The PIP2 binding mode of the C2 domains of rabphilin-3A

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a key player in the neurotransmitter release process. Rabphilin-3A is a neuronal C2 domain tandem containing protein that is involved in this process. Both its C2 domains (C2A and C2B) are able to bind PIP2. The investigation of the interactions of the two C2 domains with the PIP2 headgroup IP3 (inositol-1,4,5-trisphosphate) by NMR showed that a well-defined binding site can be described on the concave surface of each domain. The binding modes of the two domains are different. The binding of IP3 to the C2A domain is strongly enhanced by Ca(2+) and is characterized by a K(D) of 55 microM in the presence of a saturating concentration of Ca(2+) (5 mM). Reciprocally, the binding of IP3 increases the apparent Ca(2+)-binding affinity of the C2A domain in agreement with a Target-Activated Messenger Affinity (TAMA) mechanism. The C2B domain binds IP3 in a Ca(2+)-independent fashion with low affinity. These different PIP2 headgroup recognition modes suggest that PIP2 is a target of the C2A domain of rabphilin-3A while this phospholipid is an effector of the C2B domain.     

Structural preference for changes in the direction of the Ca2+-induced transition: a study of the regulatory domain of skeletal troponin-C

The determinants for specificity in the Ca(2+)-dependent response of the regulatory N-terminal domain of skeletal troponin-C are a combination of intrinsic and induced properties. We characterized computationally the intrinsic propensity of this domain for structural changes similar to those observed experimentally in the Ca(2+)-induced transition. The preference for such changes was assessed by comparing the structural effect of the harmonic and quasiharmonic vibrations specific for each Ca(2+) occupancy with crystallographic data. Results show that only the Ca(2+)-saturated form of the protein features a slow vibrational motion preparatory for the transition. From the characteristics of this mode, we identified a molecular mechanism for transition, by which residues 42-51 of helix B and of the adjacent linker move toward helices (A, D), and bind to the surface used by the protein to interact with troponin-I. By obstructing the access of the target to hydrophobic residues important in the formation of the complex, helix B and the adjacent linker act as an autoinhibitory structural element. Specific properties of the methionines at the interaction surface were found to favor the binding of the autoinhibitory region. Located over hydrophobic residues critical for binding, the methionines are easily displaceable to increase the accessibility of these residues to molecular encounter.     

Structural determinants for Ca2+ and phosphatidylinositol 4,5-bisphosphate binding by the C2A domain of rabphilin-3A

Rabphilin-3A is a neuronal C2 domain tandem containing protein involved in vesicle trafficking. Both its C2 domains (C2A and C2B) are able to bind phosphatidylinositol 4,5-bisphosphate, a key player in the neurotransmitter release process. The rabphilin-3A C2A domain has previously been shown to bind inositol-1,4,5-trisphosphate (IP3; phosphatidylinositol 4,5-bisphosphate headgroup) in a Ca2+-dependent manner with a relatively high affinity (50 microm) in the presence of saturating concentrations of Ca2+. Moreover, IP3 and Ca2+ binding to the C2A domain mutually enhance each other. Here we present the Ca2+-bound solution structure of the C2A domain. Structural comparison with the previously published Ca2+-free crystal structure revealed that Ca2+ binding induces a conformational change of Ca2+ binding loop 3 (CBL3). Our IP3 binding studies as well as our IP3-C2A docking model show the active involvement of CBL3 in IP3 binding, suggesting that the conformational change on CBL3 upon Ca2+ binding enables the interaction with IP3 and vice versa, in line with a target-activated messenger affinity mechanism. Our data provide detailed structural insight into the functional properties of the rabphilin-3A C2A domain and reveal for the first time the structural determinants of a target-activated messenger affinity mechanism.     

Synaptotagmin-like protein 1-3: a novel family of C-terminal-type tandem C2 proteins

Synaptotagmins (Syt), rabphilin-3A, and Doc2 belong to a family of carboxyl terminal type (C-type) tandem C2 proteins and are thought to be involved in vesicular trafficking. We have cloned and characterized a novel family of C-type tandem C2 proteins, designated Slp1-3 (synaptotagmin-like protein 1-3). The Slp1-3 C2 domains show high homology to granuphilin-a C2 domains, but the amino-terminal domain of Slp1-3 does not contain any known protein motifs or a transmembrane domain. A subcellular fractionation study indicated that Slp1-3 proteins are peripheral membrane proteins. Phospholipid binding experiments indicated that Slp3 is a Ca(2+)-dependent isoform, but Slp1 and Slp2 are Ca(2+)-independent isoforms, because only the Slp3 C2A domain showed Ca(2+)-dependent phospholipid binding activity. The C-terminus of Slp1-3 also bound neurexin Ialpha in vitro, in the same manner as Syt family proteins, which may be important for the membrane association of Slp1-3. In addition, Slp family proteins are differentially distributed in different mouse tissues and at different developmental stages.     

Low affinity Ca2+-binding sites of calcineurin B mediate conformational changes in calcineurin A

Limited proteolysis of calcineurin in the presence of Ca(2+) suggested that its calmodulin-binding domain, readily degraded by proteases, was unfolded while calcineurin B was compactly folded [Hubbard, M. J., and Klee, C. B. (1989) Biochemistry 28, 1868-1874]. Moreover, in the crystal structure of calcineurin, with the four Ca(2+) sites of calcineurin B occupied, the calmodulin-binding domain is not visible in the electron density map [Kissinger, C. R., et al. (1995) Nature 378, 641-644]. Limited proteolysis of calcineurin in the presence of EGTA, shows that, when the low affinity sites of calcineurin B are not occupied, the calmodulin-binding domain is completely protected against proteolytic attack. Slow cleavages are, however, detected in the linker region between the calmodulin-binding and the autoinhibitory domains of calcineurin A. Upon prolonged exposure to the protease, selective cleavages in carboxyl-terminal end of the first helix and the central helix linker of calcineurin B and the calcineurin B-binding helix of calcineurin A are also detected. Thus, Ca(2+) binding to the low-affinity sites of calcineurin B affects the conformation of calcineurin B and induces a conformational change of the regulatory domain of calcineurin A, resulting in the exposure of the calmodulin-binding domain. This conformational change is needed for the partial activation of the enzyme in the absence of calmodulin and its full activation by calmodulin. A synthetic peptide corresponding to the calmodulin-binding domain is shown to interact with a peptide corresponding to the calcineurin B-binding domain, and this interaction is prevented by calcineurin B in the presence but not the absence of Ca(2+). These observations provide a mechanism to explain the dependence on Ca(2+) binding to calcineurin B for calmodulin activation and for the 10-20-fold increase in affinity of calcineurin for Ca(2+) upon removal of the regulatory domain by limited proteolysis [Stemmer, P. M., and Klee, C. B. (1994) Biochemistry 33, 6859-6866].     

Close membrane-membrane proximity induced by Ca(2+)-dependent multivalent binding of synaptotagmin-1 to phospholipids

Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.     

Phosphatidylinositol phosphates as co-activators of Ca2+ binding to C2 domains of synaptotagmin 1

Ca2+-dependent phospholipid binding to the C2A and C2B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca2+ binding to the C2B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca2+/phospholipid binding and in release. Mutations in a polybasic sequence on the side of the C2B domain beta-sandwich or in a basic residue in a top Ca2+-binding loop of the C2A domain (R233) cause comparable decreases in the apparent Ca2+ affinity of synaptotagmin 1 and the Ca2+ sensitivity of release, whereas mutation of the residue homologous to Arg233 in the C2B domain (Lys366) has no effect. Phosphatidylinositol polyphosphates co-activate Ca2+-dependent and -independent phospholipid binding to synaptotagmin 1, but the effects of these mutations on release only correlate with their effects on the Ca2+-dependent component. These results reveal clear distinctions in the Ca2+-dependent phospholipid binding modes of the synaptotagmin 1 C2 domains that may underlie their functional asymmetry and suggest that phosphatidylinositol polyphosphates may serve as physiological modulators of Ca2+ affinity of synaptotagmin 1 in vivo.     

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 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.     

Apocalmodulin and Ca2+ calmodulin-binding sites on the CaV1.2 channel

The cardiac L-type voltage-dependent calcium channel is responsible for initiating excitation-contraction coupling. Three sequences (amino acids 1609-1628, 1627-1652, and 1665-1685, designated A, C, and IQ, respectively) of its alpha(1) subunit contribute to calmodulin (CaM) binding and Ca(2+)-dependent inactivation. Peptides matching the A, C, and IQ sequences all bind Ca(2+)CaM. Longer peptides representing A plus C (A-C) or C plus IQ (C-IQ) bind only a single molecule of Ca(2+)CaM. Apocalmodulin (ApoCaM) binds with low affinity to the IQ peptide and with higher affinity to the C-IQ peptide. Binding to the IQ and C peptides increases the Ca(2+) affinity of the C-lobe of CaM, but only the IQ peptide alters the Ca(2+) affinity of the N-lobe. Conversion of the isoleucine and glutamine residues of the IQ motif to alanines in the channel destroys inactivation (Zühlke et al., 2000). The double mutation in the peptide reduces the interaction with apoCaM. A mutant CaM unable to bind Ca(2+) at sites 3 and 4 (which abolishes the ability of CaM to inactivate the channel) binds to the IQ, but not to the C or A peptide. Our data are consistent with a model in which apoCaM binding to the region around the IQ motif is necessary for the rapid binding of Ca(2+) to the C-lobe of CaM. Upon Ca(2+) binding, this lobe is likely to engage the A-C region.     

Sr2+ binding to the Ca2+ binding site of the synaptotagmin 1 C2B domain triggers fast exocytosis without stimulating SNARE interactions

Sr(2+) triggers neurotransmitter release similar to Ca(2+), but less efficiently. We now show that in synaptotagmin 1 knockout mice, the fast component of both Ca(2+)- and Sr(2+)-induced release is selectively impaired, suggesting that both cations partly act by binding to synaptotagmin 1. Both the C(2)A and the C(2)B domain of synaptotagmin 1 bind Ca(2+) in phospholipid complexes, but only the C(2)B domain forms Sr(2+)/phospholipid complexes; therefore, Sr(2+) binding to the C(2)B domain is sufficient to trigger fast release, although with decreased efficacy. Ca(2+) induces binding of the synaptotagmin C(2) domains to SNARE proteins, whereas Sr(2+) even at high concentrations does not. Thus, triggering of the fast component of release by Sr(2+) as a Ca(2+) agonist involves the formation of synaptotagmin/phospholipid complexes, but does not require stimulated SNARE binding.     

DOC2B,C2 Domains,and Calcium: A Tale of Intricate Interactions

Ca⁺²-dependent exocytosis involves vesicle docking, priming, fusion, and recycling. This process is performed and regulated by a vast number of synaptic proteins and depends on proper protein–protein and protein–lipid interactions. Double C2 domain (DOC2) is a protein family of three isoforms found while screening DNA libraries with a C2 probe. DOC2 has three domains: the Munc13-interacting domain and tandem C2s (designated C2A and C2B) connected by a short polar linker. The C2 domain binds phospholipids in a Ca²⁺-dependent manner. This review focuses on the ubiquitously expressed isoform DOC2B. Sequence alignment of the tandem C2 protein family in mouse revealed high homology (81%) between rabphilin-3A and DOC2B proteins. We created a structural model of DOC2B's C2A based on the crystal structure of rabphilin-3A with and without calcium and found that the calcium-binding loops of DOC2B move upon calcium binding, enabling efficient plasma membrane penetration of its C2A. Here, we discuss the potential relation between the DOC2B bioinformatical model and its function and suggest a possible working model for its interaction with other proteins of the exocytotic machinery, including Munc13, Munc18, and syntaxin.     

Ca2+ activation of the cPLA2 C2 domain: ordered binding of two Ca2+ ions with positive cooperativity

During Ca(2+) activation, the Ca(2+)-binding sites of C2 domains typically bind multiple Ca(2+) ions in close proximity. These binding events exhibit positive cooperativity, despite the strong charge repulsion between the adjacent divalent cations. Using both experimental and computational approaches, the present study probes the detailed mechanisms of Ca(2+) activation and positive cooperativity for the C2 domain of cytosolic phospholipase A(2), which binds two Ca(2+) ions in sites I and II, separated by only 4.1 A. First, each of the five coordinating side chains in the Ca(2+)-binding cleft is individually mutated and the effect on Ca(2+)-binding affinity and cooperativity is measured. The results identify Asp 43 as the major contributor to Ca(2+) affinity, while the two coordinating side chains that provide bridging coordination to both Ca(2+) ions, Asp 43 and Asp 40, are observed to make the largest contributions to positive cooperativity. Electrostatic calculations reveal that Asp 43 possesses the highest pseudo-pK(a) of the coordinating acidic residues, as well as the highest general cation affinity, due to its relatively buried location within 3.5 A of seven protein oxygens with full or partial negative charges. These calculations therefore explain the greater importance of Asp 43 in defining the Ca(2+) affinity. Overall, the experimental and computational results support an activation model in which the first Ca(2+) ion binds usually to site I, thereby preordering both bridging side chains Asp 40 and 43, and partially or fully deprotonating the three coordinating Asp residues. This initial binding event prepares the conformation and protonation state of the remaining site for Ca(2+) binding, enabling the second Ca(2+) ion to bind with higher affinity than the first as required for positive cooperativity.     

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.     

X-ray reflectivity studies of cPLA2{alpha}-C2 domains adsorbed onto Langmuir monolayers of SOPC

X-ray reflectivity is used to study the interaction of C2 domains of cytosolic phospholipase A(2) (cPLA(2)alpha-C2) with a Langmuir monolayer of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) supported on a buffered aqueous solution containing Ca(2+). The reflectivity is analyzed in terms of the known crystallographic structure of cPLA(2)alpha-C2 domains and a slab model representing the lipid layer to yield an electron density profile of the lipid layer and bound C2 domains. This new method of analysis determines the angular orientation and penetration depth of the cPLA(2)alpha-C2 domains bound to the SOPC monolayer, information not available from the standard slab model analysis of x-ray reflectivity. The best-fit orientation places the protein-bound Ca(2+) ions within 1 A of the lipid phosphate group (with an accuracy of +/-3 A). Hydrophobic residues of the calcium-binding loops CBL1 and CBL3 penetrate deepest into the lipid layer, with a 2 A penetration into the tailgroup region. X-ray measurements with and without the C2 domain indicate that there is a loss of electrons in the headgroup region of the lipid monolayer upon binding of the domains. We suggest that this is due to a loss of water molecules bound to the headgroup. Control experiments with a non-calcium buffer and with domain mutants confirm that the cPLA(2)alpha-C2 binding to the SOPC monolayer is Ca(2+)-dependent and that the hydrophobic residues in the calcium-binding loops are critical for membrane binding. These results indicate that an entropic component (due to water loss) as well as electrostatic and hydrophobic interactions contributes to the binding mechanism.     

Calcium-induced conformational switching of Paramecium calmodulin provides evidence for domain coupling

Calmodulin (CaM) is an intracellular calcium-binding protein essential for many pathways in eukaryotic signal transduction. Although a structure of Ca(2+)-saturated Paramecium CaM at 1.0 A resolution (1EXR.pdb) provides the highest level of detail about side-chain orientations in CaM, information about an end state alone cannot explain driving forces for the transitions that occur during Ca(2+)-induced conformational switching and why the two domains of CaM are saturated sequentially rather than simultaneously. Recent studies focus attention on the contributions of interdomain linker residues. Electron paramagnetic resonance showed that Ca(2+)-induced structural stabilization of residues 76-81 modulates domain coupling [Qin and Squier (2001) Biophys. J. 81, 2908-2918]. Studies of N-domain fragments of Paramecium CaM showed that residues 76-80 increased thermostability of the N-domain but lowered the Ca(2+) affinity of sites I and II [Sorensen et al. (2002) Biochemistry 41, 15-20]. To probe domain coupling during Ca(2+) binding, we have used (1)H-(15)N HSQC NMR to monitor more than 40 residues in Paramecium CaM. The titrations demonstrated that residues Glu78 to Glu84 (in the linker and cap of helix E) underwent sequential phases of conformational change. Initially, they changed in volume (slow exchange) as sites III and IV titrated, and subsequently, they changed in frequency (fast exchange) as sites I and II titrated. These studies provide evidence for Ca(2+)-dependent communication between the domains, demonstrating that spatially distant residues respond to Ca(2+) binding at sites I and II in the N-domain of CaM.     

Deletions in the A(L) region of the h4xb plasma membrane Ca(2+) pump. High apparent affinity for Ca(2+) of a deletion mutant resembling the alternative spliced form h4zb

Mutants of the plasma membrane Ca(2+) pump (human isoform 4xb) with deletions in the linker between domain A and transmembrane segment M3 (A(L) region) were constructed and expressed in Chinese hamster ovary cells. The total or partial removal of the amino acid segment 300-349 did not change the maximal Ca(2+) transport activity, but mutants with deletions involving residues 300-338 exhibited a higher apparent affinity for Ca(2+) than the wild type h4xb enzyme. Deletion of the putative acidic lipid interacting sequence (residues 339-349) had no observable functional consequences. The removal of either residues 300-314 or 313-338 resulted in a similar increase in the apparent Ca(2+) affinity of the pump although the increase was somewhat lower than that obtained by the deletion 300-349 suggesting that both deletions affected the same structural determinant. The results show that alterations in the region of the alternative splicing site A change the sensitivity to Ca(2+) of the human isoform 4 of the PMCA.     

Membrane targeting of C2 domains of phospholipase C-delta isoforms.

The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. To understand the mechanisms by which the C2 domain mediates the membrane targeting of PLC-delta isoforms, we measured the in vitro membrane binding of the C2 domains of PLC-delta1, -delta3, and -delta4 by surface plasmon resonance and monolayer techniques and their subcellular localization by time-lapse confocal microscopy. The membrane binding of the PLC-delta1-C2 is driven by nonspecific electrostatic interactions between the Ca(2+)-induced cationic surface of protein and the anionic membrane and specific interactions involving Ca(2+), Asn(647), and phosphatidylserine (PS). The PS selectivity of PLC-delta1-C2 governs its specific Ca(2+)-dependent subcellular targeting to the plasma membrane. The membrane binding of the PLC-delta3-C2 also involves Ca(2+)-induced nonspecific electrostatic interactions and PS coordination, and the latter leads to specific subcellular targeting to the plasma membrane. In contrast to PLC-delta1-C2 and PLC-delta3-C2, PLC-delta4-C2 has significant Ca(2+)-independent membrane affinity and no PS selectivity due to the presence of cationic residues in the Ca(2+)-binding loops and the substitution of Ser for the Ca(2+)-coordinating Asp in position 717. Consequently, PLC-delta4-C2 exhibits unique pre-localization to the plasma membrane prior to Ca(2+) import and non-selective Ca(2+)-mediated targeting to various cellular membranes, suggesting that PLC-delta4 might have a novel regulatory mechanism. Together, these results establish the C2 domains of PLC-delta isoforms as Ca(2+)-dependent membrane targeting domains that have distinct membrane binding properties that control their subcellular localization behaviors.     

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.