Membrane orientation and position of the C2 domain from cPLA2 by site-directed spin labeling

The C2 domain is a ubiquitous Ca(2+)-binding motif that triggers the membrane docking of many key signaling proteins during intracellular Ca(2+) signals. Site-directed spin labeling was carried out on the C2 domain of cytosolic phospholipase A(2) in order to determine the depth of penetration and orientation of the domain at the membrane interface. Membrane depth parameters, Phi, were obtained by EPR spectroscopy for a series of selectively spin-labeled C2 domain cysteine mutants, and for spin-labeled lipids and spin-labeled bacteriorhodopsin cysteine mutants. Values of Phi were combined with several other constraints, including the solution NMR structure, to generate a model for the position of the C2 domain at the membrane interface. This modeling yielded an empirical expression for Phi, which for the first time defines its behavior from the bulk aqueous phase to the center of the lipid bilayer. In this model, the backbones of both the first and third Ca(2+)-binding loops are inserted approximately 10 A into the bilayer, with residues inserted as deep as 15 A. The backbone of the second Ca(2+)-binding loop is positioned near the lipid phosphate, and the two beta-sheets of the C2 domain are oriented so that the individual strands make angles of 30-45 degrees with respect to the bilayer surface. Upon membrane docking, spin labels in the Ca(2+)-binding loops exhibit decreases in local motion, suggesting either changes in tertiary contacts due to protein conformational changes and/or interactions with lipid.     

Membrane-bound orientation and position of the synaptotagmin I C2A domain by site-directed spin labeling

Site-directed spin labeling was used to determine the membrane orientation and insertion of the C2A domain from synaptotagmin I. A series of single cysteine mutants of the C2A domain of synaptotagmin I was prepared and labeled with a sulfhydryl specific spin label. Upon Ca2+ or membrane binding, the EPR line shapes of these mutants reveal dramatic decreases in label mobility within the Ca2+-binding loops. This loss in mobility is likely due in part to a reduction in local backbone fluctuations within the loop regions. Power saturation was then used to determine the position of each spin-labeled site along the bilayer normal, and these EPR distance constraints were used along with the high-resolution solution structure of C2A to generate a model for the orientation and position of the domain at the membrane interface. This model places the polypeptide backbone of both the first and third Ca2+-binding loops in contact with the membrane interface, with several labeled side chains lying within the bilayer interior. All three Ca2+-binding sites lie near a plane defined by the lipid phosphates. This model indicates that there is some desolvation of this domain upon binding and that hydrophobic as well as electrostatic interactions contribute to the binding of C2A. When compared to the C2 domain from cPLA2 (Frazier et al. (2002) Biochemistry 41, 6282), a similar orientation for the beta-sandwich region is found; however, the cPLA2 C2 domain is translocated 5-7 A deeper into the membrane hydrocarbon. This difference in depth is consistent with previous biophysical data and with the difference that long-range electrostatic interactions and desolvation are expected to make to the binding of these two C2 domains.     

Membrane-embedded synaptotagmin penetrates cis or trans target membranes and clusters via a novel mechanism

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a cytoplasmic domain largely comprised of two C2 domains designated C2A and C2B. We have determined how deep the Ca(2+)-binding loops of Ca(2+).C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca(2+)-triggered interactions with the SNARE complex. To determine whether C2A penetrates into the vesicle ("cis") or plasma ("trans") membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-TMR) into proteoliposomes. Kinetics experiments revealed that cis interactions are rapid (< or =500 micros). Binding in the trans mode was distinguished by the slow diffusion of trans target vesicles. Both modes of binding were observed, indicating that the linker between the membrane anchor and C2A domain functions as a flexible tether. C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that synaptotagmin may form clusters on the surface of synaptic vesicles. This novel mode of clustering may allow for rapid Ca(2+)-triggered oligomerization of the protein via the membrane distal C2B domain.     

Phosphatidylinositol 4,5-bisphosphate alters synaptotagmin 1 membrane docking and drives opposing bilayers closer together

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 assessed in bilayers containing phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol 4,5-bisphosphate (PIP(2)). Addition of 1 mol % PIP(2) 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 the proximity of the bilayer interface. The C2B domain is found to contact the membrane interface in two regions, the Ca(2+)-binding loops and a region opposite the Ca(2+)-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 PIP(2) and used in a simulated annealing routine to dock syt1C2AB to two membrane interfaces. The results yield an average structure different from what is found in the absence of PIP(2) and indicate that bilayer-bilayer spacing is decreased in the presence of PIP(2). The results indicate that PIP(2), 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.     

Position of synaptotagmin I at the membrane interface: cooperative interactions of tandem C2 domains

Synaptotagmin I is a synaptic vesicle associated membrane protein that appears to regulate Ca(2+)-mediated exocytosis. Here, the Ca(2+)-dependent membrane interactions of a water soluble fragment of synaptotagmin I (C2AB) that contains its two C2 domains (C2A and C2B) were determined using site-directed spin labeling. Membrane depth parameters were obtained for 19 spin-labeled mutants of C2AB when bound to phosphatidylcholine and phosphatidylserine membranes, and these distance constraints were used in combination with the high-resolution structures of C2A and C2B to generate a model for the membrane orientation and position of synaptotagmin at the bilayer interface. Both C2A and C2B bind to the membrane interface with their first and third Ca(2+) binding loops penetrating the membrane interface. The polybasic face of C2B does not interact with the membrane lipid but is available for electrostatic interaction with other components of the fusion machinery. When compared to positions determined previously for the isolated domains, both C2A and C2B have similar orientations; however, the two domains are positioned deeper into the bilayer interior when present in the tandem construct. These data indicate that C2A and C2B do not act independently but influence their mutual membrane penetration. This may explain the occurrence of multiple C2 domains in proteins that function in membrane trafficking and repair.     

Effect of PIP2 binding on the membrane docking geometry of PKC alpha C2 domain: an EPR site-directed spin-labeling and relaxation study

Protein kinase C isoform alpha (PKCalpha) is a ubiquitous, conventional PKC enzyme that possesses a conserved C2 domain. Upon activation by cytoplasmic Ca (2+) ions, the C2 domain specifically binds to the plasma membrane inner leaflet where it recognizes the target lipids phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PIP 2). The membrane penetration depth and docking angle of the membrane-associated C2 domain is not well understood. The present study employs EPR site-directed spin labeling and relaxation methods to generate a medium-resolution model of the PKCalpha C2 domain docked to a membrane of lipid composition similar to the plasma membrane inner leaflet. The approach measures EPR depth parameters for 10 function-retaining spin labels coupled to the C2 domain, and for spin labels coupled to depth calibration molecules. The resulting depth parameters, together with the known structure of the free C2 domain, provide a sufficient number of constraints to define two membrane docking geometries for C2 domain bound to physiological membranes lacking or containing PIP 2, respectively. In both the absence and presence of PIP 2, the two bound Ca (2+) ions of the C2 domain lie near the anionic phosphate plane in the headgroup region, consistent with the known ability of the Ca (2+) and membrane-binding loops (CMBLs) to bind the headgroup of the PS target lipid. In the absence of PIP 2, the polybasic lipid binding site on the beta3-beta4 hairpin is occupied with PS, but in the presence of PIP 2 this larger, higher affinity target lipid competitively displaces PS and causes the long axis of the domain to tilt 40 +/- 10 degrees toward the bilayer normal. The ability of the beta3-beta4 hairpin site to bind PS as well as PIP 2 extends the lifetime of the membrane-docked state and is predicted to enhance the kinase turnover number of PKCalpha during a single membrane docking event. In principle, PIP 2-induced tilting of the C2 domain could modulate the activity of membrane-docked PKCalpha as it diffuses between membrane regions with different local PS and PIP 2 concentrations. Finally, the results demonstrate that EPR relaxation methods are sufficiently sensitive to detect signaling-induced changes in the membrane docking geometries of peripheral membrane proteins.     

Membrane-docking loops of the cPLA2 C2 domain: detailed structural analysis of the protein-membrane interface via site-directed spin-labeling

C2 domains are protein modules found in numerous eukaryotic signaling proteins, where their function is to target the protein to cell membranes in response to a Ca(2+) signal. Currently, the structure of the interface formed between the protein and the phospholipid bilayer is inaccessible to high-resolution structure determination, but EPR site-directed spin-labeling can provide a detailed medium-resolution view of this interface. To apply this approach to the C2 domain of cytosolic phospholipase A(2) (cPLA(2)), single cysteines were introduced at all 27 positions in the three Ca(2+)-binding loops and labeled with a methanethiosulfonate spin-label. Altogether, 24 of the 27 spin-labeled domains retained Ca(2+)-activated phospholipid binding. EPR spectra of these 24 labeled domains obtained in the presence and absence of Ca(2+) indicate that Ca(2+) binding triggers subtle changes in the dynamics of two localized regions within the Ca(2+)-binding loops: one face of the loop 1 helix and the junction between loops 1 and 2. However, no significant changes in loop structure were detected upon Ca(2+) binding, nor upon Ca(2+)-triggered docking to membranes. EPR depth parameters measured in the membrane-docked state allow determination of the penetration depth of each residue with respect to the membrane surface. Analysis of these depth parameters, using an improved, generalizable geometric approach, provides the most accurate picture of penetration depth and angular orientation currently available for a membrane-docked peripheral protein. Finally, the observation that Ca(2+) binding does not trigger large rearrangements of the membrane-docking loops favors the electrostatic switch model for Ca(2+) activation and disfavors, or places strong constraints on, the conformational switch model.     

The C2B domain of synaptotagmin I is a Ca2+-binding module

Synaptotagmin I is a synaptic vesicle protein that contains two C(2) domains and acts as a Ca(2+) sensor in neurotransmitter release. The Ca(2+)-binding properties of the synaptotagmin I C(2)A domain have been well characterized, but those of the C(2)B domain are unclear. The C(2)B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca(2+)-dependent manner, leading to an attractive model whereby Ca(2+)-dependent multimerization of synaptotagmin I via the C(2)B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca(2+)-dependent C(2)B domain dimerization, as well as in analyses of other C(2)B domain interactions. To shed light on these issues, the C(2)B domain has now been studied using biophysical techniques. The recombinant C(2)B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C(2)B domain interactions, including Ca(2+)-dependent dimerization. NMR experiments show that the pure recombinant C(2)B domain binds Ca(2+) directly but does not dimerize upon Ca(2+) binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C(2)A and C(2)B domains, participates in a high molecular weight complex as a function of Ca(2+). These results show that the recombinant C(2)B domain of synaptotagmin I is a monomeric, autonomously folded Ca(2+)-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C(2)B domains or requires a posttranslational modification.     

Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4

The neuronal protein synaptotagmin 1 functions as a Ca(2+) sensor in exocytosis via two Ca(2+)-binding C(2) domains. The very similar synaptotagmin 4, which includes all the predicted Ca(2+)-binding residues in the C(2)B domain but not in the C(2)A domain, is also thought to function as a neuronal Ca(2+) sensor. Here we show that, unexpectedly, both C(2) domains of fly synaptotagmin 4 exhibit Ca(2+)-dependent phospholipid binding, whereas neither C(2) domain of rat synaptotagmin 4 binds Ca(2+) or phospholipids efficiently. Crystallography reveals that changes in the orientations of critical Ca(2+) ligands, and perhaps their flexibility, render the rat synaptotagmin 4 C(2)B domain unable to form full Ca(2+)-binding sites. These results indicate that synaptotagmin 4 is a Ca(2+) sensor in the fly but not in the rat, that the Ca(2+)-binding properties of C(2) domains cannot be reliably predicted from sequence analyses, and that proteins clearly identified as orthologs may nevertheless have markedly different functional properties.     

The top loops of the C(2) domains from synaptotagmin and phospholipase A(2) control functional specificity.

The phospholipid-binding specificities of C(2) domains, widely distributed Ca(2+)-binding modules, differ greatly despite similar three-dimensional structures. To understand the molecular basis for this specificity, we have examined the synaptotagmin 1 C(2)A domain, which interacts in a primarily electrostatic, Ca(2+)-dependent reaction with negatively charged phospholipids, and the cytosolic phospholipase A(2) (cPLA(2)) C(2) domain, which interacts by a primarily hydrophobic Ca(2+)-dependent mechanism with neutral phospholipids. We show that grafting the short Ca(2+)-binding loops from the tip of the cPLA(2) C(2) domain onto the top of the synaptotagmin 1 C(2)A domain confers onto the synaptotagmin 1 C(2)A domain the phospholipid binding specificity of the cPLA(2) C(2) domain, indicating that the functional specificity of C(2) domains is determined by their short top loops.     

C2 domain of protein kinase C alpha: elucidation of the membrane docking surface by site-directed fluorescence and spin labeling

The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca(2+)-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase C alpha (PKC alpha) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca(2+) binding loops and an anion binding site on beta-strands 3-4, such that the beta-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca(2+) binding loops and the beta-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKC alpha C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca(2+) activation or membrane docking trigger measurable fluorescence changes. Ca(2+) binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca(2+) binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Phi, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca(2+) binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca(2+) first and third Ca(2+) binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on beta-strands 3-4 lies near the headgroups. The data support a model in which the beta-strands are tilted toward the parallel orientation relative to the membrane surface.     

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).     

The C2B domain of synaptotagmin is a Ca(2+)-sensing module essential for exocytosis

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca(2+) ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca(2+)-sensing module. Here, we report that Ca(2+) drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca(2)+ are mediated by a set of conserved acidic Ca(2)+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca(2)+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.     

Activation of phospholipase C delta1 through C2 domain by a Ca(2+)-enzyme-phosphatidylserine ternary complex.

The concentration of free Ca(2+) and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C delta1 (PLCdelta1). The rate of PLCdelta1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca(2+) concentration required for half-maximal activation of PLCdelta1 from 5.4 to 0.5 microM. In the presence of Ca(2+), PLCdelta1 specifically bound to PS/PC but not to PA/PC vesicles in a dose-dependent and saturable manner. Ca(2+) also bound to PLCdelta1 and required the presence of PS/PC vesicles but not PA/PC vesicles. The free Ca(2+) concentration required for half-maximal Ca(2+) binding was estimated to be 8 microM. Surface dilution kinetic analysis revealed that the K(m) was reduced 20-fold by the presence of 25 mol % PS, whereas V(max) and K(d) were unaffected. Deletion of amino acid residues 646-654 from the C2 domain of PLCdelta1 impaired Ca(2+) binding and reduced its stimulation and binding by PS. Taken together, the results suggest that the formation of an enzyme-Ca(2+)-PS ternary complex through the C2 domain increases the affinity for substrate and consequently leads to enzyme activation.     

Structure of human synaptotagmin 1 C2AB in the absence of Ca2+ reveals a novel domain association

Release of neurotransmitter from synaptic vesicles requires the Ca2+/phospholipid-binding protein synaptotagmin 1. There is considerable evidence that cooperation between the tandem C2 domains of synaptotagmin is a requirement of regulated exocytosis; however, high-resolution structural evidence for this interaction has been lacking. The 2.7 A crystal structure of the cytosolic domains of human synaptotagmin 1 in the absence of Ca2+ reveals a novel closed conformation of the protein. The shared interface between C2A and C2B is stabilized by a network of interactions between residues on the C-terminal alpha-helix of the C2B domain and residues on loops 1-3 of the Ca2+-binding region of C2A. These interactions alter the overall shape of the Ca2+-binding pocket of C2A, but not that of C2B. Thus, synaptotagmin 1 C2A-C2B may utilize a novel regulatory mechanism whereby one C2 domain could regulate the other until an appropriate triggering event decouples them.     

Partitioning of synaptotagmin I C2 domains between liquid-ordered and liquid-disordered inner leaflet lipid phases

Synaptotagmin I is the calcium sensor in synchronous neurotransmitter release caused by fusion of synaptic vesicles with the presynaptic membrane in neurons. Synaptotagmin I interacts with acidic phospholipids, but also with soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs), at various stages in presynaptic membrane fusion. Because SNAREs can be organized into small cholesterol-dependent clusters in membranes, it is important to determine whether the C2 domains of synaptotagmin target membrane domains with different cholesterol contents. To address this question, we used a previously developed asymmetric two-phase lipid bilayer system to investigate the membrane binding and lipid phase targeting of soluble C2A and C2AB domains of synaptotagmin. We found that both domains target more disordered cholesterol-poor domains better than highly ordered cholesterol-rich domains. The selectivity is greatest (～3-fold) for C2A binding to disordered domains that are formed in the presence of 5 mol % PIP(2) and 15 mol % PS. It is smallest (～1.4-fold) for C2AB binding to disordered domains that are formed in the presence of 40 mol % PS. In the course of these experiments, we also found that C2A domains in the presence of Ca(2+) and C2AB domains in the absence of Ca(2+) are quite reliable reporters of the acidic lipid distribution between ordered and disordered lipid phases. Accordingly, PS prefers the liquid-disordered phase over the liquid-ordered phase by ～2-fold, but PIP(2) has an up to 3-fold preference for the liquid-disordered phase.     

Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of acidic fibroblast growth factor

Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER)–Golgi secretory pathway. FGF1 is released through a Cu²⁺-mediated nonclassical secretion pathway. The secretion of FGF1 involves the formation of a Cu²⁺-mediated multiprotein release complex (MRC) including FGF1, S100A13 (a calcium-binding protein) and p40 synaptotagmin (Syt1). It is believed that the binding of Cu²⁺ to the C2B domain is important for the release of FGF1 into the extracellular medium. In this study, using a variety of biophysical studies, Cu²⁺ and lipid interactions of the C2B domain of Syt1 were characterized. Isothermal titration calorimetry (ITC) experiments reveal that the C2B domain binds to Cu²⁺ in a biphasic manner involving an initial endothermic and a subsequent exothermic phase. Fluorescence energy transfer experiments using Tb³⁺ show that there are two Cu²⁺-binding pockets on the C2B domain, and one of these is also a Ca²⁺-binding site. Lipid-binding studies using ITC demonstrate that the C2B domain preferentially binds to small unilamellar vesicles of phosphatidyl serine (PS). Results of the differential scanning calorimetry and limited trypsin digestion experiments suggest that the C2B domain is marginally destabilized upon binding to PS vesicles. These results, for the first time, suggest that the main role of the C2B domain of Syt1 is to serve as an anchor for the FGF1 MRC on the membrane bilayer. In addition, the binding of the C2B domain to the lipid bilayer is shown to significantly decrease the binding affinity of the protein to Cu²⁺. The study provides valuable insights on the sequence of structural events that occur in the nonclassical secretion of FGF1.     

Structure of the Janus-faced C2B domain of rabphilin

C2 domains are widespread protein modules that often occur as tandem repeats in many membrane-trafficking proteins such as synaptotagmin and rabphilin. The first and second C2 domains (C2A and C2B, respectively) have a high degree of homology but also specific differences. The structure of the C2A domain of synaptotagmin I has been extensively studied but little is known about the C2B domains. We have used NMR spectroscopy to determine the solution structure of the C2B domain of rabphilin. The overall structure of the C2B domain is very similar to that of other C2 domains, with a rigid beta-sandwich core and loops at the top (where Ca2+ binds) and the bottom. Surprisingly, a relatively long alpha-helix is inserted at the bottom of the domain and is conserved in all C2B domains. Our results, together with the Ca(2+)-independent interactions observed for C2B domains, indicate that these domains have a Janus-faced nature, with a Ca(2+)-binding top surface and a Ca(2+)-independent bottom surface.     

Mutations in the effector binding loops in the C2A and C2B domains of synaptotagmin I disrupt exocytosis in a nonadditive manner

The secretory vesicle protein synaptotagmin I (syt) plays a critical role in Ca2+-triggered exocytosis. Its cytoplasmic domain is composed of tandem C2 domains, C2A and C2B; each C2 domain binds Ca2+. Upon binding Ca2+, positively charged residues within the Ca2+-binding loops are thought to interact with negatively charged phospholipids in the target membrane to mediate docking of the cytoplasmic domain of syt onto lipid bilayers. The C2 domains of syt also interact with syntaxin and SNAP-25, two components of a conserved membrane fusion complex. Here, we have neutralized single positively charged residues at the membrane-binding interface of C2A (R233Q) and C2B (K366Q). Either of these mutations shifted the Ca2+ requirements for syt-liposome interactions from approximately 20 to approximately 40 microm Ca2+. Kinetic analysis revealed that the reduction in Ca2+-sensing activity was associated with a decrease in affinity for membranes. These mutations did not affect sytsyntaxin interactions but resulted in an approximately 50% loss in SNAP-25 binding activity, suggesting that these residues lie at an interface between membranes and SNAP-25. Expression of full-length versions of syt that harbored these mutations reduced the rate of exocytosis in PC12 cells. In both biochemical and functional assays, effects of the R233Q and K366Q mutations were not additive, indicating that mutations in one domain affect the activity of the adjacent domain. These findings indicate that the tandem C2 domains of syt cooperate with one another to trigger release via loop-mediated electrostatic interactions with effector molecules.     

Synaptotagmins form a hierarchy of exocytotic Ca(2+) sensors with distinct Ca(2+) affinities

Synaptotagmins constitute a large family of membrane proteins implicated in Ca(2+)-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca(2+) affinities of synaptotagmin C2-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca(2+) affinities, with plasma membrane synaptotagmins binding Ca(2+) with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca(2+) affinities are functionally important, we examined the effects of synaptotagmin C2-domains on Ca(2+)-triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca(2+) affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr(2+), which was also selectively affected by high-affinity C2-domains of synaptotagmins. Together, our results suggest that Ca(2+) triggering of exocytosis involves tandem Ca(2+) sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca(2+) sensors involved in slow Ca(2+)-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca(2+) sensors specialized for fast Ca(2+)-dependent exocytosis.