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1.
Res Sq ; 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38463964

RESUMO

Self-recognition is a fundamental cellular process across evolution and forms the basis of neuronal self-avoidance1-4. Clustered protocadherins (Pcdh), comprising a large family of isoform-specific homophilic recognition molecules, play a pivotal role in neuronal self-avoidance required for mammalian brain development5-7. The probabilistic expression of different Pcdh isoforms confers unique identities upon neurons and forms the basis for neuronal processes to discriminate between self and non-self5,6,8. Whether this self-recognition mechanism exists in astrocytes, the other predominant cell type of the brain, remains unknown. Here, we report that a specific isoform in the Pcdhγ cluster, γC3, is highly enriched in human and murine astrocytes. Through genetic manipulation, we demonstrate that γC3 acts autonomously to regulate astrocyte morphogenesis in the mouse visual cortex. To determine if γC3 proteins act by promoting recognition between processes of the same astrocyte, we generated pairs of γC3 chimeric proteins capable of heterophilic binding to each other, but incapable of homophilic binding. Co-expressing complementary heterophilic binding isoform pairs in the same γC3 null astrocyte restored normal morphology. By contrast, chimeric γC3 proteins individually expressed in single γC3 null mutant astrocytes did not. These data establish that self-recognition is essential for astrocyte development in the mammalian brain and that, by contrast to neuronal self-recognition, a single Pcdh isoform is both necessary and sufficient for this process.

2.
Cell ; 186(4): 821-836.e13, 2023 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-36750096

RESUMO

The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2 or megalin) is representative of the phylogenetically conserved subfamily of giant LDL receptor-related proteins, which function in endocytosis and are implicated in diseases of the kidney and brain. Here, we report high-resolution cryoelectron microscopy structures of LRP2 isolated from mouse kidney, at extracellular and endosomal pH. The structures reveal LRP2 to be a molecular machine that adopts a conformation for ligand binding at the cell surface and for ligand shedding in the endosome. LRP2 forms a homodimer, the conformational transformation of which is governed by pH-sensitive sites at both homodimer and intra-protomer interfaces. A subset of LRP2 deleterious missense variants in humans appears to impair homodimer assembly. These observations lay the foundation for further understanding the function and mechanism of LDL receptors and implicate homodimerization as a conserved feature of the LRP receptor subfamily.


Assuntos
Endocitose , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Animais , Humanos , Camundongos , Microscopia Crioeletrônica , Rim/metabolismo , Ligantes , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo
3.
Elife ; 112022 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-35253643

RESUMO

The stochastic expression of fewer than 60 clustered protocadherin (cPcdh) isoforms provides diverse identities to individual vertebrate neurons and a molecular basis for self-/nonself-discrimination. cPcdhs form chains mediated by alternating cis and trans interactions between apposed membranes, which has been suggested to signal self-recognition. Such a mechanism requires that cPcdh cis dimers form promiscuously to generate diverse recognition units, and that trans interactions have precise specificity so that isoform mismatches terminate chain growth. However, the extent to which cPcdh interactions fulfill these requirements has not been definitively demonstrated. Here, we report biophysical experiments showing that cPcdh cis interactions are promiscuous, but with preferences favoring formation of heterologous cis dimers. Trans homophilic interactions are remarkably precise, with no evidence for heterophilic interactions between different isoforms. A new C-type cPcdh crystal structure and mutagenesis data help to explain these observations. Overall, the interaction characteristics we report for cPcdhs help explain their function in neuronal self-/nonself-discrimination.


Assuntos
Caderinas , Protocaderinas , Caderinas/metabolismo , Comunicação Celular , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo
4.
Cell Rep ; 37(3): 109828, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34686348

RESUMO

Synaptic connectivity within adult circuits exhibits a remarkable degree of cellular and subcellular specificity. We report that the axon guidance receptor Robo2 plays a role in establishing synaptic specificity in hippocampal CA1. In vivo, Robo2 is present and required postsynaptically in CA1 pyramidal neurons (PNs) for the formation of excitatory (E) but not inhibitory (I) synapses, specifically in proximal but not distal dendritic compartments. In vitro approaches show that the synaptogenic activity of Robo2 involves a trans-synaptic interaction with presynaptic Neurexins, as well as binding to its canonical extracellular ligand Slit. In vivo 2-photon Ca2+ imaging of CA1 PNs during spatial navigation in awake behaving mice shows that preventing Robo2-dependent excitatory synapse formation cell autonomously during development alters place cell properties of adult CA1 PNs. Our results identify a trans-synaptic complex linking the establishment of synaptic specificity to circuit function.


Assuntos
Região CA1 Hipocampal/metabolismo , Células Piramidais/metabolismo , Receptores Imunológicos/metabolismo , Sinapses/metabolismo , Animais , Região CA1 Hipocampal/citologia , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Potenciais Pós-Sinápticos Excitadores , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , Células de Lugar/metabolismo , Receptores Imunológicos/genética , Proteínas Roundabout
5.
Elife ; 92020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33021200

RESUMO

Sex-specific synaptic connectivity is beginning to emerge as a remarkable, but little explored feature of animal brains. We describe here a novel mechanism that promotes sexually dimorphic neuronal function and synaptic connectivity in the nervous system of the nematode Caenorhabditis elegans. We demonstrate that a phylogenetically conserved, but previously uncharacterized Doublesex/Mab-3 related transcription factor (DMRT), dmd-4, is expressed in two classes of sex-shared phasmid neurons specifically in hermaphrodites but not in males. We find dmd-4 to promote hermaphrodite-specific synaptic connectivity and neuronal function of phasmid sensory neurons. Sex-specificity of DMD-4 function is conferred by a novel mode of posttranslational regulation that involves sex-specific protein stabilization through ubiquitin binding to a phylogenetically conserved but previously unstudied protein domain, the DMA domain. A human DMRT homolog of DMD-4 is controlled in a similar manner, indicating that our findings may have implications for the control of sexual differentiation in other animals as well.


Assuntos
Proteínas de Caenorhabditis elegans , Neurônios/metabolismo , Caracteres Sexuais , Fatores de Transcrição , Ubiquitina/metabolismo , Animais , Comportamento Animal , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Transtornos do Desenvolvimento Sexual , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Transmissão Sináptica/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Nat Commun ; 11(1): 2125, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358559

RESUMO

Differential binding affinities among closely related protein family members underlie many biological phenomena, including cell-cell recognition. Drosophila DIP and Dpr proteins mediate neuronal targeting in the fly through highly specific protein-protein interactions. We show here that DIPs/Dprs segregate into seven specificity subgroups defined by binding preferences between their DIP and Dpr members. We then describe a sequence-, structure- and energy-based computational approach, combined with experimental binding affinity measurements, to reveal how specificity is coded on the canonical DIP/Dpr interface. We show that binding specificity of DIP/Dpr subgroups is controlled by "negative constraints", which interfere with binding. To achieve specificity, each subgroup utilizes a different combination of negative constraints, which are broadly distributed and cover the majority of the protein-protein interface. We discuss the structural origins of negative constraints, and potential general implications for the evolutionary origins of binding specificity in multi-protein families.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Sequência de Aminoácidos , Animais , Evolução Biológica , Drosophila , Proteínas de Drosophila/genética , Evolução Molecular , Estrutura Secundária de Proteína , Análise de Sequência de Proteína
7.
Cell Rep ; 30(8): 2655-2671.e7, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32101743

RESUMO

Non-clustered δ1- and δ2-protocadherins, close relatives of clustered protocadherins, function in cell adhesion and motility and play essential roles in neural patterning. To understand the molecular interactions underlying these functions, we used solution biophysics to characterize binding of δ1- and δ2-protocadherins, determined crystal structures of ectodomain complexes from each family, and assessed ectodomain assembly in reconstituted intermembrane junctions by cryoelectron tomography (cryo-ET). Homophilic trans (cell-cell) interactions were preferred for all δ-protocadherins, with additional weaker heterophilic interactions observed exclusively within each subfamily. As expected, δ1- and δ2-protocadherin trans dimers formed through antiparallel EC1-EC4 interfaces, like clustered protocadherins. However, no ectodomain-mediated cis (same-cell) interactions were detectable in solution; consistent with this, cryo-ET of reconstituted junctions revealed dense assemblies lacking the characteristic order observed for clustered protocadherins. Our results define non-clustered protocadherin binding properties and their structural basis, providing a foundation for interpreting their functional roles in neural patterning.


Assuntos
Fenômenos Biofísicos , Caderinas/química , Caderinas/metabolismo , Animais , Caderinas/genética , Sequência Conservada , Feminino , Células HEK293 , Humanos , Cinética , Lipossomos , Modelos Moleculares , Mutação/genética , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Soluções , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Xenopus
8.
Proc Natl Acad Sci U S A ; 116(49): 24517-24526, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31748271

RESUMO

Exonic DNA sequence variants in the Tbk1 gene associate with both sporadic and familial amyotrophic lateral sclerosis (ALS). Here, we examine functional defects in 25 missense TBK1 mutations, focusing on kinase activity and protein-protein interactions. We identified kinase domain (KD) mutations that abolish kinase activity or display substrate-specific defects in specific pathways, such as innate immunity and autophagy. By contrast, mutations in the scaffold dimerization domain (SDD) of TBK1 can cause the loss of kinase activity due to structural disruption, despite an intact KD. Familial ALS mutations in ubiquitin-like domain (ULD) or SDD display defects in dimerization; however, a subset retains kinase activity. These observations indicate that TBK1 dimerization is not required for kinase activation. Rather, dimerization seems to increase protein stability and enables efficient kinase-substrate interactions. Our study revealed many aspects of TBK1 activities affected by ALS mutations, highlighting the complexity of disease pathogenicity and providing insights into TBK1 activation mechanism.


Assuntos
Esclerose Lateral Amiotrófica/genética , Mutação de Sentido Incorreto , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Esclerose Lateral Amiotrófica/enzimologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ativação Enzimática , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Fosforilação , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Estabilidade Proteica , Serina/metabolismo , Especificidade por Substrato
9.
Neuron ; 100(6): 1369-1384.e6, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30467079

RESUMO

Drosophila Dpr (21 paralogs) and DIP proteins (11 paralogs) are cell recognition molecules of the immunoglobulin superfamily (IgSF) that form a complex protein interaction network. DIP and Dpr proteins are expressed in a synaptic layer-specific fashion in the visual system. How interactions between these proteins regulate layer-specific synaptic circuitry is not known. Here we establish that DIP-α and its interacting partners Dpr6 and Dpr10 regulate multiple processes, including arborization within layers, synapse number, layer specificity, and cell survival. We demonstrate that heterophilic binding between Dpr6/10 and DIP-α and homophilic binding between DIP-α proteins promote interactions between processes in vivo. Knockin mutants disrupting the DIP/Dpr binding interface reveal a role for these proteins during normal development, while ectopic expression studies support an instructive role for interactions between DIPs and Dprs in circuit development. These studies support an important role for the DIP/Dpr protein interaction network in regulating cell-type-specific connectivity patterns.


Assuntos
Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurópilo/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Drosophila , Proteínas de Drosophila/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Bulbo/citologia , Bulbo/crescimento & desenvolvimento , Mutação/genética , Mapas de Interação de Proteínas , Ressonância de Plasmônio de Superfície , Fatores de Transcrição/genética , Transfecção , Vias Visuais/metabolismo
10.
Neuron ; 100(6): 1385-1400.e6, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30467080

RESUMO

Binding between DIP and Dpr neuronal recognition proteins has been proposed to regulate synaptic connections between lamina and medulla neurons in the Drosophila visual system. Each lamina neuron was previously shown to express many Dprs. Here, we demonstrate, by contrast, that their synaptic partners typically express one or two DIPs, with binding specificities matched to the lamina neuron-expressed Dprs. A deeper understanding of the molecular logic of DIP/Dpr interaction requires quantitative studies on the properties of these proteins. We thus generated a quantitative affinity-based DIP/Dpr interactome for all DIP/Dpr protein family members. This revealed a broad range of affinities and identified homophilic binding for some DIPs and some Dprs. These data, along with full-length ectodomain DIP/Dpr and DIP/DIP crystal structures, led to the identification of molecular determinants of DIP/Dpr specificity. This structural knowledge, along with a comprehensive set of quantitative binding affinities, provides new tools for functional studies in vivo.


Assuntos
Proteínas de Drosophila/metabolismo , Bulbo/citologia , Neurônios/metabolismo , Vias Visuais/citologia , Animais , Animais Geneticamente Modificados , Comunicação Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Ligação Proteica , Ressonância de Plasmônio de Superfície , Transfecção
11.
Neuron ; 99(3): 480-492.e5, 2018 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-30057206

RESUMO

The tip link, a filament formed by protocadherin 15 (PCDH15) and cadherin 23, conveys mechanical force from sound waves and head movement to open hair-cell mechanotransduction channels. Tip-link cadherins are thought to have acquired structural features critical for their role in mechanotransduction. Here, we biophysically and structurally characterize the unusual cis-homodimeric architecture of PCDH15. We show that PCDH15 molecules form double-helical assemblies through cis-dimerization interfaces in the extracellular cadherin EC2-EC3 domain region and in a unique membrane-proximal domain. Electron microscopy studies visualize the cis-dimeric PCDH15 assembly and reveal the PCDH15 extracellular domain as a parallel double helix with cis cross-bridges at the two locations we defined. The helical configuration suggests the potential for elasticity through helix winding and unwinding. Functional studies in hair cells show that mutations that perturb PCDH15 dimerization contacts affect mechanotransduction. Together, these data reveal the cis-dimeric architecture of PCDH15 and show that dimerization is critical for sensing mechanical stimuli.


Assuntos
Caderinas/química , Caderinas/fisiologia , Mecanotransdução Celular/fisiologia , Multimerização Proteica/fisiologia , Animais , Proteínas Relacionadas a Caderinas , Cristalização/métodos , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
12.
Cell Rep ; 23(6): 1840-1852, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29742438

RESUMO

Type II cadherins are cell-cell adhesion proteins critical for tissue patterning and neuronal targeting but whose molecular binding code remains poorly understood. Here, we delineate binding preferences for type II cadherin cell-adhesive regions, revealing extensive heterophilic interactions between specific pairs, in addition to homophilic interactions. Three distinct specificity groups emerge from our analysis with members that share highly similar heterophilic binding patterns and favor binding to one another. Structures of adhesive fragments from each specificity group confirm near-identical dimer topology conserved throughout the family, allowing interface residues whose conservation corresponds to specificity preferences to be identified. We show that targeted mutation of these residues converts binding preferences between specificity groups in biophysical and co-culture assays. Our results provide a detailed understanding of the type II cadherin interaction map and a basis for defining their role in tissue patterning and for the emerging importance of their heterophilic interactions in neural connectivity.


Assuntos
Caderinas/metabolismo , Sequência de Aminoácidos , Animais , Caderinas/química , Adesão Celular , Linhagem Celular , Sequência Conservada , Análise Mutacional de DNA , Humanos , Camundongos , Mutação/genética , Filogenia , Ligação Proteica , Multimerização Proteica
13.
Proc Natl Acad Sci U S A ; 114(46): E9829-E9837, 2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29087338

RESUMO

Clustered protocadherins (Pcdhs) mediate numerous neural patterning functions, including neuronal self-recognition and non-self-discrimination to direct self-avoidance among vertebrate neurons. Individual neurons stochastically express a subset of Pcdh isoforms, which assemble to form a stochastic repertoire of cis-dimers. We describe the structure of a PcdhγB7 cis-homodimer, which includes the membrane-proximal extracellular cadherin domains EC5 and EC6. The structure is asymmetric with one molecule contributing interface surface from both EC5 and EC6, and the other only from EC6. Structural and sequence analyses suggest that all Pcdh isoforms will dimerize through this interface. Site-directed mutants at this interface interfere with both Pcdh cis-dimerization and cell surface transport. The structure explains the known restrictions of cis-interactions of some Pcdh isoforms, including α-Pcdhs, which cannot form homodimers. The asymmetry of the interface approximately doubles the size of the recognition repertoire, and restrictions on cis-interactions among Pcdh isoforms define the limits of the Pcdh recognition unit repertoire.


Assuntos
Caderinas/química , Caderinas/metabolismo , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Caderinas/genética , Cristalografia por Raios X , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Isoformas de Proteínas/genética , Multimerização Proteica , Alinhamento de Sequência , Análise de Sequência de Proteína
14.
Elife ; 52016 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-27782885

RESUMO

Stochastic cell-surface expression of α-, ß-, and γ-clustered protocadherins (Pcdhs) provides vertebrate neurons with single-cell identities that underlie neuronal self-recognition. Here we report crystal structures of ectodomain fragments comprising cell-cell recognition regions of mouse γ-Pcdhs γA1, γA8, γB2, and γB7 revealing trans-homodimers, and of C-terminal ectodomain fragments from γ-Pcdhs γA4 and γB2, which depict cis-interacting regions in monomeric form. Together these structures span the entire γ-Pcdh ectodomain. The trans-dimer structures reveal determinants of γ-Pcdh isoform-specific homophilic recognition. We identified and structurally mapped cis-dimerization mutations to the C-terminal ectodomain structures. Biophysical studies showed that Pcdh ectodomains from γB-subfamily isoforms formed cis dimers, whereas γA isoforms did not, but both γA and γB isoforms could interact in cis with α-Pcdhs. Together, these data show how interaction specificity is distributed over all domains of the γ-Pcdh trans interface, and suggest that subfamily- or isoform-specific cis-interactions may play a role in the Pcdh-mediated neuronal self-recognition code.


Assuntos
Caderinas/química , Caderinas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Animais , Proteínas Relacionadas a Caderinas , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica
15.
Elife ; 52016 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-27644106

RESUMO

Sidekick (Sdk) 1 and 2 are related immunoglobulin superfamily cell adhesion proteins required for appropriate synaptic connections between specific subtypes of retinal neurons. Sdks mediate cell-cell adhesion with homophilic specificity that underlies their neuronal targeting function. Here we report crystal structures of Sdk1 and Sdk2 ectodomain regions, revealing similar homodimers mediated by the four N-terminal immunoglobulin domains (Ig1-4), arranged in a horseshoe conformation. These Ig1-4 horseshoes interact in a novel back-to-back orientation in both homodimers through Ig1:Ig2, Ig1:Ig1 and Ig3:Ig4 interactions. Structure-guided mutagenesis results show that this canonical dimer is required for both Sdk-mediated cell aggregation (via trans interactions) and Sdk clustering in isolated cells (via cis interactions). Sdk1/Sdk2 recognition specificity is encoded across Ig1-4, with Ig1-2 conferring the majority of binding affinity and differential specificity. We suggest that competition between cis and trans interactions provides a novel mechanism to sharpen the specificity of cell-cell interactions.


Assuntos
Adesão Celular , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Neurônios/fisiologia , Retina/fisiologia , Cristalografia por Raios X , Análise Mutacional de DNA , Imunoglobulina G/genética , Proteínas de Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Multimerização Proteica
16.
Proc Natl Acad Sci U S A ; 113(26): 7160-5, 2016 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-27298358

RESUMO

Desmosomes are intercellular adhesive junctions that impart strength to vertebrate tissues. Their dense, ordered intercellular attachments are formed by desmogleins (Dsgs) and desmocollins (Dscs), but the nature of trans-cellular interactions between these specialized cadherins is unclear. Here, using solution biophysics and coated-bead aggregation experiments, we demonstrate family-wise heterophilic specificity: All Dsgs form adhesive dimers with all Dscs, with affinities characteristic of each Dsg:Dsc pair. Crystal structures of ectodomains from Dsg2 and Dsg3 and from Dsc1 and Dsc2 show binding through a strand-swap mechanism similar to that of homophilic classical cadherins. However, conserved charged amino acids inhibit Dsg:Dsg and Dsc:Dsc interactions by same-charge repulsion and promote heterophilic Dsg:Dsc interactions through opposite-charge attraction. These findings show that Dsg:Dsc heterodimers represent the fundamental adhesive unit of desmosomes and provide a structural framework for understanding desmosome assembly.


Assuntos
Adesivos/química , Desmocolinas/química , Desmogleínas/química , Adesivos/metabolismo , Desmocolinas/metabolismo , Desmogleínas/metabolismo , Dimerização , Humanos , Cinética , Conformação Proteica
17.
Neuron ; 90(4): 709-23, 2016 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-27161523

RESUMO

Clustered protocadherin proteins (α-, ß-, and γ-Pcdhs) provide a high level of cell-surface diversity to individual vertebrate neurons, engaging in highly specific homophilic interactions to mediate important roles in mammalian neural circuit development. How Pcdhs bind homophilically through their extracellular cadherin (EC) domains among dozens of highly similar isoforms has not been determined. Here, we report crystal structures for extracellular regions from four mouse Pcdh isoforms (α4, α7, ß6, and ß8), revealing a canonical head-to-tail interaction mode for homophilic trans dimers comprising primary intermolecular EC1:EC4 and EC2:EC3 interactions. A subset of trans interface residues exhibit isoform-specific conservation, suggesting roles in recognition specificity. Mutation of these residues, along with trans-interacting partner residues, altered the specificities of Pcdh interactions. Together, these data show how sequence variation among Pcdh isoforms encodes their diverse strict homophilic recognition specificities, which are required for their key roles in neural circuit assembly.


Assuntos
Sequência de Aminoácidos/fisiologia , Caderinas/química , Caderinas/metabolismo , Neurônios/metabolismo , Células Cultivadas , Humanos , Rede Nervosa/metabolismo , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/metabolismo
18.
Cell ; 163(3): 629-42, 2015 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-26478182

RESUMO

Self-avoidance, a process preventing interactions of axons and dendrites from the same neuron during development, is mediated in vertebrates through the stochastic single-neuron expression of clustered protocadherin protein isoforms. Extracellular cadherin (EC) domains mediate isoform-specific homophilic binding between cells, conferring cell recognition through a poorly understood mechanism. Here, we report crystal structures for the EC1-EC3 domain regions from four protocadherin isoforms representing the α, ß, and γ subfamilies. All are rod shaped and monomeric in solution. Biophysical measurements, cell aggregation assays, and computational docking reveal that trans binding between cells depends on the EC1-EC4 domains, which interact in an antiparallel orientation. We also show that the EC6 domains are required for the formation of cis-dimers. Overall, our results are consistent with a model in which protocadherin cis-dimers engage in a head-to-tail interaction between EC1-EC4 domains from apposed cell surfaces, possibly forming a zipper-like protein assembly, and thus providing a size-dependent self-recognition mechanism.


Assuntos
Caderinas/química , Caderinas/metabolismo , Neurônios/citologia , Neurônios/fisiologia , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fenômenos Fisiológicos do Sistema Nervoso , Estrutura Terciária de Proteína , Alinhamento de Sequência
19.
Nat Struct Mol Biol ; 22(7): 522-31, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26098315

RESUMO

As the sole viral antigen on the HIV-1-virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1-Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Antígenos CD4/imunologia , Cristalografia por Raios X , Epitopos/imunologia , Células HEK293 , Infecções por HIV/virologia , HIV-1/química , HIV-1/imunologia , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Internalização do Vírus
20.
Proc Natl Acad Sci U S A ; 111(40): E4175-84, 2014 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-25253890

RESUMO

Type I cadherin cell-adhesion proteins are similar in sequence and structure and yet are different enough to mediate highly specific cell-cell recognition phenomena. It has previously been shown that small differences in the homophilic and heterophilic binding affinities of different type I family members can account for the differential cell-sorting behavior. Here we use a combination of X-ray crystallography, analytical ultracentrifugation, surface plasmon resonance and double electron-electron resonance (DEER) electron paramagnetic resonance spectroscopy to identify the molecular determinants of type I cadherin dimerization affinities. Small changes in sequence are found to produce subtle structural and dynamical changes that impact relative affinities, in part via electrostatic and hydrophobic interactions, and in part through entropic effects because of increased conformational heterogeneity in the bound states as revealed by DEER distance mapping in the dimers. These findings highlight the remarkable ability of evolution to exploit a wide range of molecular properties to produce closely related members of the same protein family that have affinity differences finely tuned to mediate their biological roles.


Assuntos
Caderinas/química , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Ligação Competitiva , Caderinas/genética , Caderinas/metabolismo , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Xenopus , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
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