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1.
Cell ; 185(21): 3931-3949.e26, 2022 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-36240740

RESUMO

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.


Assuntos
Movimento Celular , Glipicanas/química , Receptores de Netrina/química , Animais , Glipicanas/metabolismo , Humanos , Camundongos , Proteínas Mutantes , Receptores de Netrina/metabolismo , Receptores de Superfície Celular/metabolismo , Anticorpos de Domínio Único , Trombospondinas
2.
Cell ; 180(2): 323-339.e19, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31928845

RESUMO

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.


Assuntos
Proteínas do Tecido Nervoso/ultraestrutura , Receptores de Peptídeos/metabolismo , Tenascina/metabolismo , Animais , Adesão Celular/fisiologia , Cristalografia por Raios X/métodos , Células HEK293 , Humanos , Células K562 , Proteínas de Repetições Ricas em Leucina , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL/embriologia , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/ultraestrutura , Ligação Proteica/fisiologia , Proteínas/metabolismo , Proteínas/ultraestrutura , Receptores de Superfície Celular/metabolismo , Receptores de Peptídeos/ultraestrutura , Sinapses/metabolismo , Tenascina/ultraestrutura
3.
Annu Rev Cell Dev Biol ; 32: 577-608, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27576119

RESUMO

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.


Assuntos
Orientação de Axônios , Proteínas do Tecido Nervoso/metabolismo , Animais , Humanos , Modelos Biológicos , Receptores de Superfície Celular/metabolismo , Transdução de Sinais
4.
Am J Hum Genet ; 105(5): 1048-1056, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31668703

RESUMO

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.


Assuntos
Proteínas Ligadas por GPI/genética , Mutação de Sentido Incorreto/genética , Netrinas/genética , Transtornos do Neurodesenvolvimento/genética , Adolescente , Adulto , Criança , Pré-Escolar , Exoma/genética , Feminino , Homozigoto , Humanos , Deficiência Intelectual/genética , Masculino , Linhagem , Sequenciamento do Exoma/métodos , Adulto Jovem
5.
Bioessays ; 36(7): 649-57, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24782340

RESUMO

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.


Assuntos
Capsídeo/imunologia , DNA Complementar/metabolismo , Células Dendríticas , Infecções por HIV/imunologia , HIV-1/imunologia , HIV-2/imunologia , Nucleotidiltransferases/metabolismo , Humanos
6.
Handb Exp Pharmacol ; 234: 67-82, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27832484

RESUMO

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.


Assuntos
Adesão Celular , Membrana Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Sítios de Ligação , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteólise , Receptores Acoplados a Proteínas G/química , Transdução de Sinais , Relação Estrutura-Atividade
7.
EMBO J ; 30(21): 4479-88, 2011 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-21946559

RESUMO

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.


Assuntos
Proteínas Ligadas por GPI/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Células HEK293 , Humanos , Ligantes , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Netrinas , Ligação Proteica/genética , Ligação Proteica/fisiologia , Conformação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Sinapses/metabolismo , Distribuição Tecidual , Transfecção
8.
Nano Lett ; 14(7): 4171-5, 2014 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-24884378

RESUMO

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.


Assuntos
Corantes Fluorescentes/análise , Proteínas Luminescentes/análise , Microscopia de Fluorescência/instrumentação , Animais , Células COS , Chlorocebus aethiops , Temperatura Baixa , Desenho de Equipamento , Congelamento , Vitrificação
9.
Neuron ; 110(22): 3656-3660, 2022 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-36356578

RESUMO

In May, an interdisciplinary group gathered in Crete for the Molecular Neurobiology Workshop. Scientists shared data acquired by vastly diverse techniques to understand how the nervous system, with only a limited number of components, is assembled to respond to infinite stimuli. Ideas of molecular cues, timing, switching, and context emerged.


Assuntos
Neurociências , Neurobiologia
10.
J Clin Invest ; 132(6)2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35104247

RESUMO

Blood vessel abnormalization alters cancer cell metabolism and promotes cancer dissemination and metastasis. However, the biological features of the abnormalized blood vessels that facilitate cancer progression and whether they can be targeted therapeutically have not been fully investigated. Here, we found that an axon guidance molecule, fibronectin leucine-rich transmembrane protein 2 (FLRT2), is expressed preferentially in abnormalized vessels of advanced colorectal cancers in humans and that its expression correlates negatively with long-term survival. Endothelial cell-specific deletion of Flrt2 in mice selectively pruned abnormalized vessels, resulting in a unique metabolic state termed "oxygen-glucose uncoupling," which suppressed tumor metastasis. Moreover, Flrt2 deletion caused an increase in the number of mature vessels, resulting in a significant increase in the antitumor effects of immune checkpoint blockers. Mechanistically, we found that FLRT2 forms noncanonical interendothelial adhesions that safeguard against oxidative stress through homophilic binding. Together, our results demonstrated the existence of tumor-specific interendothelial adhesions that enable abnormalized vessels to facilitate cancer aggressiveness. Targeting this type of adhesion complex could be a safe and effective therapeutic option to suppress cancer progression.


Assuntos
Glicoproteínas de Membrana , Neoplasias , Animais , Células Endoteliais/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Neovascularização Patológica
11.
Structure ; 30(9): 1354-1365.e5, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35700726

RESUMO

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions, which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.


Assuntos
Moléculas de Adesão Celular , Glicoproteínas de Membrana , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Dimerização , Glicoproteínas de Membrana/química , Transdução de Sinais
12.
Nat Commun ; 13(1): 6385, 2022 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-36302784

RESUMO

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.


Assuntos
Ativação de Neutrófilo , Neutrófilos , Receptor PAR-2 , Receptores Acoplados a Proteínas G , Humanos , Inflamação/patologia , Mieloblastina/metabolismo , Ativação de Neutrófilo/fisiologia , Fagocitose , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo
13.
PLoS Pathog ; 5(1): e1000277, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19119424

RESUMO

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.


Assuntos
Adenoviridae/patogenicidade , Moléculas de Adesão Celular/metabolismo , Eritrócitos/patologia , Ácido N-Acetilneuramínico/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Animais , Sítios de Ligação , Cães , Eritrócitos/virologia , Hemaglutinação , Humanos , Metaloendopeptidases
14.
EMBO Rep ; 10(7): 736-41, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19498462

RESUMO

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.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica , Sequência Conservada , Decorina , Proteínas da Matriz Extracelular/química , Heparitina Sulfato/metabolismo , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína , Proteoglicanas/química , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Relação Estrutura-Atividade
15.
Front Neurosci ; 13: 183, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30930731

RESUMO

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 X-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.

16.
Neuron ; 100(6): 1270-1272, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30571936

RESUMO

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.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Comunicação Celular , Sistema Nervoso
17.
Structure ; 26(2): 320-328.e4, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29307485

RESUMO

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.


Assuntos
Imunoglobulina G/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Dimerização , Humanos , Modelos Moleculares , Transdução de Sinais/fisiologia , Proteínas Roundabout
18.
Nat Commun ; 9(1): 1079, 2018 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-29540701

RESUMO

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.


Assuntos
Complexo Glicoproteico GPIb-IX de Plaquetas/química , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Comunicação Celular/fisiologia , Microscopia Crioeletrônica , Cristalografia por Raios X , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Estrutura Secundária de Proteína , Tenascina/química , Tenascina/genética , Tenascina/metabolismo
19.
Structure ; 26(7): 1025-1034.e2, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29887500

RESUMO

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.


Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Receptor EphA2/química , Receptor EphA2/metabolismo , Sítios de Ligação , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica
20.
Structure ; 24(2): 337-47, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26724997

RESUMO

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.


Assuntos
Bicamadas Lipídicas/metabolismo , Receptor EphA2/química , Receptor EphA2/metabolismo , Sítios de Ligação , Humanos , Bicamadas Lipídicas/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Receptor EphA2/genética
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