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STUDY GUIDE NO. 4. Phelan-McDermid Syndrome (22q11 Deletion Syndrome) and the Structures of SHANK3 Domains


Assigned Readings:

1. Garber, K. Autism’s Cause May Reside in Abnormalities at the Synapse. Science 317:190-191 (2007).

2. Park, E. et al. The Shank Family of Postsynaptic Density Proteins Interacts with and Promotes Synaptic Accumulation of the βPIX Guanine Nucleotide Exchange Factor for Rac1 and Cdc42. J. Biol. Chem. 278:19120-19128 (2003).

3. Baron, M.K. et al., An Architectural Framework That May Lie at the Core of the Postsynaptic Density. Science 311:531-535 (2006).

4. Im, Y.J. et al., Structural Basis for Asymmetric Association of the βPIX Coiled Coil and Shank PDZ.J. Mol. Biol. (2010) published on-line February 16, 2010.

5. Durand et al., Mutations in the Gene Encoding the Synaptic Scaffolding Protein SHANK3 are Associated with Autism Spectrum Disorders. Nature Genetics 39:25-27 (2007).

6. Abu-Elneel et al., Heterogeneous Dysregulation of MicroRNA’s across the Autism Spectrum. Neurogenetics 9:153-161 (2008).

7. Harbury, P.B. et al. A Switch Between Two-, Tree-, and Four-Stranded Coiled Coils in GCN4 Leucine Zipper Mutants. Science 262:1401-1407 (1993).

8. Bozdagi, O. et al., Haploinsufficiency of the autism-associated Shank3 gene leads to deficits in synaptic function, social interaction, and social communication

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.Phelan-McDermid (22q13 Deletion Syndrome).  Using the papers by Durand et al.  and Bosdagi et al. describe the domain structure of SHANK3. What is a PDZ domain? What is an ankyrin-repeat (ANK) domain? What is a SAM domain?  What is an LZ domain? What is an SH3 domain? What roles do these domains play in building up the post-synapse?

What is chromosome 22q11-13 deletion syndrome? What is its OMIM number? What is a balanced translocation? Describe the analysis of a particular balanced translocation by Durand et al. that led them to the identification of SHANK3 as a candidate gene for autism spectrum disorders. What other evidence, cited by Bosdagi et al., supports this association? Two chromosomes are involved in a balanced translocation. Why weren’t any autism-related genes implicated in the chromosome that shares the translocation breakpoint with chromosome 22? What are the phenotypes of the autism cases studied by Durand et al.? The Durand et al. study also reports on a person with a duplication of an allele of SHANK3 (trisomy), resulting in a higher gene dosage for SHANK3 compared with normal persons or those with the deletion (hemizygotes). What are the phenotypic consequences of these dosage differences for human cognition based on this individual?

2. SHANK3. Where does SHANK3 fit into the neuroligin:neurexin pathway? Image of SHANK3What is the role of the SAM sub-domain in establishing dendritic spines (Baron et al.)? The PDZ domain on SHANK3 recruits βPIX to synaptic spines. What is the in vitro evidence for the SHANK:βPIX interaction? On what basis did Park et al. suspect that βPIX should bind to SHANK? How did Park et al. establish co-localization of SHANK and βPIX in mouse brains? In what regions did they find co-localization? What regulatory role does βPIX play in the dynamics of synaptic plasticity? (HINT: what do Rac1 and Cdc42 proteins do?  What is PAK?) .

3. Neuropathology. The search for the ‘cause’ of autism spectrum disorders considered from a ‘pathway’ perspective, such as mutations in different members of the neuroligin:neurexin pathway proposed by Bourgeron, may not tell the whole story. Recall that mutations in MeCP2 (Study Guide No. 1) influence the expression of BDNF, thus affecting neuro-transmitter trafficking (for example) during experience-dependent synaptic plasticity changes. It has been proposed that micro-RNA’s (m-RNA,) which are also known to control gene expression, could influence the neuroligin:neurexin pathway. What is a m-RNA? How many have been identified in the human genome? How are their levels measured in post mortem brain samples? Did Abu-Alneel et al. discover any connection between m-RNA levels and the neuroligin:neurexin pathway? What regions of the brain were looked at for dysfunctional m-RNA levels in post mortem samples?

4. SHANK3 Structural Analysis. Using a molecular modeling program (such as SPDBV) consider the crystal structure of the SAM domain of SHANK3. Do the authors make a convincing case that the SAM:SAM contacts seen in their engineered crystal are relevant for understanding the in vivo role played by SHANK3 at the synapse? Describe the intra-chain and inter-chain protein:protein interactions in the proposed ‘architectural framework’ for the postsynaptic density. Can they explain the requirement for zinc chloride to stabilize sheet formation? Any arginines hanging about? Try to construct the sheets from the PDB file (PDB CODE: 2F44). (Note: this will require all of the modeling skills you developed in building the structures with the two crystallographically-observed neuroligin:neurexin heterotetramers. For example, you must apply crystallographic symmetry to the .pdb asymmetric unit to build up a unit cell, and then isolate the interaction of possible in vivo significance. Furthermore, to do this, you will need to learn how to apply crystallographic translations. Hint: the cryst comment in the .pdb file gives the transformations and you must learn to click on the little + and – symbols to translate molecules into neighboring unit cells, saving and merging appropriately as you go along)

Draw a topological diagram for the SHANK3 PDZ domain (PDP CODE: 1Q3O) in the unliganded (apo) form. Compare the structure with the SHANK3 PDZ domain bound to the C-terminal PDZ binding domain of βPIX (PDB CODE: 3L4F). Where do the greatest changes take place? Depict this change with SPDBV or another modeling program. Look for hinge points in the backbone that enable the conformational change to take place upon binding of the C-terminal 6 residues of βPIX.  (HINT: To carry out this exercise you must recall how to use the LAYERS Window of SPDBV. Basically, a layer is a .pdb file. More than one layer can be downloaded at a time into SPDBV, which are controlled by the LAYERS control panel. A PROJECT is defined by having more than one layer depicted on the GRAPHICS Window. It will be useful to learn how to SAVE a project for future work on it. See section 113 of the SPDBV. Be sure to learn how to disable movement of one structure so that the other can be centered, rotated, etc. during the alignment. Secondly, in order to compare structures you may need to use the ALIGNMENT Window. In this window you select structures, or selected residues of structures, for comparison. Once they have been selected, click on the FIT Tab of the GRAPHICS Window and select MAGICFIT.)

There are many PDZ domains involved in specific protein:protein interactions in eukaryotic cells. Specificity is obviously important if large-scale structures such as the PSD are to assemble properly. Engineered Using your own molecular models, show the essential determinants of specificity for the SHANK3-PDZ: βPIX interaction. Identify the critically important side-chains on both βPIX and the PDZ. (βPIX residues PAWDETNL are numbered D-8, P-7 ,…., L0). Do you see salt-bridges involving any of these side-chains? Is there an interaction that reminds you of an important determinant of specificity in the neuroligin:neurexin interaction? Create molecular images to illustrate all of the most important interactions with βPIX in the binding groove of PDZ. What amino acid fixes the orientation of the C-terminus? (If you didn’t find a critically important arginine side-chain and detailed changes in its position during binding, you are coasting through this course).

How did the authors test that the binding interactions observed in the crystal are relevant for the in vivo case? (HINT: isothermal titration calorimetry on a selected set of mutants.)

What do the authors mean by ‘asymmetric binding’ in the SHANK3-PDZ: βPIX complex? Does the structure offer an explanation for the asymmetry? Use SPDBV to compare the three different PDZ binding sites. What biophysical measurement validates their claim that the ‘asymmetric binding’ is relevant for the in vivo situation? (HINT: dynamic light scattering).

The N-terminal 51 amino acids of βPIX form an α-helix. This is separated by two amino acids from the C-terminal β-strand (WDETNL) that binds in the SHANK3-PDZ domain. The two amino acids ‘break’ the alpha helix. Explain why. The three α-helices form a coiled-coil, an extremely common mode of protein-protein interaction (along with dimer coiled-coils and tetramer coiled-coils). Coiled-coils can be recognized in a sequence by structure prediction programs by the existence of ‘heptad repeats’. The amino acids are labeled abcdefg, where the a and e positions are predominately hydrophobic for a trimer (a and d for a tetramer). Positions bcdfg are polar residues. Leucine residues are most often found in the ‘a’ position. Beginning with Leu 590, identify the heptad repeats in βPIX. Draw a ‘helical wheel’ for the βPIX α-helix. Create an image that shows the ‘hydrophobic core’ of the coiled-coil of βPIX. (HINT: see Harbury et al.). Create an image that shows the electrostatic surface potential of the βPIX coiled α-helices. (HINT: read the SPDBV tutorial).

Go to the RCSB and get 2XEE.pdb. Create a pleasing image of an ankyrin repeat domain. Check the Ramachandran diagram. Make sense? Locate all of the glycine residues. Make sense? In what region of the Ramachandran diagram are these residues found?

 

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