DETERMINATION OF THE STRUCTURAL BASIS FOR PICK1 REGULATION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Protein interacting with C-kinase 1 (PICK1) is required for ¿-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor trafficking in neurons, a crucial process involved in learning and memory. PICK1 contains an N-terminal PSD-95/Discs-large/ZO-1 (PDZ) domain followed by a central membrane-binding Bin/amphiphysin/Rvs (BAR) domain and a C-terminal acidic domain. PICK1 is regulated by Ca2+. In the absence of Ca2+, PICK1 is localized to the cytoplasm, suggesting that PICK1 is normally auto-inhibited. Upon Ca2+ stimulation PICK1 binds to the AMPA receptor tail through its PDZ domain and to the adjacent membrane via its BAR domain. Thus, a major conformational change is expected to take place between the Ca2+-bound and Ca2+-free states. We propose to use small-angle x-ray scattering (SAXS) to address how Ca2+ binding affects the structure of PICK1. Specifically, we plan to determine the overall envelope of PICK1 alone and in complex with Ca2+ and an AMPA receptor tail peptide. BAR domains are banana-shaped homodimers that interact with cellular membranes and stabilize or sense membrane curvature. There is a direct correlation between the curvature of the BAR domain and the curvature of the membrane tubules that they stabilize. In the absence of diffracting crystals of the BAR domain of PICK1, we plan to use SAXS to determine the overall shape and radius of curvature of the BAR domain. By comparing the envelop of the BAR domain with that of full length PICK1 will also learn the location of the PDZ domain with respect to the BAR domain. In summary, the SAXS data will provide us a structural understanding of the mechanism of PICK1 activation by Ca2+ and the relative positions of the various domains of PICK1.