DETERMINATION OF THE STRUCTURE OF APG7 BY MAD

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The clustering of neurotransmitter receptors at the postsynaptic terminals is a critical requirement for efficient neurotransmission and neuronal communication. This process is facilitated by adaptor proteins, which bridge the postsynaptic receptors and the underlying cytoskeleton. We have previously determined and reported the structure of one such molecule, the GABAA receptor associated protein, GABARAP, which links GABAA receptors to microtubules (Coyle et al., Neuron, 33(1):63-74, 2002). It was recently shown that GABARAP (a.k.a. Apg8) is lapidated by an ubiquitination-like system involving an E1-like protein (Apg7) and an E2-like protein (Apg3). This lipidation process provides a mechanism for dynamic regulation of the sub-cellular location of GABARAP. We have now obtained crystals of Apg7 and of the Apg7/Apg3 complex. Both Apg3 and Apg7 are novel proteins and are not predicted to share significant structural homologies with proteins of known structure. These structures, therefore, will be of great interest because they will lead to a better understanding of the molecular mechanisms of protein lipidation in eukaryotes. Our crystals of the Apg7/Apg3 complex are very small and diffract only to approximately 8-10 ¿ resolution on our home X-ray equipment, but the Apg7 crystals are large and diffract to around 4 ¿. We have also produced Se-methionine modified Apg7 and have obtained derivative crystals which diffract as well as the native ones. We, therefore, request time on CHESS beamline F2 to determine the Apg7 structure using the MAD method. We will also collect native data from the Apg7/Apg3 complex crystals. The Apg7 crystals belong to the P321 space group with a=b=170 ¿, c=160 ¿. The protein is 65 kDa and contains 10 methionines.