Structural and Functional Studies of the Munc13 MUN Domain and the RIM C2B Domain

Date

2007-12-17

Authors

Guan, Rong

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Abstract

Neurotransmitter release is essential for normal brain function and is achieved through exocytosis of synaptic vesicles. Many proteins are involved the regulation of neurotransmission. The central fusion machinery includes the SNARE proteins and Munc18- 1. Besides these universal components, many other neuronal specific proteins are also involved in regulating Ca2+-triggered neurotransmitter release, such as the key priming factors RIMs and Munc13s. Munc13s are essential for vesicle priming. RIMs form a protein scaffold in the presynaptic nerve terminal. My studies have focused on the structures and functions of the Munc13 MUN domain and the RIM C2B domain. I have studied the structure and function of the Munc13 MUN domain. On one hand, I have tried to determine the three dimensional structure of the Munc13 MUN domain by Xray crystallography. I have successfully obtained crystals of the Munc13-1 MUN domain, Munc13-3 MUN domain and a fragment containing the Munc13-1 C1, C2B and MUN domains. These crystals will be further optimized to enable structure determination. On the other hand, I have tried to identify the binding partners of the MUN domain using various methods. Cross-linking experiments revealed an interaction between the Munc13-1 MUN domain and endogenous Munc18-1. In addition, cofloatation assays revealed an interaction between MUN and reconstituted SNARE complex. Detailed analysis using cofloatation assays suggested both MUN and complexin can compete with Munc18-1 for SNARE complex binding in a membrane environment. Our studies also suggested that the membrane environment can modulate the strength of protein-protein interactions remarkably, which emphasize the importance to include membranes in the studies of protein-protein interactions involved in neurotransmission. I have also analyzed the structural and biochemical properties of the RIM1 C2B domain. NMR spectroscopy and FRET experiments demonstrated no interaction between the RIM1 C2B domain and Ca2+, phospholipids, or its putative binding partners, synaptotagmin 1 and liprins. X-ray crystallography revealed the existence of a RIM1 C2B domain homodimer, which was confirmed by analytical ultracentrifugation and NMR spectroscopy. Our results suggested a model that RIM1 C2B dimerization might facilitate the Munc13 C2A homodimer to Munc13 C2A/RIM zinc finger heterodimer switch during synaptic vesicle priming.

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