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The 8-kDa light chain of dynein (DLC8) is ubiquitously expressed in various cell types. Other than serving as a light chain of the dynein complexes, this highly conserved protein has been shown to bind a larger number of proteins with diverse biological functions. DLC8 forms a homodimer via three-dimensional domain swapping of an internal beta-strand (the beta2-strand) at neutral pH. The protein undergoes non-reversible dimer-to-monomer dissociation when the pH value of the protein solution decreases. The three-dimensional structure of the DLC8 monomer determined by NMR spectroscopy at pH 3.0 showed that the protein is well folded. The major conformational change accompanied by dimer dissociation is in the beta2-strand of the protein, which undergoes transition from a beta-strand to a nascent alpha-helix. The monomer form of DLC8 is not capable of binding to target proteins. Insertion of two flexible amino acid residues in the tight beta1/beta2-loop dramatically stabilized the monomer conformation of the protein. NMR studies showed that the mutation altered the conformation as well as the three-dimensional domain swapping-mediated assembly of the DLC8 dimer. The mutant DLC8 was unable to bind to its targets even at physiological pH. The three-dimensional structure of the mutant protein in its monomeric form provides the structural basis of the mutation-induced stabilization of the monomer conformation. Based on the experimental data, we conclude that the formation of the beta2-strand swapping-mediated dimer is mandatory for the structure and function of DLC8. We further note that the DLC8 dimer represents a novel mode of three-dimensional domain swapping.

Shank family proteins are a class of newly discovered neuronal scaffold proteins. Full-length Shank contains, from its amino-to carboxyl-end, multiple ankyrin repeats, an SH3 domain, a PDZ domain, a long proline-rich region, and a SAM domain. Each of these domains can act as a docking site for neuronal proteins via specific protein-protein interactions. For example, Shank is known to interact with three major classes of glutamate receptors (the N-methyl-D-aspartate receptor, the metabotropic Glutamate Receptor and the Alpha-amino-3-hydroxy-5-Methyl-4-isoxazole-Proprionic Acid Receptor). Due to lack of detailed biochemical and structural characterization of the various domains of Shank, the molecular basis of Shank-mediated protein interaction is largely unknown. The work of this thesis focuses on the biochemical and structural properties of the SH3 and PDZ domains of Shank. In this thesis research, an efficient expression and purification scheme was developed for obtaining large quantities of recombinant SH3 and PDZ domains of Shank. The work laid a foundation for high resolution structure determination of the domains by NMR spectroscopic techniques. The high resolution structure of the Shank SH3 domain shows that the domain contains an extra ordered N-terminal fragment that is not observed in the structure of other known SH3 domains. The structure of the Shank SH3 domain suggests that the N-terminal fragment may function as a regulatory element of the Shank SH3 domain by influencing its target-binding capacities. We further show that the SH3 domain and the PDZ domain of Shank interact with each other. Detailed biochemical analysis indicated that the association of the two domains is intramolecular in nature. It is possible that the SH3 domain is kept in an auto-inhibited conformation by such inter-domain interaction. The data presented in this thesis provide insights into the biochemical basis of the SH3- PDZ domain-mediated target interaction of Shank proteins.

PDZ domains are modular protein units that play important roles in organizing signal transduction complexes. PDZ domains mediate interactions with both C-terminal peptide ligands and other PDZ domains. Here, we used PDZ domains from neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) to explore the mechanism for PDZ-dimer formation. The nNOS PDZ domain terminates with a similar to 30 residue amino acid beta -finger peptide that is shown to be required for nNOS/PSD-95 PDZ dimer formation. Ln addition, formation of the PDZ dimer requires this beta -finger peptide to be physically anchored to the main body of the canonical nNOS PDZ domain. A buried salt bridge between the beta -finger and the PDZ domain induces and stabilizes the beta -hairpin structure of the nNOS PDZ domain. In apo-nNOS, the beta -finger peptide is partially flexible and adopts a transient beta -strand like structure that is stabilized upon PDZ dimer formation. The flexibility of the NOS PDZ beta -finger is likely to play a critical role in supporting the formation of nNOS/PSD-95 complex. The experimental data also suggest that nNOS PDZ and the second PDZ domain of PSD-95 form a "head-to-tail" dimer similar to the nNOS/syntrophin complex characterized by X-ray crystallography. (C) 2000 Academic Press.