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Release of neurotransmitter from synaptic vesicles requires the Ca2+/phospholipid-binding protein synaptotagmin 1. There is considerable evidence that cooperation between the tandem C2 domains of synaptotagmin is a requirement of regulated exocytosis; however, high-resolution structural evidence for this interaction has been lacking. The 2.7 A crystal structure of the cytosolic domains of human synaptotagmin 1 in the absence of Ca2+ reveals a novel closed conformation of the protein. The shared interface between C2A and C2B is stabilized by a network of interactions between residues on the C-terminal alpha-helix of the C2B domain and residues on loops 1-3 of the Ca2+-binding region of C2A. These interactions alter the overall shape of the Ca2+-binding pocket of C2A, but not that of C2B. Thus, synaptotagmin 1 C2A-C2B may utilize a novel regulatory mechanism whereby one C2 domain could regulate the other until an appropriate triggering event decouples them.

Synaptotagmin acts as the Ca 2+ sensor for neuronal exocytosis. The cytosolic domain of human synaptotagmin 1 is composed of tandem C2 domains: C2A and C2B. These C2 domains modulate the interaction of synaptotagmin with the phospholipid bilayer of the presynaptic terminus and effector proteins such as the SNARE complex. Human synaptotagmin C2A-C2B has been expressed as a glutathione-S-transferase fusion protein in Escherichia coli. The purification, crystallization and preliminary X-ray analysis of this protein are reported here. The crystals diffract to 2.7 Å and belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 82.37, b = 86.31, c = 140.2 Å . From selfrotation function analysis, there are two molecules in the asymmetric unit. The structure determination of the protein using this data is ongoing.

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