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Structure of Synaptic Connectors Solved Print
Wednesday, 30 April 2008 00:00

 

Establishment of neural connections at specialized intercellular junctions called synapses is critical for proper brain function, and errors in the process are thought to be associated with autism and other disorders. Researchers from Stanford University and the University of Texas Southwestern Medical Center have reported high-resolution, three-dimensional structures of the proteins, called neuroligin-1 and neurexin-1β, that form this connection. Because mutations in the neurexin and neuroligin genes are among the multiple genetic causes of autism, understanding the molecular mechanism of these proteins in synapse development is a first step towards development of novel therapeutics directed to treat and possibly cure autism.

Structures of neuroligin 1 and the neuroligin-1/neurexin-1β complex. (A) Two views of a ribbon representation of a neuroligin-1 monomer showing major structural features, such as α-helices (orange) and β-sheets (cyan). Each view is rotated by 180° relative to the other around the axis shown. (B) Ribbon representation of the neuroligin 1/neurexin 1β heterotetramer. (C) Overall view of a neuroligin 1/neurexin 1β heterodimer showing carbohydrates (yellow sticks), Ca2+ ions at the binding interface (green spheres), and splice sites SS4 of neurexin-1β and SSB of neuroligin-1 (arrows).

On the Road to Curing Autism?

Appearing in the first three years of a child's life, autism is a developmental disorder of the brain that impairs social interactions and causes communication deficits and repetitive behaviors. About 1 in every 150 children is affected by the disorder. At present, there is no cure. Genetic factors are the most significant causes of the spectrum of autism disorders, which include autism itself and the milder Asperger syndrome, as well as mental retardation. While the genetics of autism spectrum disorders are complex, it is known that mutations in the genes of two proteins called neurexin and neuroligin are among the multiple genetic causes.

To gain a better understanding of how the two proteins interact, Araç et al. have investigated the molecular structures of a specific protein, neuroligin-1, and the complex of neuroligin-1 and neurexin-1β. The structures allowed them to visualize the interfaces between the two neuroligin and two neurexin molecules in the four-molecule complex, as well as binding sites for calcium, which is required for formation of the complex. The new structural information and an understanding of how mutations affect that structure could offer clues into how the complex might have been disrupted in individuals with autism, leading to possible future pharmaceutical approaches to treating autism that stabilize such interactions and compensate for the effect of the mutations.

In the presynaptic neuron, the connector protein is called neurexin; its partner on the postsynaptic neuron is known as neuroligin. Past studies had indicated that neuroligins and neurexins establish their connection by forming a complex in which two neuroligin molecules link to each other, and a neurexin molecule attaches to each side of the pair. As neurons create new synapses during learning, they must form this neuroligin–neurexin connection for those synapses to become functionally mature.

Problems with this process of synapse development are thought to be a major cause of brain disorders such as autism and mental retardation. To gain a better understanding of how the two proteins interact, the Stanford–Texas team investigated the molecular structure of neuroligin-1 itself, as well as the complex of neuroligin-1 and neurexin-1β, which allowed them to visualize the interfaces between the two neuroligin and two neurexin molecules in the four-molecule complex, as well as binding sites for calcium, which is required for formation of the complex. X-ray crystallography data were gathered at ALS Beamlines 8.2.1 and 8.2.2 and Stanford Synchrotron Radiation Laboratory Beamline 11-1.

The structure of the neuroligin-1 bound to neurexin-1β revealed that two neurexin-1β molecules bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1β complex exhibits high affinity, and includes a large binding interface that contains bound Ca2+. Spliced sites (SS4 and SSB) in neurexin-1β and in neuroligin-1 are positioned near the binding interface and regulate the strength of the interaction. The neuroligin point mutations that were found in some autism patients are located away from the binding interface and face the inside of the protein, implying that these mutations most likely affect the folding of neuroligin rather than its interaction with neurexin.

Based on this structure, the researchers suggest a model of the structural organization of these cell-adhesion proteins at the synaptic junction: The arrangement of the neuroligin-1/neurexin-1β complex positions features called the C-terminal stalk regions of neuroligin-1 and neurexin-1β at opposite faces of the heterotetramer. In this way, the complex bridges the 15-to-20-nm distance between the pre- and post-synapic membranes by tethering to these membranes through the stalk regions of neuroligin-1 and neurexin-1β.

Model for the arrangement of the neuroligin-1/neurexin-1β complex at the synapse. Neurexins are tethered to the pre-synaptic cell membrane and neuroligins are tethered to the post-synaptic cell membrane by their stalk regions. Their interaction provides trans-synaptic connectivity.

To determine whether the structure they had developed reflected the actual behavior of the proteins, the researchers mutated neuroligin-1 at locations they predicted would disrupt the interface between it and neurexin. The mutations not only reduced the affinity of neuroligin-1 for neurexin-1β up to three orders of magnitude and confirmed the binding interface revealed by the neuroligin-1/neurexin-1β complex structure, but also gave the researchers molecular tools to probe the details of this interaction.

The new structural information and an understanding of how mutations affect that structure could offer clues into how these proteins might have been disrupted in individuals with autism and provide molecular insights for understanding the role of the neuroligin/neurexin interaction in synapse function. Eventually, it may be possible to develop pharmaceutical approaches to treating autism by stabilizing such interactions and compensating for the effect of the mutations.

 


 

Research conducted by D. Araç, P. Strop, and A. Brunger (Howard Hughes Medical Institute and Stanford University); A.A. Boucard and T.C. Südhof (Howard Hughes Medical Institute and the University of Texas Southwestern Medical Center); and Engin Özkan and E. Newell (Stanford University)

Research Funding: Canadian Institutes of Health Research, U.S. National Institute of Mental Health, Life Sciences Research Foundation, and the Howard Hughes Medical Institute. Operation of the ALS and SSRL is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: D. Araç, A.A. Boucard, E. Özkan, P. Strop, E. Newell, T.C. Südhof, and A.T. Brunger, "Structures of Neuroligin-1 and the Neuroligin-1/Neurexin-1β complex reveal specific protein-protein and protein-Ca2+ interactions," Neuron 56, 992 (2007).