Direct Measurement of Site-Specific Binding Energetics of Synaptotagmin-1 with Anionic Lipid Membranes

CLÉMENCE GRUGET; JEFF COLEMAN; OSCAR BELLO; SHYAM KRISHNAKUMAR; FREDERIC PINCET; JAMES E. ROTHMAN; STEPHEN H. DONALDSON, JR.

San Francisco, CA

Congreso; 62st Annual Meeting of the Biophysical-Society; 2018

Biophysical Society and American Physical Society

Upon arrival of an action potential at the neuronal synapse,calcium ions (Ca2+) are released, allowing soluble N-ethylmaleimide-sensitivefactor activating protein receptor (SNARE) proteins to fully zipper, leading tofusion of pre-docked vesicles containing neurotransmitters. Regulatory proteinssuch as complexin and synaptotagmin are closely coupled to SNAREs duringsynaptic vesicle fusion and lead to synchronous fusion. Synaptotagmin-1 (Syt1)is a transmembrane protein found in synaptic vesicles and is the Ca2+ sensorfor synaptic transmission. Syt1 has two Ca2+ binding domains, C2A and C2B,along with a flexible linker from the membrane to C2A, and a linker between C2Aand C2B. A polybasic patch in C2B has been shown to bind to anionic lipids suchas phosphatidylserine (PS) and phosphatidylinositol (PIP2) in the absence ofCa2+. Ca2+ binding to Syt1 allows non-polar residues nearby the Ca2+ bindingsites to insert into the membrane. While these mechanisms are relativelywell-accepted, the precise biochemical and biophysical mechanisms for the Syt1Ca2+ trigger remain unclear. In this work, we directly measure the interactionsof Syt1-coated surfaces with anionic membranes including PS and PIP2 lipids bythe surface forces apparatus technique, in order to mimic a Syt1-coatedsynaptic vesicle membrane interacting with the anionic plasma membrane. We performsite directed mutagenesis of the Ca2+ binding sites of C2A and C2B, along withthe polybasic patch in C2B, to fully map the site-binding energetics of Syt1with membranes, both in the absence and presence of divalent ions. The additionof Ca2+ leads to a dramatic increase in the binding energy, around ∼10kBT, due to the insertion of the hydrophobic C2B Ca2+ loop into the membrane.The results illuminate a passive role for C2A and a strong-binding, active rolefor C2B.