Phosphatidylinositol 4,5-Bisphosphate Alters Synaptotagmin 1 Membrane Docking and Drives Opposing Bilayers Closer Together

Abstract

Synaptotagmin 1 (syt1) is a synaptic vesicle-anchored membrane protein that acts as the calcium sensor for the synchronous component of neuronal exocytosis. Using site-directed spin labeling, the position and membrane interactions of a fragment of syt1 containing its two C2 domains (syt1C2AB) were determined in bilayers containing phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol, 4,5-bisphosphate (PIP2). Addition of 1 mole% PIP2 to a lipid mixture of PC and PS results in a deeper membrane penetration of the C2A domain and alters the orientation of the C2B domain so that the polybasic face of C2B comes into close proximity to the bilayer interface. The C2B domain is found to contact the membrane interface in two regions, the Ca2+-binding loops and a region opposite the Ca2+-binding loops. This suggests that syt1C2AB is configured to bridge two bilayers and is consistent with a model generated previously for syt1C2AB bound to membranes of PC and PS. Point-to-plane depth restraints, obtained by progressive power saturation, and interdomain distance restraints, obtained by double electron-electron resonance, were obtained in the presence of PIP2 and used in a simulated annealing routine to dock syt1C2AB to two membrane interfaces. The results yield a different average structure than is found in the absence of PIP2 and indicate that bilayer-bilayer spacing is decreased in the presence of PIP2. The results indicate that PIP2, which is necessary for bilayer fusion, alters C2 domain orientation, enhances syt1-membrane electrostatic interactions and acts to drive vesicle and cytoplasmic membrane surfaces closer together.

Structural insights into the Ca 2+ and PI(4,5)P 2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

“…Given the close proximity of the lysine-rich cluster and the CBR, these results are compatible with a parallel orientation model (Fig. 6F) in which these two motifs would interact with a vesicle representing the plasma membrane, leaving the bottom face free to interact in trans with another lipid vesicle (19,(47)(48)(49)(50)(51). How these two C2 domains act in tandem and in cooperation with other SNARE proteins to regulate vesicle fusion is still under debate and will need further exploration.…”

“…Several studies have demonstrated that the C2B domain of syt1 induces Ca 2+ -dependent lipid aggregation as indicative of the predisposition to facilitate the fusion of synaptic membranes in cooperation with the SNARE complex (19,45,46). Critical Arg residues located at the polybasic region (Lys326/Lys327) and at the bottom face (Arg398/Arg399) of the domain are responsible for that function (19,45,47). To investigate if the reconstitution of the lysine-rich cluster in the C2A domain of synaptotagmin would participate in this process, we measured the ability of syt1 C2A, syt1 C2A-E194K, and syt1 C2B domains to induce vesicle clustering at increasing protein concentrations.…”

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

“…For the C2D, C2E, and C2F domains, association rate constants remained similar at the different calcium concentrations, whereas the dissociation rate decreased significantly at 20 M calcium. min-1 and PIP 2 via positively charged synaptotagmin lysine residues is an important step in neuronal vesicle fusion (40), and it has been shown that PIP 2 enhances calcium binding by synaptotagmin-1 (28,41,42). Light scattering results indicate that under the conditions of the present investigation otoferlin C2 domain proteins are properly folded and non-aggregating (Fig.…”

PIP2 and Talin Join Forces to Activate Integrin

“…PIP2 is able to regulate many important cellular processes including vesicular trafficking, platelet activation, and organization of the cytoskeleton, 28,29,30 and it may control several steps in focal adhesion. 19,20,31 PIP2 can also affect protein conformation, 32,33,34,35 target cytoplasmic proteins to the membrane, and stabilize protein oligomers. 31,36 In this article, we focus on clarifying the importance of lipid membrane composition in the conformational modulation of integrins.…”