Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

28-POS Board 10 CALCIUM INHIBITS MEMBRANE BINDING BY SYNAPTOTAGMIN-LIKE PROTEIN 2 THROUGH COMPETITIVE INTERACTIONS WITH ANIONIC LIPIDS

Emily M Irlbeck 1 ; Timothy A Spotts 1 ; David Flores 2,3 ; Abena Watson-Siriboe 1 ; Cisloynny Beauchamp-Perez 1 ; Markus Zweckstetter 2,3 ; Jefferson Knight 1 ;

1 University of Colorado Denver, Department of Chemistry, Denver, CO, USA 2 German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany 3 Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany

Synaptotagmin-like protein 2 (Slp-2; also called exophilin-4) is a Rab27 effector protein involved in trafficking and docking of dense-core vesicles to the plasma membrane. In contrast to canonical synaptotagmin C2 domains which bind membranes in response to increased intracellular calcium, the C2A domain of synapotagmin-like protein 2 is reported to be inhibited by calcium. Here, we use a combination of NMR spectroscopy and fluorescence-based assays of protein-liposome binding to uncover the physical-chemical origins of this unusual property. Our data confirm that the purified recombinant C2A domain binds anionic lipid membranes containing phosphatidylserine (PS) and/or phosphatidylinositol-(4,5)-bisphosphate (PIP2) in a manner that is inhibited by calcium. Like other Slp family members, membrane binding is strongest when both PS and PIP2 are present. Although the Slp-2 C2A domain has 3 of 5 conserved aspartate residues that form the canonical C2 domain calcium binding site, our data do not show any binding to calcium ions. Rather, calcium inhibits protein-membrane binding through competing with the protein for binding to anionic lipids. For example, the C2 domain binds with approximately equal affinity to PS or phosphatidylglycerol (PG) lipids, but is more sensitive to calcium inhibition on PS because Ca 2+ ions bind more strongly to PS. Furthermore, we show that the PIP2 binding site of the Slp-2 C2A domain is highly susceptible to reaction with nucleophilic carbonyls, including compounds endogenous to E. coli strains commonly used for protein expression. Such modifications can potentially lead to protein stock variability in lipid binding assays if the protein is not fully purified. Overall, the calcium independence and inhibition of this membrane trafficking protein are consistent with its role in vesicle docking prior to calcium influx.

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