Specific targeting and local organization of pre- and postsynaptic components are critical to faithful signal transmissions in neuronal synapses. Multimodular scaffold proteins have been shown to play central roles in protein targeting and signaling complex organization. These scaffold proteins often contain multiple protein-protein interaction modules arranged in tandem. Previous biochemical evidence showed that the tandem arrangement of protein-binding modules endows distinct target binding properties with respect to individual domains or simple sum of isolated domains. The main focus of this dissertation is to uncover the structural basis of the supramodular nature of selected multidomain neuronal scaffold proteins....[ Read more ]

Specific targeting and local organization of pre- and postsynaptic components are critical to faithful signal transmissions in neuronal synapses. Multimodular scaffold proteins have been shown to play central roles in protein targeting and signaling complex organization. These scaffold proteins often contain multiple protein-protein interaction modules arranged in tandem. Previous biochemical evidence showed that the tandem arrangement of protein-binding modules endows distinct target binding properties with respect to individual domains or simple sum of isolated domains. The main focus of this dissertation is to uncover the structural basis of the supramodular nature of selected multidomain neuronal scaffold proteins.

I have determined the three dimensional (3D) structure of the PDZ45 tandem of glutamate receptor binding protein, GRIP1, using NMR spectroscopy. The structure, in conjunction with mutagenesis studies, reveals that the two PDZ domains of PDZ45 pack extensively with each other forming a compact supramodule. The structure of PDZ45 tandem also shows that PDZ4 has a closed αB/βB-groove that is unlikely to bind to carboxyl peptides, whereas PDZ5 has the canonical binding groove mediating the interaction between GRIP1 and GluR2/3. Because the isolated PDZ5 is unfolded in the solution, we suggest that PDZ4 functions to stabilize the structure of PDZ5. The supramodular nature is also extended to the PDZ tandem in another neuronal scaffold protein X11α. In this study, I uncovered an auto-inhibited conformation of the PDZ domains which is mediated by an intra-molecular interaction between the conserved carboxyl terminal tail and the first PDZ domain of the X11α PDZ tandem. Further, PDZ1, PDZ2, and the carboxyl tail of X11α forms a supramodule with distinct target binding property. Alteration of the auto-inhibited conformational status of the PDZ tandem results in significant changes in the X11α-mediated stabilization of amyloid precursor protein. The structures of GRIP1 PDZ45 and X11α PDZ12 indicate that these tandemly arranged protein-protein interaction modules are not just simple attachments of "beads on a string", but often represent functional supramodules with distinct structures and biological functions with respect to individual domains.

The second part of this dissertation describes structural studies of L27 domain mediated supramolecular complex assembly. I showed that isolated L27 domains are largely unfolded. Mixing of a cognate pair of L27 domains induces de novo folding of the domains and forms a stable tetrameric complex. The structure of L27 domain complex formed by SAP97/mLin-2 L27 domains was determined by NMR spectroscopy, and this structure represents the first L27 complex structure. The structure of the SAP97/mLin-2 L27 complex further suggests a mechanistic model for polymerization of L27 domain scaffold proteins. I have also determined the prototype L27 domain complex comprised of mLin-2/mLin-7 L27 domains and L27 domain complex formed by Patj/Palsl L27 domains. The structures show that tetrameric structure is the general assembly mode for cognate pairs of L27 domains. Structural analysis of the L27 domain complex structures indicates that the central four-helix bundles are highly distinct between different pairs of L27 domain complexes. Biochemical studies reveal that C-terminal α-helix responsible for the formation of the central helix bundle is a critical specificity determinant for each L27 domain in choosing its binding partner.