The Ca2+ Affinity of Synaptotagmin 1 Is Markedly Increased by a Specific Interaction of Its C2B Domain with Phosphatidylinositol 4,5-Bisphosphate

Abstract

The synaptic vesicle protein synaptotagmin 1 is thought to convey the calcium signal onto the core secretory machinery. Its cytosolic portion mainly consists of two C2 domains, which upon calcium binding are enabled to bind to acidic lipid bilayers. Despite major advances in recent years, it is still debated how synaptotagmin controls the process of neurotransmitter release. In particular, there is disagreement with respect to its calcium binding properties and lipid preferences. To investigate how the presence of membranes influences the calcium affinity of synaptotagmin, we have now measured these properties under equilibrium conditions using isothermal titration calorimetry and fluorescence resonance energy transfer. Our data demonstrate that the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), but not phosphatidylserine, markedly increases the calcium sensitivity of synaptotagmin. PI(4,5)P 2 binding is confined to the C2B domain but is not affected significantly by mutations of a lysine-rich patch. Together, our findings lend support to the view that synaptotagmin functions by binding in a trans configuration whereby the C2A domain binds to the synaptic vesicle and the C2B binds to the PI(4,5)P 2 -enriched plasma membrane.Calcium-dependent secretion of neurotransmitter-loaded synaptic vesicles is at the heart of synaptic transmission. The underlying membrane fusion reaction between vesicle and plasma membrane has been intensively studied and found to be promoted by both protein-protein as well as protein-lipid interactions. From the multitude of proteins involved in this membrane fusion event, the Ca 2ϩ -binding protein synaptotagmin 1 is one of its central regulating factors (for review, see Refs. 1-6). Synaptotagmin 1 is anchored in the membrane of synaptic vesicles via a single transmembrane region. Its N-terminal region comprises a short luminal domain, whereas the larger cytoplasmic C-terminal region consists of tandem C2 domains, termed C2A and C2B, tethered to each other via a short linker (7) (a schematic outline of the structural features of synaptotagmin 1 is given in Fig. 1A). Several isoforms with similar domain structure have been identified (8).C2 domains are Ca 2ϩ binding modules of ϳ130 amino acids, first described as the second conserved region of protein kinase C (PKC) 2 (9). The C2A domain of synaptotagmin 1 was the first C2 domain structure to be determined (10). In subsequent studies other C2 domains, including the C2B domain of synaptotagmin, were shown to exhibit very similar three-dimensional structures. They have a conserved eight-stranded anti-parallel ␤-sandwich connected by surface loops. C2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction (PKC, phospholipases, phosphatidylinositol 3-kinases, etc.) and proteins involved in membrane trafficking (synaptotagmins, rabphilin, DOC2, etc.) (11).Calcium ions bind in a cup-shaped depression formed by the N-and C-terminal loops of the C2 key motifs of C2 domains. Nota...

Phosphatidylinositol 4,5-Bisphosphate Increases Ca2+ Affinity of Synaptotagmin-1 by 40-fold

“…The C2AB fragment of synaptotagmin-1 (rat sequence, residues 97-421) was expressed in Escherichia coli and purified as described (3,10). The single cysteine mutant (C278S/S342C) was labeled with Alexa Fluor 488-maleimide (Invitrogen) as described (3,10).…”

“…2ϩ binding to synaptotagmin-1, originally demonstrated by equilibrium dialysis using native protein (2), has been characterized by isothermal titration calorimetry (3) and NMR (4 -6) using a soluble fragment containing both C2 domains (C2AB fragment, residues 97-421). The C2A domain binds to three Ca 2ϩ ions with affinities ranging from 50 M to 10 mM.…”

“…Isothermal titration calorimetry and NMR require high (100 M to 1 mM) concentrations of protein (3)(4)(5). Therefore, high affinities well below these concentrations cannot be accurately determined with these approaches.…”

“…Binding of synaptotagmin to PIP 2 is often inferred from binding of the C2 domains to artificial membranes containing a defined fraction of PIP 2 (e.g. by FRET (3), pulldown assays (11,13), or density flotations (3,12)). However, it is difficult to quantitatively distinguish Ca 2ϩ from PIP 2 binding with these approaches.…”

Markierungsfreie “Microscale Thermophoresis” zur Bestimmung von Bindestellen und Affinitäten bei Protein‐Liganden‐Wechselwirkungen

“…20 nm [b] 44 nm (IC 50 ) [23] Thrombin [c] 15-mer (32AE15) nm 25 bis 100 nm [25,26] 29-mer (133AE42) nm 0.5 oder 100 nm [26,27] Syt1 [c] Ca 2+ (326AE26) mm 50 mm bis 3 mm [28] [a] Die gemessenen Affinitäten stimmen für alle untersuchten biomolekularen Bindungsereignissen mit den Literaturwerten überein. [ Die K D -Werte wurden berechnet, indem der Anteil gebundener Proteine mit der K D als einzigem freien Parameter an die quadratische Lçsung des Massenwirkungsgesetzes für das Bindungsgleichgewicht angepasst wurde.…”