selective binding sites; however, defined structure-encoded receptor promiscuity also enables cross talk between different receptor/ligand families and can also involve extracellular matrix components. A picture emerges in which a multitude of highly context-dependent structural assemblies determines the finely tuned cellular behavior required for nervous system development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev-cellbio-111315-125008DOI Listing
October 2016

Super-complexes of adhesion GPCRs and neural guidance receptors.

Nat Commun 2016 Apr 19;7:11184. Epub 2016 Apr 19.

Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms11184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838878PMC
April 2016

Structures of the EphA2 Receptor at the Membrane: Role of Lipid Interactions.

Structure 2016 Feb 24;24(2):337-47. Epub 2015 Dec 24.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

Ephs are transmembrane receptors that mediate cell-cell signaling. The N-terminal ectodomain binds ligands and enables receptor clustering, which activates the intracellular kinase. Relatively little is known about the function of the membrane-proximal fibronectin domain 2 (FN2) of the ectodomain. Multiscale molecular dynamics simulations reveal that FN2 interacts with lipid bilayers via a site comprising K441, R443, R465, Q462, S464, S491, W467, F490, and P459-461. FN2 preferentially binds anionic lipids, a preference that is reduced in the mutant K441E + R443E. We confirm these results by measuring the binding of wild-type and mutant FN2 domains to lipid vesicles. In simulations of the complete EphA2 ectodomain plus the transmembrane region, we show that FN2 anchors the otherwise flexible ectodomain at the surface of the bilayer. Altogether, our data suggest that FN2 serves a dual function of interacting with anionic lipids and constraining the structure of the EphA2 ectodomain to adopt membrane-proximal configurations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2015.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744086PMC
February 2016

Analysis of the Resistance Mechanism of a Benzoxaborole Inhibitor Reveals Insight into the Leucyl-tRNA Synthetase Editing Mechanism.

ACS Chem Biol 2015 Oct 31;10(10):2277-85. Epub 2015 Jul 31.

European Molecular Biology Laboratory, Grenoble Outstation and Unit of Virus Host-Cell Interactions, UJF-EMBL-CNRS, UMI 3265 , 71 Avenue des Martyrs, BP181, 38042 Grenoble Cedex 9, France.

A new class of antimicrobial benzoxaborole compounds was identified as a potent inhibitor of leucyl-tRNA synthetase (LeuRS) and therefore of protein synthesis. In a novel mechanism, AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole) blocks fungal cytoplasmic LeuRS by covalently trapping tRNA(Leu) in the editing site of the enzyme's CP1 domain. However, some resistant mutation sites are located outside of the CP1 hydrolytic editing active site. Thus, their mode of action that undermines drug inhibition was not understood. A combination of X-ray crystallography, molecular dynamics, metadynamics, biochemical experiments, and mutational analysis of a distal benzoxaborole-resistant mutant uncovered a eukaryote-specific tyrosine "switch" that is critical to tRNA-dependent post-transfer editing. The tyrosine "switch" has three states that shift between interactions with a lysine and the 3'-hydroxyl of the tRNA terminus, to inhibit or promote post-transfer editing. The oxaborole's mechanism of action capitalizes upon one of these editing active site states. This tunable editing mechanism in eukaryotic and archaeal LeuRSs is proposed to facilitate precise quality control of aminoacylation fidelity. These mechanistic distinctions could also be capitalized upon for development of the benzoxaboroles as a broad spectrum antibacterial.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.5b00291DOI Listing
October 2015

Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins.

Sci Rep 2015 Mar 31;5:9583. Epub 2015 Mar 31.

1] Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK [2] Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. We identified key aspects of the sample preparation procedure of high pressure freezing, freeze substitution and resin embedding that are critical for preserving fluorescence and photo-switching of standard fluorescent proteins, such as mGFP, mVenus and mRuby2. This enabled us to combine single molecule localization microscopy with transmission electron microscopy imaging of standard fluorescent proteins in cryo-fixed resin embedded cells. We achieved a structural resolution of 40-50 nm (~17 nm average single molecule localization accuracy) in the fluorescence images without the use of chemical fixation or special fluorophores. Using this approach enabled the correlation of fluorescently labeled structures to the ultrastructure in the same cell at the nanometer level and superior structural preservation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep09583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379466PMC
March 2015

Structural basis of latrophilin-FLRT interaction.

Structure 2015 Apr 26;23(4):774-81. Epub 2015 Feb 26.

Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2015.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396693PMC
April 2015

Production of cell surface and secreted glycoproteins in mammalian cells.

Methods Mol Biol 2015 ;1261:115-27

The Division of Structural Biology, The Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Oxford, OX3 7BN, UK.

Mammalian protein expression systems are becoming increasingly popular for the production of eukaryotic secreted and cell surface proteins. Here we describe methods to produce recombinant proteins in adherent or suspension human embryonic kidney cell cultures, using transient transfection or stable cell lines. The protocols are easy to scale up and cost-efficient, making them suitable for protein crystallization projects and other applications that require high protein yields.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-2230-7_6DOI Listing
August 2015

FLRT structure: balancing repulsion and cell adhesion in cortical and vascular development.

Neuron 2014 Oct 22;84(2):370-85. Epub 2014 Oct 22.

Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany; Munich Cluster for Systems Neurology (SyNergy), 80336 Munich, Germany. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2014.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210639PMC
October 2014

Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions.

Nano Lett 2014 Jul 4;14(7):4171-5. Epub 2014 Jun 4.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics and ‡Department of Biochemistry, University of Oxford , Oxford, United Kingdom.

We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400-500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. This enabled us to perform super-resolution imaging of vitrified biological samples and to visualize structures in unperturbed fast frozen cells for the first time with a structural resolution of ∼125 nm (average single molecule localization accuracy ∼40 nm), corresponding to a 3-5 fold resolution improvement.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/nl501870pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092024PMC
July 2014

Keeping your armour intact: how HIV-1 evades detection by the innate immune system: HIV-1 capsid controls detection of reverse transcription products by the cytosolic DNA sensor cGAS.

Bioessays 2014 Jul 29;36(7):649-57. Epub 2014 Apr 29.

Medical Research Council Human Immunology Unit, Medical Research Council Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

HIV-1 infects dendritic cells (DCs) without triggering an effective innate antiviral immune response. As a consequence, the induction of adaptive immune responses controlling virus spread is limited. In a recent issue of Immunity, Lahaye and colleagues show that intricate interactions of HIV capsid with the cellular cofactor cyclophilin A (CypA) control infection and innate immune activation in DCs. Manipulation of HIV-1 capsid to increase its affinity for CypA results in reduced virus infectivity and facilitates access of the cytosolic DNA sensor cGAS to reverse transcribed DNA. This in turn induces a strong host response. Here, we discuss these findings in the context of recent developments in innate immunity and consider the implications for disease control and vaccine design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bies.201400019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4190646PMC
July 2014

Structurally encoded intraclass differences in EphA clusters drive distinct cell responses.

Nat Struct Mol Biol 2013 Aug 30;20(8):958-64. Epub 2013 Jun 30.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.2617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3941021PMC
August 2013

Structural basis for cell surface patterning through NetrinG-NGL interactions.

EMBO J 2011 Sep 23;30(21):4479-88. Epub 2011 Sep 23.

Division of Structural Biology, CR-UK Receptor Structure Research Group, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/emboj.2011.346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230378PMC
September 2011

An extracellular steric seeding mechanism for Eph-ephrin signaling platform assembly.

Nat Struct Mol Biol 2010 Apr 14;17(4):398-402. Epub 2010 Mar 14.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

Erythropoetin-producing hepatoma (Eph) receptors are cell-surface protein tyrosine kinases mediating cell-cell communication. Upon activation, they form signaling clusters. We report crystal structures of the full ectodomain of human EphA2 (eEphA2) both alone and in complex with the receptor-binding domain of the ligand ephrinA5 (ephrinA5 RBD). Unliganded eEphA2 forms linear arrays of staggered parallel receptors involving two patches of residues conserved across A-class Ephs. eEphA2-ephrinA5 RBD forms a more elaborate assembly, whose interfaces include the same conserved regions on eEphA2, but rearranged to accommodate ephrinA5 RBD. Cell-surface expression of mutant EphA2s showed that these interfaces are critical for localization at cell-cell contacts and activation-dependent degradation. Our results suggest a 'nucleation' mechanism whereby a limited number of ligand-receptor interactions 'seed' an arrangement of receptors which can propagate into extended signaling arrays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.1782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3672960PMC
April 2010

Structure and functional relevance of the Slit2 homodimerization domain.

EMBO Rep 2009 Jul 5;10(7):736-41. Epub 2009 Jun 5.

European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France.

Slit proteins are secreted ligands that interact with the Roundabout (Robo) receptors to provide important guidance cues in neuronal and vascular development. Slit-Robo signalling is mediated by an interaction between the second Slit domain and the first Robo domain, as well as being dependent on heparan sulphate. In an effort to understand the role of the other Slit domains in signalling, we determined the crystal structure of the fourth Slit2 domain (D4) and examined the effects of various Slit2 constructs on chick retinal ganglion cell axons. Slit2 D4 forms a homodimer using the conserved residues on its concave face, and can also bind to heparan sulphate. We observed that Slit2 D4 frequently results in growth cones with collapsed lamellipodia and that this effect can be inhibited by exogenously added heparan sulphate. Our results show that Slit2 D4-heparan sulphate binding contributes to a Slit-Robo signalling mechanism more intricate than previously thought.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/embor.2009.95DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2693880PMC
July 2009

Crystal structures of the human and fungal cytosolic Leucyl-tRNA synthetase editing domains: A structural basis for the rational design of antifungal benzoxaboroles.

J Mol Biol 2009 Jul 6;390(2):196-207. Epub 2009 May 6.

European Molecular Biology Laboratory, Grenoble Outstation rue Jules Horowitz, France.

Leucyl-tRNA synthetase (LeuRS) specifically links leucine to the 3' end of tRNA(leu) isoacceptors. The overall accuracy of the two-step aminoacylation reaction is enhanced by an editing domain that hydrolyzes mischarged tRNAs, notably ile-tRNA(leu). We present crystal structures of the editing domain from two eukaryotic cytosolic LeuRS: human and fungal pathogen Candida albicans. In comparison with previous structures of the editing domain from bacterial and archeal kingdoms, these structures show that the LeuRS editing domain has a conserved structural core containing the active site for hydrolysis, with distinct bacterial, archeal, or eukaryotic specific peripheral insertions. It was recently shown that the benzoxaborole antifungal compound AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-1,2-benzoxaborole) inhibits LeuRS by forming a covalent adduct with the 3' adenosine of tRNA(leu) at the editing site, thus locking the enzyme in an inactive conformation. To provide a structural basis for enhancing the specificity of these benzoxaborole antifungals, we determined the structure at 2.2 A resolution of the C. albicans editing domain in complex with a related compound, AN3018 (6-(ethylamino)-5-fluorobenzo[c][1,2]oxaborol-1(3H)-ol), using AMP as a surrogate for the 3' adenosine of tRNA(leu). The interactions between the AN3018-AMP adduct and C. albicans LeuRS are similar to those previously observed for bacterial LeuRS with the AN2690 adduct, with an additional hydrogen bond to the extra ethylamine group. However, compared to bacteria, eukaryotic cytosolic LeuRS editing domains contain an extra helix that closes over the active site, largely burying the adduct and providing additional direct and water-mediated contacts. Small differences between the human domain and the fungal domain could be exploited to enhance fungal specificity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2009.04.073DOI Listing
July 2009

The cell adhesion molecule "CAR" and sialic acid on human erythrocytes influence adenovirus in vivo biodistribution.

PLoS Pathog 2009 Jan 2;5(1):e1000277. Epub 2009 Jan 2.

European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France.

Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad-erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1000277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2607015PMC
January 2009

Structure of the C-terminal head domain of the fowl adenovirus type 1 short fibre.

Virology 2008 Aug 17;378(1):169-76. Epub 2008 Jun 17.

Unit for Virus Host Cell Interactions, UMR5233 UJF-EMBL-CNRS, CIBB, 6 rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France.

There are more than 100 known adenovirus serotypes, including 50 human serotypes. They can infect all 5 major vertebrate classes but only Aviadenovirus infecting birds and Mastadenovirus infecting mammals have been well studied. CELO (chicken embryo lethal orphan) adenovirus is responsible for mild respiratory pathologies in birds. Most studies on CELO virus have focussed on its genome sequence and organisation whereas the structural work on CELO proteins has only recently started. Contrary to most adenoviruses, the vertices of CELO virus reveal pentons with two fibres of different lengths. The distal parts (or head) of those fibres are involved in cellular receptor binding. Here we have determined the atomic structure of the short-fibre head of CELO (amino acids 201-410) at 2.0 A resolution. Despite low sequence identity, this structure is conserved compared to the other adenovirus fibre heads. We have used the existing CELO long-fibre head structure and the one we show here for a structure-based alignment of 11 known adenovirus fibre heads which was subsequently used for the construction of an evolutionary tree. Both the fibre head sequence and structural alignments suggest that enteric human group F adenovirus 41 (short fibre) is closer to the CELO fibre heads than the canine CAdV-2 fibre head, that lies closer to the human virus fibre heads.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2008.05.011DOI Listing
August 2008

Structural and mutational analysis of human Ad37 and canine adenovirus 2 fiber heads in complex with the D1 domain of coxsackie and adenovirus receptor.

J Biol Chem 2006 Nov 21;281(44):33704-16. Epub 2006 Aug 21.

Grenoble Outstation, European Molecular Biology Laboratory, Grenoble, France.

Adenovirus fibers from most serotypes bind the D1 domain of coxsackie and adenovirus receptor (CAR), although the binding residues are not strictly conserved. To understand this further, we determined the crystal structures of canine adenovirus serotype 2 (CAV-2) and the human adenovirus serotype 37 (HAd37) in complex with human CAR D1 at 2.3 and 1.5A resolution, respectively. Structure comparison with the HAd12 fiber head-CAR D1 complex showed that the overall topology of the interaction is conserved but that the interfaces differ in number and identity of interacting residues, shape complementarity, and degree of conformational adaptation. Using surface plasmon resonance, we characterized the binding affinity to CAR D1 of wild type and mutant CAV-2 and HAd37 fiber heads. We found that CAV-2 has the highest affinity but fewest direct interactions, with the reverse being true for HAd37. Moreover, we found that conserved interactions can have a minor contribution, whereas serotype-specific interactions can be essential. These results are discussed in the light of virus evolution and design of adenovirus vectors for gene transfer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M605316200DOI Listing
November 2006

Crystal structure of enteric adenovirus serotype 41 short fiber head.

J Virol 2005 Nov;79(22):14088-94

EMBL Grenoble Outstation, France.

Human enteric adenoviruses of species F contain two fibers in the same virion, a long fiber which binds to coxsackievirus and adenovirus receptor (CAR) and a short fiber of unknown function. We have determined the high-resolution crystal structure of the short fiber head of human adenovirus serotype 41 (Ad41). The short fiber head has the characteristic fold of other known fiber heads but has three unusual features. First, it has much shorter loops between the beta-strands. Second, one of the usually well-ordered beta-strands on the distal face of the fiber head is highly disordered and this same region is sensitive to digestion with pepsin, an enzyme occurring naturally in the intestinal tract, the physiological environment of Ad41. Third, the AB loop has a deletion giving it a distinct conformation incompatible with CAR binding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.79.22.14088-14094.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1280240PMC
November 2005
-->