Recent experimental evidence indicates that many scaffolding proteins localize at and make up synapses. These scaffolding proteins play important roles in both the maintenance of the architecture of synapses and the organization of neurotransmitters signaling complexes within synapses. Many scaffolding proteins are multidomain in nature, and these multimodular scaffold proteins often contain several repeat domains, which mainly mediate protein-protein interactions, arranged in tandem. The functional significances of such tandem arrangements of protein-protein interaction domains in scaffold proteins are largely unknown. In this thesis, I have presented the supramodular structure of the first two PDZ domains of PSD-95 (PDZ12). The structure of PDZ12 reveals that the two tandem PDZ domain...[ Read more ]

Recent experimental evidence indicates that many scaffolding proteins localize at and make up synapses. These scaffolding proteins play important roles in both the maintenance of the architecture of synapses and the organization of neurotransmitters signaling complexes within synapses. Many scaffolding proteins are multidomain in nature, and these multimodular scaffold proteins often contain several repeat domains, which mainly mediate protein-protein interactions, arranged in tandem. The functional significances of such tandem arrangements of protein-protein interaction domains in scaffold proteins are largely unknown. In this thesis, I have presented the supramodular structure of the first two PDZ domains of PSD-95 (PDZ12). The structure of PDZ12 reveals that the two tandem PDZ domains are connected by a short and conserved inter-domain linker sequence ("KPPAE"). The PDZ 12 tandem structure further showes that the C-terminal peptide-binding groove on each of the two PDZ domains are aligned with a near parallel orientation. The supramodular arrangement of PSD-95 PDZ12 thereby affords the synergistic binding of the scaffold protein to multimerized carboxyl peptides. Therefore, my data provide a mechanistic explanation for PSD-95 mediated clustering of receptors and ion-channels. Indeed, abolition of the inter-domain orientation by insertion of nine amino acids ("GSSGSSGSS") into the linker region of PDZ1 and PDZ2 lowered the binding affinity of PDZ12 to its multimeric targets. I further went to demonstrate this insertion could decrease the clustering of potassium channel Kv4.1 in heterologous cells.

The second part of this thesis addresses the supramodular nature of the tandem PDZ domains found in another neuronal scaffold protein, X11α. The three dimensional structures of the isolated PDZ domains as well as the tandem PDZ domains of X11α were determined. The structure shows that the first PDZ domain of X11α (PDZ1) binds to a carboxy1 peptide by recognizing only the last two amino acid residues. Such PDZ domain-mediated interaction with its target peptide is novel when compared to the canonical PDZ/target interaction. Most importantly, the structure of the tandem PDZ domain from X11α reveals that the C-terminus folds back and inserts into the target-recognition groove of PDZ1, thus keeping X11α in an auto-inhibited conformation. I further showed that the auto-inhibited conformation of the tandem PDZ domain from X11α was functionally important in synaptogenesis.

The third part of this thesis describes the structural and biochemical basis of L27 domain-mediated supramodular complex organization. The three-dimensional structure of the tetrameric L27 domain composed of two L27S (L27 domain of SAP97) and two L27N (N-terminal L27 domain of mLin-2/CASK) were determined. The structure shows that each L27 domain contains three α-helices. The first two helices of each domain are packed against each other to form a four-helical bundle in the heterodimer. The third helix of each L27 domain forms another four-helical bundle that assembles the two heterodimers into a tetramer. The structure of the complex provides a mechanistic explanation for L27 domain-mediated polymerization of scaffold proteins. To systematically characterize L27 domain-mediated scaffold protein assembly, I have determined the three-dimensional structure of another L27-domain complex composed of L27M (L27 domain of Mals2/mLin-7) and L27C (the C-terminal L27 domain of mLin-2/CASK). Comparison of the two L27-domain complexes sheds light into the molecular specificity of L27 domain-mediated supramodular protein assembly. In summary, the work described in this thesis shows the supramolecular nature of neuronal scaffold proteins. This data provide many insights into the molecular mechanism of neuronal signal transduction.