PSD95 is a multi-domain scaffold protein localized in the postsynaptic density of neurons. PSD95 domains modulate protein-protein interactions at the membrane and specifically bind to short peptide motifs of target proteins. In our studies the target protein is the strong inwardly rectifying potassium channel, Kir2.1. Under the direction of PSD95’s multivalent domain interactions, the Kir channels tend to cluster forming ordered tetrad complexes. Our aim is to elucidate the precise detail of the PSD95-mediated Kir2.1 clustering mechanism, thus improving on the low resolution of our current model. We will carry out this aim by studying components that are involved in these interactions, namely the Kir2.1 cytoplasmic domains, PSD95 and sub-domains of PSD95 comprising single and multiple PDZ domains. Using these components a number of Biophysical approaches including; X-ray crystallography, small angle X-ray scattering, single particle cryo-electron microscopy and ligand interaction measurements, will be undertaken to obtain a clear understanding of the inter-domain interactions.
Our current work focuses on the preparation of these individual components. Kir2.1 cytoplasmic domains are found to favour an oligomer/monomer dynamic equilibrium in a wide range of buffer conditions. Purified PSD95 shows a tendency for auto-aggregation. Crystals of PDZ1-2 have been obtained in Spacegroup I41 diffracting to 2.0Å. The data quality from the structural techniques that will be deployed is highly dependent on the purity and homogeneity of the components: Therefore the optimized preparative steps under development are critical for the success of the study.