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Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling
Thursday Speaker Abstracts
Synaptic Vesicle Lipids Reveal Structured State and Antagonistic Allosteric Mechanisms of
Intrinsic Disorder within Synaptotagmin I
Michael E. Fealey
1,2
, Anne Hinderliter
2
.
1
University of Minnesota, Minneapolis, MN, USA,
2
University of Minnesota Duluth, Duluth,
MN, USA.
Synaptotagmin I (Syt I) is a vesicle-localized integral membrane protein responsible for sensing
the calcium influx that triggers neurotransmitter release. Syt I consists of a transmembrane helix,
a cytosolic 60 residue tether region, and two C2 domains that bind calcium and acidic
phospholipids. Until recently, the role of the 60 residue tether region in Syt I function received
little attention. We noticed that this tether region has features of intrinsic disorder and
hypothesized that it exerts allosteric control over the adjacent calcium binding C2 domains to
tune protein function. In testing this hypothesis, we first assessed the impact of local lipid
environment on the structure of the isolated tether region. Using differential scanning
calorimetry (DSC) and nuclear magnetic resonance, we found that a lipid composition
mimicking a synaptic vesicle selects for ordered conformers in the intrinsically disordered
sequence. A simple binary lipid mixture did not have any apparent ordering impact. Knowing the
intrinsically disorder region was influenced by a membrane that mimics its native organelle, we
next assessed its allosteric impact on the first C2 domain (C2A) using DSC. Strikingly, we found
that discrete regions of the disordered tether had opposite effects on C2A unfolding. Calcium
binding to C2A in the presence of the entire disordered region shifts the unfolding transition to
low temperature. In contrast, when the 13 most N-terminal residues (mostly lysines) are
removed, calcium binding dramatically increases the temperature of the unfolding transition. In
both cases, calcium binding stabilizes the protein but the extent differs as do the contributions of
each thermodynamic parameter underlying the transition. These results indicate that the combine
intrinsic disorder and synaptic vesicle lipids have the ability to evoke antagonistic responses of
Syt I to calcium ligation.