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STUDY GUIDE NO. 2. Tuberous Sclerosis and the Structure of Eph4A:Ephrin

Assigned Readings:

1. Nie et al., Tsc2-Rheb signaling regulates Eph-A-mediated axon guidance. Nat. Neuroscience 13:163-19 (2010).

2. Bowden et al., Structural Plasticity of Eph Receptor A4 Facilitates Cross-Class Ephrin Signaling. Structure 17:1386-1397 (2009).

3. Hinanen et al., Architecture of Eph receptor clusters, P.N.A.S. (USA) 107:10860-10865 (2010).

4. Carmona et al., Glial ephrin-A3 regulates hippocampal spine morphology and glutamate transport. PNAS 106:1252-12529 (2009).

5. Ehninger et al., From mTOR to cognition: molecular and cellular mechanisms of cognitive impairments in tuberous sclerosis. J. Int. Disabilities Res. 53:838-851 (2009).

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.Tuberous Sclerosis. Describe the clinical symptoms of tuberous sclerosis. What is a hamartoma? What are the functions of the gene products of TSC1/TSC2 (hamartin & tuberin)? What are the chromosomal locations of TSC1 and TSC2?  What is the ‘misconnection hypothesis’ of autism spectrum disorders (ASDs)?

2.Eph:Ephrin. Describe the organization into structural domains of the proteins comprising the ephrin:eph system of  tyrosine kinase receptors. Image of Eph-Ephrin What are the differences between the two general classes (A&B)? What ideas have been put forth to explain the role of ephrin receptors (ephs) in forming functional clusters in the post-synaptic density? What is meant by ‘bi-directional signaling’ in the ephrin:eph signaling system? Give an example (hint: hippocampal glial cells control local spine morphology). What is the role of ephrin in dictating axonal path-finding (hint: describe the mechanism by which growth cones are repelled)? What are the key phosphorylation steps in transducing the signal from ephrin receptor (eph) to the hamartin:tuberin complex? What are the downstream effects of inactivating TSC1? What is mTOR? Does this explain ‘growth cone collapse’? How do Nie et al. use mouse models to demonstrate that the ephrin:eph system controls axon guidance? Is it likely that these observations generalize to the development of other pathways in the nervous system (thalamocortical e.g.)? Why does rapamycin have therapeutic potential for treating adults with tuberous sclerosis (hint: how does mTOR gets its name?)?

3. Mouse model. In the ephrin-A3 knockout mice described by Carmona et al., what are the changes seen in overall hippocampal cytoarchitecture? How does the appearance of dendritic spines differ from wild-type mice (Fig. 1)?  How does this phenotype compare with observations of spines in eph4A-knockout mice? Does the lack of ephrin-A3 signaling influence the structure of the post-synaptic density? How is this demonstrated? Are there any changes in the levels of eph4A phosphorylation? Is this surprising? How do Carmona et al. prove that ephrin-A3 co-localises with GLAST, the glutamic acid/aspartate transporter? Ephrin-3A does not appear to co-localize with PSD-95 or VGLUT-1 (vesicular glutamate transporter 1). Instead cells stained for ephrin-3A surround PSD-95/eph4A puncta. What is the nature of the ephrin bi-directional signal into the glial cell? Carmona et al. are rather specific about the type of cognitive deficit seen in the ephrin-3A knockout mice. On what basis do they conclude that learning from cues is normal in the KO mice, whereas contextual learning is impaired? What brain circuits are altered in the KO compared with the WT?

4. Structure. What does it mean that EphA4 is a ‘structural chameleon’? Bowden et al. present the results of three separate crystal structure determinations: the free ligand binding domain of ephrin-A3, and two bound forms with ephrinA2 and ephrinB2. This enables comparisons between the bound and unbound states, and a determination of structural hallmarks that distinguish between class A and class B ephrins. Describe the topology of the ephrin ligands. Describe the topology of the eph4A receptor (hint: what is a Swiss jelly-roll fold?). What is a CRD? Extract the CRD coordinates from the Eph2A coordinate file and display the structure in a manner that emphasizes the disulphide bonds.

Use SPDBV or other modeling program to depict the interactions that stabilize the extended loop of ephrinB2 in the binding pocket of ephA2. In particular show how W122 (ephrinB2) stacks against R162 (Eph4A). Does R162 make any salt-bridges? What other hydrophobic residues form this stabilizing cluster? Carry out the same analysis for the binding of ephrinA2 to ephA4. What interactions does R122 make? Is the salt-bridge still there? What are the key hydrophobic residues on ephrinA2 that occupy the same position as W122?  These two residues are separated by one amino acid, what role (if any) does this intervening residue play in stabilizing the interaction (HINT: H-bond)?

Describe the changes that occur on the two sides of the binding jaws of eph4A (DE and JK loops).Using the Magic Fit Option in the Display Window of SPDBV reproduce Figures 1B and 1D of Bowden et al. This will reveal the structural plasticity in the Eph2A binding loop (JK).

EphA2:ephrinA1 forms heterotetramers in solution (two molecules each of eph and ephrin).

Using the Hinanen et al. paper as your guide, create an EphA2:ephrinA1 ‘heterotetramerization assembly’. Begin by loading 3MBW twice. Rename the second copy (say, 3MBWB) and activate it in the Control Panel (i.e., select all atoms in chains A & B). Click on the dog-eared page symbol at the left of the control icons. This displays the pdb file in text format. Scroll to REMARK 350 and copy down the transformation matrix BIOMT. Then use the Display Window TOOLS option ‘Apply Transformation Matrix’. This should result in the rotation of 3MBWB on the screen. You have now created the symmetry mates for the 3MBW molecules. To create a heterotetramer coordinate file, use the MERGE option in the Display window (Edit>create merged layer from selection), then rename the _MERGE_ file (say, Eph:Ephrin heterotetramer).  SEE NOTE 110 in the SWPDB Users Manual for further help.

The heterotetramerization assembly occurs naturally in the crystal unit cell. There is a second assembly, called the ‘clustering assembly’ by Hinanen et al., that also appears in the crystal. To see this load the 3MBW coordinates. Be sure to click on the crystal symmetry line at the top of the pdb coordinates. Then use the TOOLS option ‘Apply crystallographic symmetry’. All molecules in the unit cell will appear on the display panel. Select the ones you want and save them in a merged file. Rename the merged file ‘clustering assembly’. HINT: you want the molecules obtained by applying the symmetry operators ½+x,1/2-y,-z and ½-x,1/2-y,1/2+z.

You are now in a position to reproduce the clustered assembly shown in figure 2A. First, load your two hetero tetramers. Second, superpose one of the Eph4A molecules of the ‘heterotetramerization assembly’ with one of the Eph4A molecules of the ‘clustering assembly’. You will now see three Ephs and three ephrins on the display. Third, load up another ‘hetrerotetramerization assembly’ and rename it. Follow this with superposition of the other of the two Eph4A molecules with the alternative Eph4A of the ‘clustering assembly’ from the previous step. 

 

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