Structural characterization of synaptotagmin I



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Synaptotagmin I is the most abundant Ca+2 binding protein present on synaptic vesicles accounting for 7% of total vesicle protein and is widely accepted as the Ca2+ sensor in fast synchronous neurotransmitter release. The protein is composed of one trans-membrane domain, an unstructured linker followed by two C2A domains identified as C2A and C2B. Each C2 domain is composed of an 8 stranded β-sandwich joined by a 9 amino acid linker. The Ca+2 binding pocket is composed of three loops located at the apex of the protein. In the Syt I C2AB structure, we see evidence of a domain structural change in the absence of Ca2+. Analysis of interacting residues between C2A and C2B show a network of highly conserved residues within the C2 domain that regulates Ca+2/phohspholipid binding in C2A. Analysis of the Syt I C2A structure, as well as, previous C2A structures shows a strong H-bond between Tyr 180 and His 237 in C2A. By removing this H-bond, disorder of Loop 3 is increased and the thermodynamic stability of the C2 domain decreases. Our hypothesis is that the absolute position of the Ca2+ binding loops of C2 domains affects Ca+2 affinity and, and ultimately domain stability. We used several different biochemical approaches to test the hypothesis. We assessed the importance of Loop 3 mutations using X-ray crystallography methods, bulk thermodynamic measurements using lifetime fluorescence, and analyzed the mechanical properties of the C2-domains using single molecule force spectroscopy.\r\nWe studied the mechanical stability of the C2A and C2B domains of human Syt1 using single-molecule atomic force microscopy. We found that stretching the C2AB domains of Syt1 resulted in two distinct unfolding force peaks. The larger force peak of ~100pN was identified as C2B and the second peak of ~50pN as C2A. Further, a significant fraction of C2A domains unfolded through a low force intermediate that was not observed in C2B. We conclude that these domains have different mechanical properties. We hypothesize that a relatively small stretching force may be sufficient to deform the effector-binding regions of C2A domain and modulate the affinity for SNAREs, phospholipids and Ca+2.\r\n



X-ray crystallography, synaptotagmin, protein structure, exocytosis