Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors are glutamate-gated cation channels and mediators of fast excitatory neurotransmission in the mammalian central nervous system. Trafficking and functional regulation of AMPA receptors GluA1-4 is carried out through numerous intracellular protein interactions and post-translational modifications. The aim of this thesis work was to study the selective interaction between AMPA receptor subunit GluA1 and synapse-associated protein 97 (SAP97), a protein scaffold belonging to the protein family of membrane associated guanylate kinase homologs. The interaction between SAP97 and GluA1 has been implicated in AMPA receptor trafficking, neuronal development and synaptic plasticity, while disturbances in normal levels of both GluA1 and SAP97 have been linked to neuropathologies such as Alzheimer s disease and schizophrenia. In the present study, a combination of biochemical and structural work was employed to gain detailed information on the selective interaction of GluA1 with SAP97 identifying molecular determinants involved in and regulating the interaction. X-ray crystallization screens of the second PDZ domain of SAP97 (SAP97PDZ2) yielded well-diffracting crystals both for the apo and ligand bound form. The solved crystal structure of the SAP97PDZ2-GluA1 peptide complex conformed to a conventional class I PDZ interaction with hydrogen bonds forming between the carboxylate group of the ultimate C-terminal residue of the peptide and main chain nitrogens in the carboxylate binding loop of the PDZ domain, and a hydrogen bond between the antepenultimate residue of the peptide and a conserved histidine in the αB helix lining the peptide binding groove. Beside these typical PDZ interactions, as a novel finding we observed contacts within the PDZ domain reorganizing upon peptide binding leading to a slight opening of the peptide binding groove facilitating better accommodation of the ligand. In vitro binding analysis of isolated PDZ domains and short GluA1 peptides showed that, in addition to the prototypic PDZ interaction, a C-terminal cysteine, C893 located upstream from the short PDZ binding motif on GluA1 participated in the interaction through a disulfide bond formed with cysteine C378 of SAP97 under in vitro conditions. Streptavidin pull-down experiments with full-length molecules expressed in cultured cells showed that the C893S mutation leads to a substantial reduction in binding of GluA1 to SAP97, confirming the involvement of C893 in the regulation of the interaction in live cells. Reactive cysteines, like C893, can in addition to disulfide bonds participate in other thiol modifications. In our work we constructed several deletion and cysteine replacement mutants of GluA1 and tested their sensitivity to S-nitrosylating agents nitrosoglutathione and nitrosocysteine. Out of the three C-terminal cysteine residues in GluA1, we identified C893 as the sole cysteine residue sensitive to a post-translational modification by nitric oxide (NO). Furthermore, we found evidence of a physical link between GluA1 and the NO generating neuronal enzyme nitric oxide synthase nNOS via SAP97. The results of the present study provide, for the first time, detailed structural information on the interaction between GluA1 AMPA receptor and SAP97. In addition to a canonical PDZ interaction, the association with SAP97 involves a reactive cysteine residue, C893, in GluA1 C-terminal tail, a potential regulatory target for nitric oxide and oxidative conditions.
|Award date||3 Feb 2017|
|Place of Publication||Helsinki|
|Publication status||Published - 3 Feb 2017|
|MoE publication type||G5 Doctoral dissertation (article)|
Fields of Science
- 1182 Biochemistry, cell and molecular biology