RESUMO
The cell-surface attached glycoprotein contactin 2 is ubiquitously expressed in the nervous system and mediates homotypic cell-cell interactions to organize cell guidance, differentiation, and adhesion. Contactin 2 consists of six Ig and four fibronectin type III domains (FnIII) of which the first four Ig domains form a horseshoe structure important for homodimerization and oligomerization. Here we report the crystal structure of the six-domain contactin 2Ig1-6 and show that the Ig5-Ig6 combination is oriented away from the horseshoe with flexion in interdomain connections. Two distinct dimer states, through Ig1-Ig2 and Ig3-Ig6 interactions, together allow formation of larger oligomers. Combined size exclusion chromatography with multiangle light scattering (SEC-MALS), small-angle X-ray scattering (SAXS) and native MS analysis indicates contactin 2Ig1-6 oligomerizes in a glycan dependent manner. SAXS and negative-stain electron microscopy reveals inherent plasticity of the contactin 2 full-ectodomain. The combination of intermolecular binding sites and ectodomain plasticity explains how contactin 2 can function as a homotypic adhesion molecule in diverse intercellular environments.
Assuntos
Moléculas de Adesão Celular Neuronais , Contactina 2 , Espalhamento a Baixo Ângulo , Difração de Raios X , Sítios de Ligação , Conformação Molecular , Moléculas de Adesão Celular Neuronais/química , Adesão Celular/fisiologiaRESUMO
Cell-surface expressed contactin 1 and neurofascin 155 control wiring of the nervous system and interact across cells to form and maintain paranodal myelin-axon junctions. The molecular mechanism of contactin 1 - neurofascin 155 adhesion complex formation is unresolved. Crystallographic structures of complexed and individual contactin 1 and neurofascin 155 binding regions presented here, provide a rich picture of how competing and complementary interfaces, post-translational glycosylation, splice differences and structural plasticity enable formation of diverse adhesion sites. Structural, biophysical, and cell-clustering analysis reveal how conserved Ig1-2 interfaces form competing heterophilic contactin 1 - neurofascin 155 and homophilic neurofascin 155 complexes whereas contactin 1 forms low-affinity clusters through interfaces on Ig3-6. The structures explain how the heterophilic Ig1-Ig4 horseshoe's in the contactin 1 - neurofascin 155 complex define the 7.4 nm paranodal spacing and how the remaining six domains enable bridging of distinct intercellular distances.
Assuntos
Moléculas de Adesão Celular , Contactina 1 , Moléculas de Adesão Celular/metabolismo , Fatores de Crescimento Neural/metabolismo , Contactinas , Axônios/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismoRESUMO
Type-I transmembrane proteins represent a large group of 1,412 proteins in humans with a multitude of functions in cells and tissues. They are characterized by an extracellular, or luminal, N-terminus followed by a single transmembrane helix and a cytosolic C-terminus. The domain composition and structures of the extracellular and intercellular segments differ substantially amongst its members. Most of the type-I transmembrane proteins have roles in cell signaling processes, as ligands or receptors, and in cellular adhesion. The extracellular segment often determines specificity and can control signaling and adhesion. Here we focus on recent structural understanding on how the extracellular segments of several diverse type-I transmembrane proteins engage in interactions and can undergo conformational changes for their function. Interactions at the extracellular side by proteins on the same cell or between cells are enhanced by the transmembrane setting. Extracellular conformational domain rearrangement and structural changes within domains alter the properties of the proteins and are used to regulate signaling events. The combination of structural properties and interactions can support the formation of larger-order assemblies on the membrane surface that are important for cellular adhesion and intercellular signaling.
RESUMO
Signaling of SorCS receptors by proneurotrophin ligands regulates neuronal plasticity, induces apoptosis and is associated with mental disorders. The detailed structure of SorCS2 and its extracellular specificity are unresolved. Here we report crystal structures of the SorCS2-NGF complex and unliganded SorCS2 ectodomain, revealing cross-braced SorCS2 homodimers with two NGF dimers bound in a 2:4 stoichiometry. Five out of six SorCS2 domains directly contribute to dimer formation and a C-terminal membrane proximal unreported domain, with an RNA recognition motif fold, locks the dimer in an intermolecular head-to-tail interaction. The complex structure shows an altered SorCS2 conformation indicating substantial structural plasticity. Both NGF dimer chains interact exclusively with the top face of a SorCS2 ß-propeller. Biophysical experiments reveal that NGF, proNGF, and proBDNF bind at this site on SorCS2. Taken together, our data reveal a structurally flexible SorCS2 receptor that employs the large ß-propeller as a ligand binding platform.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/química , Fator de Crescimento Neural/química , Proteínas do Tecido Nervoso/química , Receptores de Superfície Celular/química , Motivos de Aminoácidos , Animais , Biofísica , Cristalografia por Raios X , Células HEK293 , Humanos , Ligantes , Camundongos , Mutação , Plasticidade Neuronal , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Transdução de Sinais , Ressonância de Plasmônio de SuperfícieRESUMO
Whirlin is a protein essential to sensory neurons. Its defects are responsible for nonsyndromic deafness or for the Usher syndrome, a condition associating congenital deafness and progressive blindness. This large multidomain scaffolding protein is expressed in three isoforms with different functions and localizations in stereocilia bundles of hearing hair cells or in the connecting cilia of photoreceptor cells. The HHD2 domain of whirlin is the only domain shared by all isoforms, but its function remains unknown. In this article, we report its crystal structure in two distinct conformations, a monomeric five-helix bundle, similar to the known structure of other HHD domains, and a three-helix bundle organized as a swapped dimer. Most of the hydrophobic contacts and electrostatic interactions that maintain the globular monomeric form are conserved at the protomer interface of the dimer. NMR experiments revealed that the five-helix conformation is predominant in solution, but exhibits increased dynamics on one face encompassing the hinge loops. Using NMR and SAXS, we also show that HHD2 does not interact with its preceding domains. Our findings suggest that structural plasticity might play a role in the function of the HHD2 domain.
Assuntos
Proteínas de Membrana/química , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Espalhamento a Baixo Ângulo , Homologia de SequênciaRESUMO
Calexcitin was first identified in the marine snail Hermissenda crassicornis as a neuronal-specific protein that becomes upregulated and phosphorylated in associative learning. Calexcitin possesses four EF-hand motifs, but only the first three (EF-1 to EF-3) are involved in binding metal ions. Past work has indicated that under physiological conditions EF-1 and EF-2 bind Mg(2+) and Ca(2+), while EF-3 is likely to bind only Ca(2+). The fourth EF-hand is nonfunctional owing to a lack of key metal-binding residues. The aim of this study was to use a crystallographic approach to determine which of the three metal-binding sites of calexcitin is most readily replaced by exogenous metal ions, potentially shedding light on which of the EF-hands play a `sensory' role in neuronal calcium signalling. By co-crystallizing recombinant calexcitin with equimolar Gd(3+) in the presence of trace Ca(2+), EF-1 was shown to become fully occupied by Gd(3+) ions, while the other two sites remain fully occupied by Ca(2+). The structure of the Gd(3+)-calexcitin complex has been refined to an R factor of 21.5% and an Rfree of 30.4% at 2.2â Å resolution. These findings suggest that EF-1 of calexcitin is the Ca(2+)-binding site with the lowest selectivity for Ca(2+), and the implications of this finding for calcium sensing in neuronal signalling pathways are discussed.