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STUDY GUIDE NO. 3. Autism and the Structure of Neurexin:Neuroligin.

Assigned Readings:

1. Sudhof, T. Neuroligins and Neurexins Link Synaptic Function to Cognitive Disease Nature 455, 903-911 (2008).

2. Arac, D. at al.  Structures of Neuroligin-1 and the Neuroligin-1/Neurexin-1β Complex Reveal Specific Protein-Protein and Protein-Ca2+ Interactions. Neuron 56:992-1003 (2007).

3. Fabrichny et al. Structural Analysis of the Synaptic Protein Neuroligin and Its β-Neurexin Complex: Determinants for Folding and Cell Adhesion. Neuron 56:979-991 (2007).

4. Chih et al. Control of Excitatory and Inhibitory Synapse Formation by Neuroligins. Science 307:1324-1328 (2005).

5. Jamain et al. Reduced Social Interaction and Ultrasonic Communication in a Mouse Model of Monogenic Heritable Autism. PNAS 105:1710-1715 (2008).

Resources:

www.rcsb.org   Rutgers Center for Structural Biology. Database of biological structures.

www.ncbi.nlm.nih.gov/omim/  Online Mendelian Disorders in Man. Database of  medical and genetic information on inherited human diseases.

www.proteopedia.org Proteopedia

Concise answers to the Study Guide questions and molecular images resulting from the modeling exercises are available on the Proteopedia website. Simply type in ‘autism’ in the search bar.

Questions:

1. Autism. Read carefully the review article by Thomas Sudhof. As you read his review, make note of how he uses the insights of structural biology to make his case that autism may be due to subtle alterations in synaptic chemistry. Autism has been described as a condition due to a disturbance in the ratio of excitatory to inhibitory neurotransmission (E/I) in the brain. Natural questions arise: Is this disturbed ratio found throughout the brain? How can it be measured? Does this theory potentially explain the triad of social, communication, and behavioral dysfunctions in autism? Do the gene mutations and copy number variants that have been linked to autism offer any explanations? How can a disturbed E/I influence the course of neurological and cognitive development? Is it possible to develop a mouse model mirroring the complex communication (language) and social abilities (Facebook participation) of the human being?

2. Neurexin:Neuroligin. Describe the neuroligin family of synaptic adhesion proteins. What role does each member play in the specification of synaptic type (E or I) and spine number? Image of NRXN-NRGLNWhat is the topological fold of the extracellular domain of neuroligin? How does it differ in structure from acetylcholinesterase (AchE), the canonical member of the α/β hydroylase superfamily? What is the main functional difference between NLGN-1 and NLGN-2?

Describe the domain structure for the neurexin-α family? What is the canonical LNS domain? What is an EGF-binding domain? What are the extracellular matrix proteins that bind to the neurexins? What do they recognize on the surface of the neurexins? What does the simpler neurexin-β structure have in common with the neurexin-α family?

It is estimated that there are over 2000 isoforms of neurexin expressed in the human brain. Where is this structural variation in the amino sequence and how do these map onto in the structure of the protein? What is the molecular mechanism for generating this diversity? Sudhof argues that cognitive disorders could be due to subtle abnormalities at the synapse attributable to mutations or deletions that localize to the neuroligin:neurexin interaction. What examples does he give? If these proteins regulate the E/I neurotransmission ratio, how could signaling in local microcircuits be affected? (HINT: microcircuits consist of neurons that feed back on one another to generate rhythmic bursting).

3. Mouse models.  Go to OMIM and key in 300336.001 or neuroligin R451C. This will open up a wealth of information about the specifics of this mutation and a publication trail to match. Tabuchi et al. generated a NLGN3-1 mouse  model with an Arg451 to Cys451 mutation that has been seen in a small number of individuals with autism. R451C is a gain-of-function mutation. What is the gained function? That is, what cognitive tests were used to demonstrate gain of function? What is unusual about protein expression levels of NLGN-3 in R451C mutated mice? How was the homology to AchE used to work out the explanation for this phenomenon? In what crucial way is the R451C mouse different from other ASD models in terms of synaptic function? Does the R451C mutant have normal dendritic spine morphology?  How does one prove that there is an increase or a decrease in inhibitory synapses?  Excitatory synapses? Referring back to Huda Zoghbi’s paper (Study Guide for Week No. 1), do the present results support her hypothesis for the etiology of autism?

Jamain et al. have engineered a NLGN-4 knockout mouse that appears to have two of  the  three hallmarks of autism: reduced reciprocal social interaction and impairments in vocal communication. List all of the behavioral tests performed on these animals and give a brief (three sentence) description of the results. Describe the observational basis for the claim that the NLGN-4 KO mice have impaired social vocalization. In what regions of the brain is NLGN-4 most highly expressed? What morphological or cytoarchitectonic changes in the brain occur in the KO mice? Are these consistent with autism in humans? What arguments do the authors make for this being a superior model for non-syndromic  autism compared with models of Rett syndrome, Fragile-X, Tuberous Sclerosis and neurofibromatosis?

4. Neurexin:Neuroligin Structures. Using a molecular modeling program (such as SPDBV) consider the structure of the LNS domain from neurexin-1β (PDP Code 1C4R) described by Rudenko et al. (Cell 99,  pp 93-101, (1999)) A special feature of this structure compared to other β-jelly-rolls is a salt-bridge between Asp-111 and Arg-180. Check out this interaction for other special stabilizing interactions (hydrophobic or H-bonds). Another unusual feature of neuurexins is a cluster of three amino acids Asp-170, His-174 and Asp-190. Note how the histidine is sandwiched between the two aspartic acid residues. Examine the relationship between this highly charged cluster and the β-arch connecting β7 and β8 and the tight turn between β9 and β10. These forces constrain the positions of the amino acids in the vicinity. Label with color strands β7 and β8, and with another color strands β9 and β10.

Build the hydrophobic core of neurexin-1β:

CORE RESIDUES: (L113,I115,V125,V126,L127,L139,I148,V150,V176,I244,I245,Y263)

Now, take a look at the crystal structure of the NLGN-4:NRXN-1β complex (Fabrichny et al.; PDB code 2WQZ). NLGN is a member of the α/β hydroylase superfamily exemplified by AchE. Compared with the other members of this superfamily NLGN has a unique disulfide bridge (Cys476-Cys510) that constrains the structure of LOOP 4 (Gly503-Ser523). LOOP 4 in turn stabilizes LOOP 3 (Gln477-Ser487).  The significance of this is that LOOP 4 contains Trp 484 which makes a neat stacking interaction with Arg 437 and Val 377.  (Arg 437 in NLGN4 is homologous to Arg 451 in NLGN3 discussed by Tabuchi et al.). Locate these residues on the molecular model. An R377C mutant is associated (rare case) with autism. Create a molecular image of this interaction. You may wish to consider Asp388, Glu434, Lys338. Another mutation related to autism is Lys 378. Where is it in relation to the above-mentioned structures?

Another neuroligin mutation V403M is linked to a rare case of autism. Where does Val403 reside in the structure? Does the mutation help explain the occurrence of autism in a person harboring this mutation? Build a molecular model showing the environment around Val403. (HINT: disruption of a key hydrophobic ‘core’).

The neuroligin:neurexin interface is stabilized by two Ca++ cations. Using the crystal structure of the complex (Arac et al.; PDB Code 3BIW), build this interfacial contact. Start with a stick figure representation of the loop Q395 to D402 in the context of ribbons for the rest of the two molecules. (NOTE: use chain A and Chain E in the PDB file, the calcium coordinates are found at the end of the E file). Describe the coordination about each of the calcium cations. What is the architectural relationship between this interface and splice site B on NLGN? (HINT: consider the effect of mutations E397A, K306A). What are the effects of the mutations L399A,N400A,D402N on the structure of the interface? Now build the other half of the interface using NRXN residues N103,S107,R109,L135,R232,I236,N238. Does R232 form a salt-bridge?

What is an EF-hand? What is unusual about the EF-hand in the neuroligin structure?

 

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