Ca2+ releases E‐Syt1 autoinhibition to couple ER‐plasma membrane tethering with lipid transport

The extended synaptotagmins (E‐Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and transport lipids between them via SMP domains. E‐Syt1 tethers and transports lipids in a Ca²⁺‐dependent manner, but the role of Ca²⁺ in this regulation is unclear. Of the five C2 domains of E‐Syt1, only C2A and C2C contain Ca²⁺‐binding sites. Using liposome‐based assays, we show that Ca²⁺ binding to C2C promotes E‐Syt1‐mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P₂‐rich membranes, as previously suggested by studies in semi‐permeabilized cells. Importantly, Ca²⁺ binding to C2A enables lipid transport by releasing a charge‐based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E‐Syt1 constructs defective in Ca²⁺ binding in either C2A or C2C failed to rescue two defects in PM lipid homeostasis observed in E‐Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca²⁺‐triggered phosphatidylserine scrambling. Thus, a main effect of Ca²⁺ on E‐Syt1 is to reverse an autoinhibited state and to couple membrane tethering with lipid transport.     

Quantitation of the Calcium and Membrane Binding Properties of the C2 Domains of Dysferlin

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca²⁺ sensitive, the Ca²⁺ binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca²⁺ and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca²⁺ with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with K_d values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar K_d value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca²⁺ enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca²⁺ albeit with varying affinity and stoichiometry.     

Quantitation of the Calcium and Membrane Binding Properties of the C2 Domains of Dysferlin

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca²⁺ sensitive, the Ca²⁺ binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca²⁺ and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca²⁺ with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with K_d values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar K_d value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca²⁺ enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca²⁺ albeit with varying affinity and stoichiometry.     

Oligomerization and Ca2+/calmodulin control binding of the ER Ca2+-sensors STIM1 and STIM2 to plasma membrane lipids

Ca²⁺ (calcium) homoeostasis and signalling rely on physical contacts between Ca²⁺ sensors in the ER (endoplasmic reticulum) and Ca²⁺ channels in the PM (plasma membrane). STIM1 (stromal interaction molecule 1) and STIM2 Ca²⁺ sensors oligomerize upon Ca²⁺ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca²⁺ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca²⁺ have been studied but responses towards elevated cytosolic Ca²⁺ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P₂-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P₂, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca²⁺ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca²⁺ and PI(4,5)P₂ concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca²⁺ concentration down-regulates STIM2-mediated ER–PM contacts via CaM binding.     

The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca²⁺ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.     

Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca²⁺ sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb²⁺, a structural and functional surrogate of Ca²⁺, to generate the protein states with well-defined protein-metal ion stoichiometry. NMR experiments revealed that binding of one divalent metal ion per C2 domain results in loss of conformational plasticity of the loop regions, potentially pre-organizing them for additional metal-ion and membrane-binding events. In C2A, a divalent metal ion in site 1 is sufficient to drive its weak association with phosphatidylserine-containing membranes, whereas in C2B, it enhances the interactions with the signaling lipid phosphatidylinositol-4,5-bisphosphate. In full-length Syt1, both Pb²⁺-complexed C2 domains associate with phosphatidylserine-containing membranes. Electron paramagnetic resonance experiments show that the extent of membrane insertion correlates with the occupancy of the C2 metal ion sites. Together, our results indicate that upon partial metal ion saturation of the intra-loop region, Syt1 adopts a dynamic, partially membrane-bound state. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, “prime” Syt1 for cooperative binding of a full complement of metal ions and deeper membrane insertion.     

Interaction of synaptotagmin with lipid bilayers, analyzed by single-molecule force spectroscopy

Synaptotagmin I is the major Ca²⁺ sensor for membrane fusion during neurotransmitter release. The cytoplasmic domain of synaptotagmin consists of two C2 domains, C2A and C2B. On binding Ca²⁺, the tips of the two C2 domains rapidly and synchronously penetrate lipid bilayers. We investigated the forces of interaction between synaptotagmin and lipid bilayers using single-molecule force spectroscopy. Glutathione-S-transferase-tagged proteins were attached to an atomic force microscope cantilever via a glutathione-derivatized polyethylene glycol linker. With wild-type C2AB, the force profile for a bilayer containing phosphatidylserine had both Ca²⁺-dependent and Ca²⁺-independent components. No force was detected when the bilayer lacked phosphatidylserine, even in the presence of Ca²⁺. The binding characteristics of C2A and C2B indicated that the two C2 domains cooperate in binding synaptotagmin to the bilayer, and that the relatively weak Ca²⁺-independent force depends only on C2A. When the lysine residues K189-192 and K326, 327 were mutated to alanine, the strong Ca²⁺-dependent binding interaction was either absent or greatly reduced. We conclude that synaptotagmin binds to the bilayer via C2A even in absence of Ca²⁺, and also that positively charged regions of both C2A and C2B are essential for the strong Ca²⁺-dependent binding of synaptotagmin to the bilayer.     

A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release

The function of synaptotagmin as a Ca²⁺ sensor in neurotransmitter release involves Ca²⁺-dependent phospholipid binding to its two C₂ domains, but this activity alone does not explain why Ca²⁺ binding to the C₂B domain is more critical for release than Ca²⁺ binding to the C₂A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca²⁺ induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca²⁺ binding to the C₂B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C₂B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.     

The Calcium-Dependent and Calcium-Independent Membrane Binding of Synaptotagmin 1: Two Modes of C2B Binding

The Ca²⁺-independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine and phosphatidylcholine in a Ca²⁺-independent manner with a lipid partition coefficient of approximately 3.0 × 10² M^− 1. A soluble fragment containing the first and second C2 domains of syt1, C2A and C2B, has a similar affinity, but C2A alone has no detectable affinity to phosphatidylcholine/phosphatidylserine bilayers in the absence of Ca²⁺. Although the Ca²⁺-independent membrane affinity of C2B is modest, it indicates that this domain will never be free in solution within the cell. Site-directed spin labeling was used to obtain bilayer depth restraints, and a simulated annealing routine was used to generate a model for the membrane docking of C2B in the absence of Ca²⁺. In this model, the polybasic strand of C2B forms the membrane binding surface for the domain; however, this face of C2B does not penetrate the bilayer but is localized within the aqueous double layer when C2B is bound. This double-layer location indicates that C2B interacts in a purely electrostatic manner with the bilayer interface. In the presence of Ca²⁺, the membrane affinity of C2B is increased approximately 20-fold, and the domain rotates so that the Ca²⁺-binding loops of C2B insert into the bilayer. This Ca²⁺-triggered conformational change may act as a switch to modulate the accessibility of the polybasic face of C2B and control interactions of syt1 with other components of the fusion machinery.     

Membrane Docking Geometry and Target Lipid Stoichiometry of Membrane-Bound PKCα C2 Domain: A Combined Molecular Dynamics and Experimental Study

Protein kinase Cα (PKCα) possesses a conserved C2 domain (PKCα C2 domain) that acts as a Ca²⁺-regulated membrane targeting element. Upon activation by Ca²⁺, the PKCα C2 domain directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca²⁺ concentrations, binding of the C2 domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association; however, at typical physiological Ca²⁺ concentrations, binding to both PS and phosphoinositidyl-4,5-bisphosphate (PIP₂) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of the PKCα C2 domain docked to (i) PS alone and (ii) both PS and PIP₂ simultaneously. These two EPR docking geometries exhibit significantly different tilt angles relative to the plane of the membrane, presumably induced by the large size of the PIP₂ headgroup. The present study utilizes the two EPR docking geometries as starting points for molecular dynamics simulations that investigate atomic features of the protein-membrane interaction. The simulations yield approximately the same PIP₂-triggered change in tilt angle observed by EPR. Moreover, the simulations predict a PIP₂:C2 stoichiometry approaching 2:1 at a high PIP₂ mole density. Direct binding measurements titrating the C2 domain with PIP₂ in lipid bilayers yield a 1:1 stoichiometry at moderate mole densities and a saturating 2:1 stoichiometry at high PIP₂ mole densities. Thus, the experiment confirms the target lipid stoichiometry predicted by EPR-guided molecular dynamics simulations. Potential biological implications of the observed docking geometries and PIP₂ stoichiometries are discussed.     

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the “fusion peptide” (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca²⁺ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca²⁺ binding to the SARS-CoV-2-FP, the role of Ca²⁺ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca²⁺-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca²⁺-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca²⁺ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca²⁺ ions. These findings provide novel mechanistic insights regarding the role of Ca²⁺ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.     

Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca2+ concentration

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca²⁺-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-β-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-β-cyclodextrin complexes restored the Ca²⁺-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca²⁺, annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca²⁺ concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca²⁺ concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.     

The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival

Cardiac contractility is regulated through the activity of various key Ca²⁺-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca²⁺ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca²⁺ by SR membranes during relaxation. Recently, the antiapoptotic HS-1–associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575–594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca²⁺ levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca²⁺ stores.     

NMR analysis of free and lipid nanodisc anchored CEACAM1 membrane proximal peptides with Ca2+/CaM

CEACAM1, a homotypic transmembrane receptor with 12 or 72 amino acid cytosolic domain isoforms, is converted from inactive cis-dimers to active trans-dimers by calcium-calmodulin (Ca²⁺/CaM). Previously, the weak binding of Ca²⁺/CaM to the human 12 AA cytosolic domain was studied using C-terminal anchored peptides. We now show the binding of ¹⁵N labeled Phe-454 cytosolic domain peptides in solution or membrane anchored using NMR demonstrates a significant role for the lipid bilayer. Although binding is increased by the mutation Phe454Ala, this mutation was previously shown to abrogate actin binding. On the other hand, Ca²⁺/CaM binding is abrogated by phosphorylation of nearby Thr-457, a post-translation modification required for actin binding and subsequent in vitro lumen formation. Binding of Ca²⁺/CaM to a membrane proximal peptide from the long 72 AA cytosolic domain anchored to lipid nanodiscs was very weak compared to lipid free conditions, suggesting membrane specific effects between the two isoforms. NMR analysis of ¹⁵N labeled Ca²⁺/CaM with unlabeled peptides showed the C-lobe of Ca²⁺/CaM is involved in peptide interactions, and hydrophobic residues such as Met-109, Val-142 and Met-144 play important roles in binding peptide. This information was incorporated into transmembrane models of CEACAM1 binding to Ca²⁺/CaM. The lack of Ca²⁺/CaM binding to phosphorylated Thr-457, a residue we have previously shown to be phosphorylated by CaMK2D, also dependent on Ca²⁺/CaM, suggests stepwise binding of the cytosolic domain first to Ca²⁺/CaM and then to actin.     

SNARE complex alters the interactions of the Ca2+ sensor synaptotagmin 1 with lipid bilayers

Release of neuronal transmitters from nerve terminals is triggered by the molecular Ca²⁺ sensor synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca²⁺ binding. Before fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which is thought to bind the C2B domain in vivo. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. We investigated how the isolated C2 modules and the C2AB tandem of Syt1 interact with membranes mimicking either SV or PM. The simulations showed that the C2AB tandem can either bridge SV and PM or insert into PM with its Ca²⁺-bound tips and that the latter configuration is more favorable. Surprisingly, C2 domains did not cooperate in penetrating into PM but instead mutually hindered their insertion into the bilayer. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the C2AB-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains and promotes their insertion into PM. We performed the MD simulations of the prefusion protein complex positioned between the lipid bilayers mimicking PM and SV, and our results demonstrated in silico that the presence of the Ca²⁺ bound C2AB tandem promotes lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex.     

Ca-induced stimulation of the membrane binding of Escherichia coli SecA and its association with signal peptides of secretory proteins

Previously, it was found that Ca²⁺ stimulates the intrinsic Escherichia coli SecA ATPase activity [Kim et al., FEBS Lett. 493 (2001) 12-16]. Now, we suggest that Ca²⁺ is required for efficient interaction of SecA with membranes and the signal peptide of ribose-binding protein. When the amount of external Ca²⁺ was enhanced, the amounts of membrane-bound SecA and its lipid/ATPase activity increased. In the presence of entrapped Ca²⁺ in liposomes, the binding was also stimulated in a Ca²⁺ concentration-dependent manner. The effect of Ca²⁺ on the functional regulation of SecA was also evident in the presence of the signal peptides of secretory proteins, which the interaction of SecA with the signal peptide increased with increasing Ca²⁺ concentration in the presence of membranes. However, other divalent cations including Mg²⁺, Mn²⁺, and Zn²⁺ had inhibitory or no effect, suggesting a specific role of Ca²⁺ in SecA interaction with lipid bilayers and signal peptides.     

A Highlights from MBoC Selection: Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1

C2 domains are widespread motifs that often serve as Ca²⁺-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca²⁺ sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca²⁺, and shifted the Ca²⁺ dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation–secretion coupling.     

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

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)P₂), but not phosphatidylserine, markedly increases the calcium sensitivity of synaptotagmin. PI(4,5)P₂ 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)P₂-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 Ca²⁺-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).Open in a separate windowFIGURE 1.Structure of synaptotagmin 1. Synaptotagmin 1 protein consists of two C2 domains, C2A and C2B, that coordinate three and two calcium ions, respectively (16). The acidic residues that coordinate calcium binding is shown schematically, with the residues mutated in the calcium binding mutants (i.e. C2Ab*, C2a*B, and C2a*b*) shown in red. The Lys-rich patch is represented as a ball-and-stick model colored blue with the single cysteine site for the FRET assay (S342C) colored in green (A). The different mutants and constructs used in the study are schematically depicted (B).C2 domains are Ca²⁺ binding modules of ∼130 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 β-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. Notably, the coordination spheres for the Ca²⁺ ions are incomplete (12, 13). In canonical C2 domains, this incomplete coordination sphere can be occupied by anionic and neutral (14, 15) phospholipids, enabling the C2 domain to be attached to the membrane. Hence, it is thought that the general function of C2 domains is to mediate Ca²⁺-triggered binding of the protein to a membrane. In fact, upon rise of the intracellular calcium level, C2 domain-containing enzymes are translocated to the membrane so that the catalytic domains can interact with lipids or membrane-anchored protein substrates (11). Yet synaptotagmin 1 does not contain such a catalytic domain, suggesting that the properties of its tandem C2 domains are the sole key to understanding its molecular function. In neurotransmission, synaptotagmin is thought to transmit the Ca²⁺ signal onto the core membrane fusion machinery, composed of the three SNARE (soluble N-ethylmaleimide sensitive factor attachment receptor) proteins syntaxin 1, SNAP-25 (Q-SNAREs, residing on the plasma membrane), and synaptobrevin 2 (also referred to as VAMP2 (vesicle-associated membrane protein) (R-SNARE, residing on the synaptic vesicle)). So far the multifarious interplay between the SNARE machinery, the two fusing membranes, and synaptotagmin 1 is not well understood. The crystal structure of the entire cytosolic domain of synaptotagmin in the absence of Ca²⁺ has revealed an interesting domain arrangement with the two C2 domains facing in opposite directions (16), hinting at the possibility that the molecule might interact with two opposing membranes upon rise of intracellular Ca²⁺.Although the underlying processes of Ca²⁺ binding and Ca²⁺-dependent membrane binding of synaptotagmin 1 have been studied by a multitude of structural and biochemical investigations, they have not revealed features of synaptotagmin C2 domains that are different from those of other C2 domain-containing proteins. Calcium binding to synaptotagmin in the absence of membranes has been studied by NMR. These studies showed that the isolated C2A domain of synaptotagmin 1 binds three calcium ions with an apparent affinity of ∼60–75 μm, ∼400–500 μm, and more than 1 mm (17). The isolated C2B domain binds two calcium ions with similar calcium affinities in the range of ∼300–600 μm (18). The relatively low intrinsic Ca²⁺ affinities of both C2 domains are difficult to reconcile with the role of synaptotagmin 1 as the Ca²⁺ sensor for fast and synchronous neurotransmitter release, suggesting that interaction with phospholipids contributes to its Ca²⁺ sensitivity. Indeed, Ca²⁺-triggered binding of isolated C2 domains to lipid membranes was first shown in an in vitro study of synaptotagmin 1 using a fluorescence-based approach (19). Subsequent equilibrium fluorescence studies have shed more light on the molecular process underlying membrane binding of synaptotagmin 1, for example by demonstrating that the isolated C2A domain dips into the membrane bilayer upon Ca²⁺ binding (20). This penetration was corroborated by electro-paramagnetic resonance (EPR) spectroscopy studies, which also showed that the penetration depth increased when both C2 domains of synaptotagmin 1 were attached to each other (21) as compared with the single domains (22, 23). However, a variety of different Ca²⁺ and lipid preferences for the individual C2 domains of synaptotagmin has been reported (3, 5, 6).To resolve these discrepancies and to shed more light on the molecular interactions of synaptotagmin 1, we have now used quantitative approaches to study the Ca²⁺ concentration and the lipid composition needed for synaptotagmin to bind to membranes. We employed isothermal titration calorimetry (ITC) to measure the intrinsic calcium binding affinities of synaptotagmin 1 C2 domains both as isolated domains as well as in the context of the tandem C2AB protein. Then, we investigated whether the intrinsic calcium affinity is modulated in the presence of lipids using a newly developed fluorescence resonance energy transfer (FRET) approach. In addition, we investigated how Ca²⁺ and phospholipid binding of synaptotagmin is affected when the Ca²⁺ binding sites in both C2 domains and the putative phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)-interacting site in the C2 domain are inactivated. We found that the two C2 domains bind calcium largely independently but cooperate in membrane binding. Furthermore, we confirmed that the C2B domain interacts specifically with PI(4,5)P₂. Remarkably, in the presence of PI(4,5)P₂, drastically lower amounts of calcium were needed for membrane binding.     

Relation between phosphorylation and adenosine triphosphate-dependent Ca2+ binding of swine and bovine erythrocyte membranes

The correlation between the ATP-dependent Ca²⁺ binding and the phosphorylation of the membranes from swine and bovine erythrocytes was studied. The Ca²⁺ binding was measured by using ⁴⁵CaCl₂, and the phosphorylation by [γ-³²P]ATP was studied with the technique of SDS polyacrylamide gel electrophoresis. 200 mM NaCl and KCl markedly repressed the Ca²⁺ binding of swine erythrocyte membranes. The radioactivity of ³²P-labelled membranes was revealed mainly in 250 000 dalton protein and a lipid fraction. NaCl and KCl also repressed the phosphorylation of the lipid which was identified as triphosphoinositide by paper chromatography. The membranes prepared from trypsin-digested erythrocytes completely retained the Ca²⁺-binding activity, and lost 30% of $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity. The Ca²⁺-binding and ATPase activity of isolated membranes decreased to 55% and to 0%, respectively, by tryptic digestion. Neither the Ca²⁺ binding nor the phosphorylation of polyphosphoinositides were detected in bovine erythrocyte membranes.These results suggest that the formation of triphosphoinositide rather than the $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ of membranes is linked to the ATP-dependent Ca²⁺ binding of erythrocyte membranes.     

Triggered Ca2+ influx is required for extended synaptotagmin 1-induced ER-plasma membrane tethering

The extended synaptotagmins (E-Syts) are ER proteins that act as Ca²⁺-regulated tethers between the ER and the plasma membrane (PM) and have a putative role in lipid transport between the two membranes. Ca²⁺ regulation of their tethering function, as well as the interplay of their different domains in such function, remains poorly understood. By exposing semi-intact cells to buffers of variable Ca²⁺ concentrations, we found that binding of E-Syt1 to the PI(4,5)P₂-rich PM critically requires its C2C and C2E domains and that the EC₅₀ of such binding is in the low micromolar Ca²⁺ range. Accordingly, E-Syt1 accumulation at ER-PM contact sites occurred only upon experimental manipulations known to achieve these levels of Ca²⁺ via its influx from the extracellular medium, such as store-operated Ca²⁺ entry in fibroblasts and membrane depolarization in β-cells. We also show that in spite of their very different physiological functions, membrane tethering by E-Syt1 (ER to PM) and by synaptotagmin (secretory vesicles to PM) undergo a similar regulation by plasma membrane lipids and cytosolic Ca²⁺.