Structures of Neuroligin-1 and the Neuroligin-1/Neurexin-1β Complex Reveal Specific Protein-Protein and Protein-Ca 2+ Interactions (original) (raw)
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Neuron, 2007
Neurexins and neuroligins provide trans-synaptic connectivity by the Ca 2+-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1b. Neuroligin-1 forms a constitutive dimer, and two neurexin-1b monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1b complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca 2+. Alternatively spliced sites in neurexin-1b and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1b, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1b complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.
Neuron, 2007
The neuroligins are postsynaptic cell adhesion proteins whose associations with presynaptic neurexins participate in synaptogenesis. Mutations in the neuroligin and neurexin genes appear to be associated with autism and mental retardation. The crystal structure of a neuroligin reveals features not found in its catalytically active relatives, such as the fully hydrophobic interface forming the functional neuroligin dimer; the conformations of surface loops surrounding the vestigial active center; the location of determinants that are critical for folding and processing; and the absence of a macromolecular dipole and presence of an electronegative, hydrophilic surface for neurexin binding. The structure of a β-neurexin-neuroligin complex reveals the precise orientation of the bound neurexin and, despite a limited resolution, provides substantial information on the Ca 2+ -dependent interactions network involved in trans-synaptic neurexin-neuroligin association. These structures exemplify how an α/β-hydrolase fold varies in surface topography to confer adhesion properties and provide templates for analyzing abnormal processing or recognition events associated with autism.
Neuron, 2017
Membrane-associated mucin domain-containing glycosylphosphatidylinositol anchor proteins (MDGAs) bind directly to neuroligin-1 (NL1) and neuroligin-2 (NL2), thereby respectively regulating excitatory and inhibitory synapse development. However, the mechanisms by which MDGAs modulate NL activity to specify development of the two synapse types remain unclear. Here, we determined the crystal structures of human NL2/MDGA1 Ig1-3 complex, revealing their stable 2:2 arrangement with three interaction interfaces. Cell-based assays using structure-guided, site-directed MDGA1 mutants showed that all three contact patches were required for the MDGA's negative regulation of NL2-mediated synaptogenic activity. Furthermore, MDGA1 competed with neurexins for NL2 via its Ig1 domain. The binding affinities of both MDGA1 and MDGA2 for NL1 and NL2 were similar, consistent with the structural prediction of similar binding interfaces. However, MDGA1 selectively associated with NL2, but not NL1, in v...
Structural insights into the exquisite selectivity of neurexin/neuroligin synaptic interactions
The EMBO Journal, 2010
The extracellular domains of neuroligins and neurexins interact through Ca 2 þ to form flexible trans-synaptic associations characterized by selectivity for neuroligin or neurexin subtypes. This heterophilic interaction, essential for synaptic maturation and differentiation, is regulated by gene selection, alternative mRNA splicing and post-translational modifications. A new, 2.6 Å-resolution crystal structure of a soluble neurexin-1b-neuroligin-4 (Nrx1b-NL4) complex permits a detailed description of the Ca 2 þcoordinated interface and unveils concerted positional rearrangements of several residues of NL4, not observed in neuroligin-1, associated with Nrx1b binding. Surface plasmon resonance analysis of the binding of structureguided Nrx1b mutants towards NL4 and neuroligin-1 shows that flexibility of the Nrx1b-binding site in NL4 is reflected in a greater dissociation constant of the complex and higher sensitivity to ionic strength and pH variations. Analysis of neuroligin mutants points to critical functions for two respective residues in neuroligin-1 and neuroligin-2 in governing the affinity of the complexes. Although neuroligin-1 and neuroligin-2 have pre-determined conformations that respectively promote and prevent Nrx1b association, unique conformational reshaping of the NL4 surface is required to permit Nrx1b association.
Mutational analysis of the neurexin/neuroligin complex reveals essential and regulatory components
Proceedings of the National Academy of Sciences, 2008
Neurexins are cell-surface molecules that bind neuroligins to form a heterophilic, Ca 2؉ -dependent complex at central synapses. This transsynaptic complex is required for efficient neurotransmission and is involved in the formation of synaptic contacts. In addition, both molecules have been identified as candidate genes for autism. Here we performed mutagenesis experiments to probe for essential components of the neurexin/neuroligin binding interface at the single-amino acid level. We found that in neurexins the contact area is sharply delineated and consists of hydrophobic residues of the LNS domain that surround a Ca 2؉ binding pocket. Point mutations that changed electrostatic and shape properties leave Ca 2؉ coordination intact but completely inhibit neuroligin binding, whereas alternative splicing in ␣and -neurexins and in neuroligins has a weaker effect on complex formation. In neuroligins, the contact area appears less distinct because exchange of a more distant aspartate completely abolished binding to neurexin but many mutations of predicted interface residues had no strong effect on binding. Together with calculations of energy terms for presumed interface hot spots that complement and extend our mutagenesis and recent crystal structure data, this study presents a comprehensive structural basis for the complex formation of neurexins and neuroligins and their transsynaptic signaling between neurons.
Direct Visualization of Trans-Synaptic Neurexin-Neuroligin Interactions during Synapse Formation
The Journal of Neuroscience, 2014
Neurexins and neuroligins are synaptic cell-adhesion molecules that are essential for normal synapse specification and function and are thought to bind to each other trans-synaptically, but such interactions have not been demonstrated directly. Here, we generated neurexin-1 and neuroligin-1 and neuroligin-2 fusion proteins containing complementary "split" GFP fragments positioned such that binding of neurexin-1 to neuroligin-1 or neuroligin-2 allowed GFP reconstitution without dramatically changing their binding affinities. GFP fluorescence was only reconstituted from split-GFP-modified neurexin-1 and neuroligin-1 if and after neurexin-1 bound to its neuroligin partner; reassociation of the split-GFP components with each other did not mediate binding. Using trans-cellular reconstitution of GFP fluorescence from split-GFP-modified neurexin-1 and neuroligins as an assay, we demonstrate that trans-synaptic neurexin/neuroligin binding indeed occurred when mouse hippocampal neurons formed synapses onto non-neuronal COS-7 cells expressing neuroligins or when mouse hippocampal neurons formed synapses with each other. This visualization of synapses by neurexin/ neuroligin binding prompted us to refer to this approach as "SynView." Our data demonstrate that neurexin-1 forms a trans-synaptic complex with neuroligin-1 and neuroligin-2 and that this interaction can be used to label synapses in a specific fashion in vivo.
Characterization of the solution structure of a neuroligin/β-neurexin complex
Chemico-Biological Interactions, 2008
Neuroligins are postsynaptic cell adhesion molecules that promote synaptic maturation and stabilization upon binding with presynaptic partners, the αand β-neurexins. Using a combination of analytical ultracentrifugation, small angle x-ray, and neutron scattering, we have characterized the low-resolution three-dimensional structure of the extracellular domain of the neuroligins, free in solution, and in complex with β-neurexin. The globular extracellular domain of the neuroligins forms stable homodimers through a four-helix bundle typical of the cholinesterases and other members of the α/β-hydrolase fold family. The presence of the stalk region adds to the extracellular domain of neuroligin-1 an elongated structure, suggesting a rod-like nature of the stalk domain. Sedimentation equilibrium coupled with solution scattering data of the β-neurexin/neuroligin-1 complex indicated a 2:2 stoichiometry where two β-neurexin molecules bind to a neuroligin-1 dimer. Deuteration of neurexin allowed us to collect neutron scattering data that, in combination with other biochemical techniques, provide a basis for optimizing the positioning of each component in a higher resolution computational model of the neuroligin/neurexin complex. As several mutations of both neurexin and neuroligin genes have been linked to autism spectrum disorders and mental retardation, these new structures provide an important framework for the study of altered structure and function of these synaptic proteins.
Neuron, 2005
Previous studies suggested that postsynaptic neuroligins form a trans-synaptic complex with presynaptic b-neurexins, but not with presynaptic a-neurexins. Unexpectedly, we now find that neuroligins also bind a-neurexins and that a-and b-neurexin binding by neuroligin 1 is regulated by alternative splicing of neuroligin 1 (at splice site B) and of neurexins (at splice site 4). In neuroligin 1, splice site B is a master switch that determines a-neurexin binding but leaves b-neurexin binding largely unaffected, whereas alternative splicing of neurexins modulates neuroligin binding. Moreover, neuroligin 1 splice variants with distinct neurexin binding properties differentially regulate synaptogenesis: neuroligin 1 that binds only b-neurexins potently stimulates synapse formation, whereas neuroligin 1 that binds to both a-and b-neurexins more effectively promotes synapse expansion. These findings suggest that neuroligin binding to a-and b-neurexins mediates trans-synaptic cell adhesion but has distinct effects on synapse formation, indicating that expression of different neuroligin and neurexin isoforms specifies a trans-synaptic signaling code.
Structure, 2007
Neuroligins are postsynaptic cell adhesion proteins that associate with their presynaptic partners, the neurexins. Using small-angle X-ray scattering, we determined the shapes of the extracellular region of several neuroligin isoforms in solution. We conclude that the neuroligins dimerize via the characteristic four-helix bundle observed in other cholinesterases and that the connecting sequence between the globular lobes of the dimer and the cell membrane is elongated, projecting away from the dimer interface. X-ray scattering and neutron contrast variation data show that two neurexin monomers bind at symmetric locations on opposite sides of the long axis of the neuroligin dimer, separated by 107 Å. Using these data, we develop structural models that delineate the spatial arrangements of different neuroligin domains and their partnering molecules. As mutations of neurexin and neuroligin genes appear linked to autism, these models provide a structural framework for understanding altered recognition by these proteins in neuro-developmental disorders.