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J Biol Chem, Vol. 273, Issue 26, 15879-15882, June 26, 1998

MINIREVIEW
C₂-domains, Structure and Function of a Universal Ca²⁺-binding Domain^*

Josep Rizo^§ and Thomas C. Südhof^¶

From the Departments of  Biochemistry, Pharmacology, and ^¶ Molecular Genetics, the Center for Basic Neuroscience, and the Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235

	INTRODUCTION

Top Introduction References

A vast amount of protein sequence data accumulated over recent years has revealed that protein modules are widespread in nature. Many intracellular and extracellular proteins consist, in part or fully, of combinations of protein modules. C₂-domains, together with SH2, PTB, PH, SH3, WW, and PDZ domains, are typical examples of intracellular protein modules. These modules form independently folding domains of 80-160 residues with characteristic binding properties; C₂-domains bind Ca²⁺ and phospholipids, SH2 and PTB domains phosphotyrosine-containing sequences, PH domains phosphatidylinositol phosphates, SH3 and WW domains proline-rich sequences, and PDZ domains C-terminal sequences. C₂-domains are unique among these modules because phospholipid binding to many C₂-domains is regulated by Ca²⁺. For this reason, C₂-domains are sometimes referred to as Ca²⁺-dependent lipid binding domains. However, C₂-domains are not obligatory Ca²⁺- and phospholipid-binding modules. C₂-domains have diverged evolutionarily into Ca²⁺-dependent and Ca²⁺-independent forms that interact with multiple targets. Thus, although most C₂-domains are probably Ca²⁺-binding domains, they represent a family of versatile protein modules with diverse functions.

C₂-domains comprise approximately 130 residues and were first identified in protein kinase C (1). Close to 100 C₂-domain sequences are listed in the current data banks. Although reviews of several C₂-domain proteins have been published (2-11), recent results on the structure and interactions of C₂-domains by x-ray crystallography and NMR spectroscopy offer a new opportunity to rationalize the properties of C₂-domains in structural terms. In this minireview, we will attempt to use this opportunity and correlate the functional properties of C₂-domains with their structures.

	Proteins Containing C2-domains

Most proteins with C₂-domains function in signal transduction or membrane traffic. The first category includes proteins involved in the generation of lipid second messengers (e.g. cPLA₂¹ (12), PLCs (13), and phosphatidylinositol 3-kinases (14)), in protein phosphorylation (e.g. PKC (15, 16)), in activation of GTPases (e.g. Ras-GAP (17)), and in ubiquitin ligation (e.g. Nedd4 (18)). The second category contains synaptotagmins (19, 20), rabphilin-3 (21), RIM (22), and Munc13 (2). In addition to many well characterized proteins, several open reading frames with C₂-domains are reported in GenBank^TM. For example, open reading frames in Caenorhabditis elegans and yeast encode transmembrane proteins with three or four C₂-domains whose biological roles have not been identified. This suggests that additional interesting functions for C₂-domain proteins remain to be discovered.

Much of the current data on the structures and interactions of C₂-domains were derived from studies of PLC1, PKC, cPLA₂, and, in particular, synaptotagmin I. PLC1 hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (5). PKCs represent a family of protein kinases that are regulated by diacylglycerol and other lipids (1, 4). In addition, activation of the classical isoforms of PKC (PKC, -, and -) depends on Ca²⁺. cPLA₂ hydrolyzes glycerophospholipids to produce arachidonic acid, a precursor of prostaglandins and leukotrienes, which are involved in inflammation (7). In PLC1, PKC, and cPLA₂, the C₂-domain is believed to play a regulatory role by mediating the Ca²⁺-dependent recruitment of these enzymes to phospholipid membranes. In contrast, synaptotagmin I functions in membrane traffic. Synaptotagmin I belongs to a family of at least 12 transmembrane proteins containing two C₂-domains, the C₂A- and C₂B-domains. The C₂-domains occupy most of the cytoplasmic region of the synaptotagmins and probably act as Ca²⁺ effector domains. Synaptotagmin I is localized to synaptic vesicles where it is essential for the fast, Ca²⁺-dependent component of neurotransmitter release (23, 24). Synaptotagmin I is believed to function as the main Ca²⁺ sensor in synaptic vesicle exocytosis by a mechanism involving Ca²⁺ binding to both C₂-domains (see below). The functions of the other synaptotagmins and membrane trafficking proteins with C₂-domains are less well characterized but may be similar.

	Diverse Ca2+-dependent Properties of C2-domains

The notion that C₂-domains act as Ca²⁺-binding motifs arose from the observation that the classical isoforms of PKC, which contain a C₂-domain (PKC, -, and -), were regulated by Ca²⁺. In contrast, isoforms apparently lacking a C₂-domain (PKC, -, -, and -) were Ca²⁺-independent (1). Activation of classical PKCs by Ca²⁺ involves the translocation of PKCs to the membrane by Ca²⁺-dependent phospholipid binding (4, 25). These observations led to the hypothesis that C₂-domains may mediate Ca²⁺-dependent phospholipid binding.

This notion was first actually demonstrated for the C₂A-domain of synaptotagmin I. The isolated C₂A-domain was shown to represent an autonomously folding module that binds phospholipids in a Ca²⁺-dependent manner (26). The C₂A-domain bound all negatively charged phospholipids independent of headgroup structure. Mg²⁺, Ba²⁺, and Sr²⁺ were unable to stimulate phospholipid binding. Ca²⁺ acted cooperatively with a Hill coefficient of 3 and an apparent affinity in the low micromolar range. Further studies revealed that the C₂A-domain binds Ca²⁺ directly without phospholipids but with a lower apparent Ca²⁺ affinity (0.2 mM compared with 5 µM free Ca²⁺) (27). The C₂A-domain of synaptotagmin I also bound syntaxin 1 as a function of Ca²⁺, with a low apparent Ca²⁺ affinity resembling that of intrinsic Ca²⁺ binding (28). These data suggested that the functions of C₂-domains may be diverse and include Ca²⁺-dependent interactions with proteins in addition to phospholipids. Analysis of the C₂A-domains from other synaptotagmins revealed that those from synaptotagmins II, III, V, and VII bind phospholipids and syntaxin 1, but the C₂A-domains of synaptotagmins IV, VI, X, and XI do not (28). Although the C₂A-domains of synaptotagmins I, II, III, V, and VII bind to phospholipids with similar Ca²⁺ affinities, they exhibit distinct cation specificities (29). All of the C₂A-domains from synaptotagmins that bind to phospholipids also interact with syntaxin 1 as a function of Ca²⁺ but with different Ca²⁺ dependences: synaptotagmins I, II, and V require Ca²⁺ concentrations of >0.2 mM whereas synaptotagmins III and VII bind at <1 µM Ca²⁺ (28).

Sequence analyses of the C₂A- and C₂B-domain of synaptotagmin I revealed that they contain evolutionarily conserved differences. The C₂B-domain does not exhibit the same Ca²⁺-dependent phospholipid binding properties as the C₂A-domain. Nevertheless, most C₂B-domains probably bind Ca²⁺ because they contain the requisite Ca²⁺-binding sequences defined in C₂A-domains (see below), and because the C₂B-domains of synaptotagmins I and II mediate the Ca²⁺-dependent self-association of synaptotagmins (30). These results led to a model whereby the C₂A- and C₂B-domains of most synaptotagmins are Ca²⁺-binding domains that are specialized for different Ca²⁺-dependent activities.

Similar to the C₂A-domains of synaptotagmins, the C₂-domains from cPLA₂ (31, 32), PKC (27), and Nedd4 (18) bind phospholipids at micromolar Ca²⁺ concentrations. However, the C₂A-domains of synaptotagmins and the C₂-domain of PKC preferentially bind to negatively charged phospholipids whereas the C₂-domain from cPLA₂ interacts with neutral phospholipids (26, 33). Furthermore, although the C₂-domain from PKC is similar to the synaptotagmin C₂A-domains, it does not bind to syntaxin 1 as a function of Ca²⁺. Therefore, even among C₂-domains that share Ca²⁺-dependent phospholipid binding, there are functional distinctions that may be important for their biological roles.

To complicate matters, some C₂-domains that are Ca²⁺-regulated simultaneously bind other molecules in a Ca²⁺-independent manner. For example, the C₂B-domain of synaptotagmin I interacts with AP-2 (34), inositol polyphosphates (35), -SNAP (36), and Ca²⁺ channels (37). Finally, many C₂-domains may not bind Ca²⁺ at all. Several synaptotagmins appear to be unable to bind Ca²⁺, as may be the case with the C₂-domains of RIM. Interestingly, PKC isoforms that initially were not thought to have a C₂-domain and are Ca²⁺-independent (PKC, -, -, and -) actually contain a C₂-domain that is located at the N terminus and probably does not bind Ca²⁺ (2, 11, 38). Thus, as a group C₂-domains perform multiple biological functions.

	Three-dimensional Structures of C2-domains

X-ray diffraction analysis of the synaptotagmin I C₂A-domain yielded the first structure of a C₂-domain (39). The structure consists of a compact -sandwich composed of two four-stranded -sheets (Fig. 1A). Three loops at the top of the domain and four at the bottom connect the eight -strands. Ca²⁺ binding occurs exclusively at the top three loops (see below). NMR spectroscopy showed that the solution structure of the C₂A-domain is identical to the crystal structure (27).²

View larger version (107K): [in this window] [in a new window]   Fig. 1.   Ribbon diagrams of the structures of the C2A-domain of synaptotagmin I (A) and the C2-domain of PLC1 (B) and schematic drawing of their -strand topologies (C). In A and B, the locations of the N and C termini and of the Ca2+-binding loops are indicated. Each C2-domain is shown complexed to three Ca2+ ions (orange) (27, 40, 41, 44).2,3 The diagrams were prepared with the program MOLSCRIPT (51). In C, -strands in the C2-domains from synaptotagmin I and PKC (left) and from PLC1 and cPLA2 (right) are numbered in the order of the primary sequences. The three Ca2+-binding loops at the top of the C2-domains are indicated.

Determination of the structures of three other C₂-domains (from PKC, cPLA₂, and PLC1) revealed similar designs and interesting differences. PLC1 is a modular protein composed of PH-, EF-hand, C₂-, and catalytic domains. X-ray diffraction studies of crystals from PLC1 lacking the N-terminal PH domain provided the three-dimensional structure of a C₂-domain in the context of a nearly full-length protein (40, 41). The three-dimensional structure of the PLC1 C₂-domain (Fig. 1B) is very similar to that of the synaptotagmin I C₂A-domain, with a root mean square deviation of 1.4 Å for 109 equivalent -carbons. The topology of the -strands, however, is strikingly different (40). The arrangement of -strands in the PLC1 C₂-domain constitutes a circular permutation of the topology observed in the C₂A-domain of synaptotagmin I (Fig. 1C). As a result, strand 1 of the synaptotagmin I C₂A-domain occupies the same position as strand 8 of the PLC1 C₂-domain. The N and C termini are at the top of the C₂-domain in synaptotagmin I but at the bottom in PLC1 (Fig. 1). The two types of topology are referred to as topology I (synaptotagmin I C₂A-domain) or topology II (PLC1 C₂-domain). The crystal structures of the C₂-domains from PKC³ and cPLA₂ (42) are also similar to those of synaptotagmin I and PLC1 and exhibit topologies I and II, respectively. It is unclear why C₂-domains occur in two topologies. One reason may be that the topology influences the relative orientation of a C₂-domain with respect to its neighboring domains.

There is a high degree of structural homology between C₂-domains in the core -sandwich and less similarity in the top and bottom loops (Fig. 1). Accordingly, the C₂-domain sequences involved in the core -sandwich are highly conserved between C₂-domains whereas the sequences of the loops, particularly loop 1 and the three bottom loops, are not. The high degree of structural identity between the core -sandwiches of C₂-domains suggests that the -sandwich represents a scaffold. This scaffold allows the emergence of variable loops at the top and bottom of the domain. As discussed below, the loops are involved in Ca²⁺ binding and may determine the functional specificity of a C₂-domain. The C₂-domain structures provide a framework to interpret the properties of C₂-domains and at the same time allow us to predict the minimum sequences required for a complete, well folded -sandwich. Thus results from experiments performed with incomplete C₂-domain fragments or with mutants containing deletions in a -strand should be interpreted with caution since misfolding is likely.

	How Do C2-domains Bind Ca2+?

The Ca²⁺ binding modes of the C₂-domains from synaptotagmin I, PKC, PLC-1, and cPLA₂ were analyzed by x-ray diffraction and NMR spectroscopy (27, 39-44).^2,3 In all C₂-domains, multiple Ca²⁺ ions bind in a cluster exclusively at the top loops (Fig. 1). These loops are widely separated in the primary sequences (Fig. 2). The Ca²⁺-binding sites are formed primarily by aspartate side chains that serve as bidentate ligands for two or three Ca²⁺ ions.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Sequences of the C2-domains from synaptotagmin I (S), PLC1 (C), and cPLA2 (A). Identical residues are shown on a yellow background. Aspartate, asparagine, and serine residues that coordinate the Ca2+ ions in the different C2-domains are shown on a pink background. Residues whose backbone carbonyl groups coordinate Ca2+ ions are shown on a blue background. Sequences that are structurally almost identical in the three-dimensional structures are indicated by a green bar below the alignment, and locations of -strands by an orange bar. The positions of the three loops involved in Ca2+ binding are shown above the alignment.

In the C₂A-domain of synaptotagmin I, loops 1 and 3 contain three Ca²⁺-binding sites (Ca1, Ca2, and Ca3 in Fig. 3). The Ca²⁺-binding sites are formed by five aspartate side chains, one serine side chain, and three carbonyl groups (27, 39, 44)² (Figs. 2 and 3). The presence of three Ca²⁺-binding sites in the C₂A-domain correlates well with the Hill coefficient of 3 observed in Ca²⁺-dependent phospholipid binding experiments (26). Ca²⁺ binding to all three sites is necessary for syntaxin 1 and phospholipid binding (44).⁴ The coordination spheres of the bound Ca²⁺ ions in the C₂A-domain are incomplete, especially for Ca3. This results in the low apparent intrinsic Ca²⁺ affinity of this site (>1.0 mM). When phospholipids bind, they probably fill unsatisfied coordination sites on the bound Ca²⁺ ions, resulting in a 1000-fold increase in the apparent affinity of the C₂A-domain for Ca²⁺. The Ca²⁺ binding mode of the C₂-domain of PKC, as determined by x-ray crystallography, is very similar to that of the C₂A-domain.³

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Summary of the Ca2+-binding sites in the C2-domains of synaptotagmin I (A) and of PLC1 (B). The side chains and carbonyl groups involved in binding are indicated. Sites actually observed in the structures are shown by solid circles, and potential additional sites in each C2-domain by open circles. For the PLC1 C2-domain, site Ca2 was only occupied in the La3+ complex, and the serine 650 side chain (S650) was only involved in metal ion coordination in this complex. Although not observed experimentally, a site could exist in the synaptotagmin I C2A-domain that is analogous to site Ca4 in PLC1, and a site analogous to site Ca3 could be formed by the PLC1 C2-domain.

The C₂-domain of PLC1 shares two of the Ca²⁺-binding sites of the synaptotagmin I C₂A-domain (Ca1 and Ca2) but contains a distinct third Ca²⁺-binding site (Ca4) (41, 43) (Fig. 3). Site Ca4 involves one aspartate, one asparagine, and one serine side chain in addition to one backbone carbonyl group. Sites Ca1 and Ca4 were occupied in all complexes of PLC1 with Ca²⁺ and Ca²⁺ analogs (La³⁺, Sm³⁺, and Ba²⁺). Site Ca2 was only filled in the La³⁺ complex, but it seems likely that Ca²⁺ also binds to this site at Ca²⁺ concentrations above 1 mM or at lower Ca²⁺ concentrations in the presence of phospholipids (43). In addition, all side chains from site Ca3 in the synaptotagmin I C₂A-domain are conserved in the PLC1 C₂-domain and have similar orientations in the structures of both C₂-domains (44).² This strongly suggests that Ca²⁺ may also occupy this site and that the PLC1 C₂-domain may contain a total of four Ca²⁺-binding sites. Bound Ca²⁺ ions in the C₂-domain of PLC1 have unsatisfied coordination sites, suggesting that in the absence of phospholipids they may also exhibit low apparent affinities. The C₂-domain of cPLA₂ has a Ca²⁺ binding mode similar to that of the PLC1 C₂-domain but apparently with only sites Ca1 and Ca4 occupied (42).

The Ca²⁺ binding modes summarized above can be used to anticipate the Ca²⁺ binding properties of other C₂-domains. The aspartate residues involved in Ca²⁺ binding in the synaptotagmin I C₂A-domain are conserved in many C₂-domains. Based on this observation, we proposed that the motif formed by these aspartate residues is widespread and named it the C₂-motif (27). Sites Ca1 and Ca2 are probably the most common Ca²⁺-binding sites in C₂-domains, and additional Ca²⁺-binding sites are likely to exist in many C₂-domains depending on the side chains present in loops 1-3. Ca²⁺-dependent C₂-domains thus appear to have been designed to concentrate multiple Ca²⁺ ions in a small region. The Ca²⁺ ions contain unsatisfied coordination sites that remain available for interaction with target molecules.

	Mechanisms of C2-domain Function

The three-dimensional structures of C₂-domains determined so far show no evidence that Ca²⁺ induces a substantial change from one well defined conformation to another well defined conformation. Comparison of the NMR solution structure of the Ca²⁺-bound form of the synaptotagmin I C₂A-domain with the crystal structure of the Ca²⁺-free form demonstrated that Ca²⁺ binding involves rotations of some side chains but causes no substantial backbone rearrangements (27).² The NMR data indicate that the Ca²⁺-binding region is flexible in the absence of Ca²⁺ and is stabilized after Ca²⁺ binding. Structural stabilization by Ca²⁺ binding is consistent with decreased B-factors in the crystal structure of the C₂A-domain after partial Ca²⁺ saturation (39) and with the observations that Ca²⁺ causes a large change in denaturation temperature (27) and increases the resistance of the synaptotagmin I C₂A-domain against proteolysis (45). With regard to the PLC1 C₂-domain, 10 of 12 x-ray structures obtained in the presence or absence of Ca²⁺ or Ca²⁺ analogs are very similar, suggesting that Ca²⁺ binding does not cause conformational changes (40, 41, 43).

If Ca²⁺ does not induce a major conformational change in C₂-domains, how does Ca²⁺ regulate their function? The structural stabilization induced by Ca²⁺ probably does not account for Ca²⁺ regulation because the conformations compatible with binding to target molecules are also available in the absence of Ca²⁺. However, Ca²⁺ binding causes a major change in the electrostatic potential of the synaptotagmin I C₂A-domain that may be important for regulating interactions. Analysis by NMR spectroscopy showed that the region around the Ca²⁺-binding sites of the C₂A-domain is responsible for Ca²⁺-dependent binding to syntaxin (46). This region contains the cluster of aspartate residues that coordinate Ca²⁺ and a ring of basic amino acids surrounding it. Binding to syntaxin, a negatively charged protein, could therefore be driven by the change in electrostatic potential caused by Ca²⁺ binding and could be mediated by the basic side chains surrounding the Ca²⁺-binding site. Two exposed hydrophobic side chains in the region and coordination of the unsatisfied Ca²⁺ valences by acidic residues of syntaxin may contribute to the interaction (46).

The mode of interaction between synaptotagmin I and syntaxin 1 suggested that synaptotagmin I acts as an electrostatic switch in neurotransmitter release. The binding of phospholipids by the C₂A-domain is also best explained by this model. Mutations in basic and acidic amino acids that disrupt syntaxin binding (44, 46) also inhibit phospholipid binding.⁴ Phospholipid binding correlates with the density of negative charges on the surface of the phospholipid bilayer rather than with a specific chemical structure. Furthermore, binding is inhibited by high salt. These results support the importance of electrostatic interactions for the Ca²⁺-dependent binding of the synaptotagmin I C₂A-domain to phospholipids (26). Other C₂-domains that bind to negatively charged phospholipids in a Ca²⁺-dependent manner such as those of classical PKCs may share this mechanism of binding. Substitutions in two of the aspartate residues that bind Ca²⁺ in PKC have shown, however, that lipid binding is probably not purely electrostatic (47). It is likely that coordination of the Ca²⁺ ions bound to C₂-domains by the phosphate groups of the lipids may provide a major contribution to the binding energy, which is supported by the observation that the apparent Ca²⁺ affinities are much higher in the presence of phospholipids than in their absence. Insertion of highly exposed hydrophobic side chains into the lipid bilayer may contribute to binding as proposed for the Ca²⁺-dependent binding of phosphatidylcholine to the C₂-domain of cPLA₂ (33). The side chains in the Ca²⁺-binding loops are likely to influence the preference for types of lipids. Thus, the preference of cPLA₂ for neutral rather than negatively charged phospholipids may be because of the presence of two acidic residues in loops 1 and 3, in addition to the Ca²⁺-binding residues, and to the absence of basic residues.

	Evolution of C2-domains: Example of Synaptotagmins

The differences between C₂-domains in synaptotagmins may give clues about how this domain adapted to diverse functions. In evolution, the C₂-domains of the more than 12 different synaptotagmins developed distinct Ca²⁺ affinities, or in some cases, Ca²⁺ independence (28, 29). Interestingly synaptotagmins IV and XI have a single, evolutionarily conserved amino acid change in the Ca²⁺-binding residues of the C₂A-motif. These C₂A-domains are unable to bind phospholipids as a function of Ca²⁺ (48). Reversal of this amino acid change restored the ability of synaptotagmins IV and XI to bind phospholipids as a function of Ca²⁺. Thus all other structural requirements for Ca²⁺-dependent phospholipid binding were evolutionarily retained in these synaptotagmins, and a single amino acid substitution was selected to abolish Ca²⁺-dependent phospholipid binding. This finding supports the notion that at least the C₂A-domain in synaptotagmins performs other functions in addition to Ca²⁺-dependent phospholipid binding.

	Conclusion

C₂-domains are remarkable modules present in a wide variety of proteins that can participate in different types of interactions. Two widespread Ca²⁺-binding motifs defined by structural characteristics are known: EF-hands as the most widely distributed motifs with little structural autonomy; and C₂-domains representing autonomous modules present in probably more than 100 proteins. Although more EF-hands than C₂-domains have been described, the growing number of C₂-domains in the data banks suggests that C₂-domains are universal Ca²⁺-binding domains. The Ca²⁺-binding sites formed by C₂-motifs and EF-hands have different architectures and function by distinct mechanisms. The EF-hand is formed by a contiguous helix-turn-helix sequence that binds a single Ca²⁺ ion and usually is a substructure in an -helical protein domain (49, 50). Multiple EF-hands may be present in a protein. Ca²⁺ binding to EF-hands in contiguous domains often occurs in a concerted manner, causing conformational changes that expose hydrophobic surfaces. In contrast, C₂-domains are autonomously folding modules with a stable -sheet scaffold. Multiple Ca²⁺ ions bind in a cluster at the tip of the domain in a region formed by loops that are distant in the sequence. The Ca²⁺ binding properties of C₂-domains confer onto them the ability to act as electrostatic switches without requiring large conformational changes. The Ca²⁺ binding mode of C₂-domains may be particularly useful for fast Ca²⁺-triggered reactions, such as neurotransmitter release. We expect that the number of C₂-domains and the variety of their interactions will continue to grow, with new developments and surprises for years to come.

	ACKNOWLEDGEMENTS

We would like to thank Drs. Bryan Sutton, Steven Sprang, Joseph L. Goldstein, and Michael S. Brown for valuable discussions and communication of data before publication.

	FOOTNOTES

^* This minireview will be reprinted in the 1997 Minireview Compendium, which will be available in December, 1997. Original research from the authors' laboratories was supported by National Institutes of Health Grants NS33731 and NS37200, the Welch Foundation, and the HFSP.

^§ To whom correspondence may be addressed. Tel.: 214-648-9026; Fax: 214-648-8673; E-mail: jose{at}arnie.swmed.edu.

To whom correspondence may be addressed. Tel. 214-648-5022; Fax: 214-648-6426; E-mail: TSudho{at}MEDNET.swmed.edu.

¹ The abbreviations used are: cPLA₂, cytoplasmic phospholipase A₂; C₂A- and C₂B-domains, first and second C₂-domains of synaptotagmins; PKC, protein kinase C; PLC, phosphoinositide-specific phospholipase C.

² X. Shao, I. Fernandez, T. C. Südhof, and J. Rizo, submitted for publication.

³ B. Sutton and S. Sprang, submitted for publication.

⁴ X. Zhang, J. Rizo, and T. C. Südhof, submitted for publication.

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