C2A activates a cryptic Ca²⁺-triggered membrane penetration activity within the C2B domain of synaptotagmin I

Materials and Methods

Results

A lone Trp residue, placed at the distal tip of Ca²⁺-binding loop 3 of C2A, shows membrane penetration of the loop as an increase in intensity and a blue shift in the emission spectrum (9–11). To explore the possibility that C2B, in the context of C2A-C2B, also penetrates into membranes, we placed a fluorescent reporter at the analogous position in its Ca²⁺-binding loop 3 (position Ile-367 as determined using the crystal structures of syt I and III as templates) (20, 21). Because C2A-C2B contains a number of Trp residues that are critical for its function (data not shown), we substituted Ile-367 with a cysteine (Cys) residue and labeled it with an AEDANS. As a positive control, the equivalent position in C2A (Phe-234 in loop 3) was also changed to a Cys and labeled with AEDANS (Fig. 1A).

Consistent with previous studies using Trp probes, the label on the C2A domain (designated C2A*-C2B) exhibited a Ca²⁺-triggered increase in the fluorescence intensity and blue shift in its emission spectrum when mixed with liposomes containing acidic phospholipids (25% PS/75% PC unless otherwise indicated). These changes depended on addition of both liposomes and Ca²⁺ (Fig. 1B Left) (9–11) and result from the Ca²⁺-driven penetration of the Ca²⁺-bound loop, harboring the fluorescent label, into the hydrophobic phase of the lipid bilayer (refs. 9 and 11; also described in detail below). Membrane penetration requires the presence of acidic phospholipids (25% PS); the fluorescent reporters did not penetrate liposomes composed of only PC (refs. 6, 7, and 11 and data not shown). This is likely because of the need for acidic phospholipid head groups to complete the Ca²⁺ coordination sites; anionic lipids probably interact with basic residues in the Ca²⁺-binding loops (9, 21, 25, 26).

We then used this approach to address the possibility that C2B might also penetrate bilayers. As shown in Fig. 1B (Right), the fluorescence of an AEDANS label on Ca²⁺-binding loop 3 of C2B (designated C2A-C2B*) exhibited strikingly similar fluorescence changes upon addition of Ca²⁺ and liposomes; i.e., an increase in fluorescence intensity and a blue shift in the emission spectra [in the absence of liposomes, Ca²⁺ causes a slight (<1%) decrease in fluorescence]. These data support a model in which C2A activates a cryptic membrane penetration activity within C2B.

We next determined whether the interactions of C2A*-C2B and C2A-C2B* with membranes are rapid enough to couple Ca²⁺ influx to fusion using a stopped-flow rapid mixing approach. Rapid mixing of liposomes with C2A*-C2B or C2A-C2B* in EGTA resulted in an unchanging baseline fluorescence signal (Fig. 1C). Rapid mixing of liposomes with C2A*-C2B or C2A-C2B* in Ca²⁺ resulted in a rapid and marked increase in the fluorescence signal (Fig. 1C). These increases were well fitted with single exponential functions to determine the observed rate, k_obs. We then measured k_obs as a function of the [liposome]; these data are plotted in Fig. 1D; the Y-intercept yields k_off and the slope yields k_on. These values were 146.6 ± 6.9 s⁻¹/1.06 ± 0.09 × 10¹⁰ M⁻¹⋅s⁻¹ for C2A*-C2B and 134.6 ± 9.3 s⁻¹/1.07 ± 0.12 × 10¹⁰ M⁻¹⋅s⁻¹ for C2A-C2B*. If C2A and C2B function as independent domains, these studies indicate that they exhibit similar affinities for membranes; the calculated dissociation constants from these kinetics data are 13–14 nM for each of the reporter constructs. As we reported for the isolated C2A domain, the on-rates for C2A*-C2B or C2A-C2B* membrane interactions approach the collisional limit and more than satisfy the kinetic constraints of exocytosis (27, 28).

The optical reporters were also used to measure steady-state Ca²⁺ dependencies for the penetration of C2A and C2B into membranes, again in the context of C2A-C2B. When a reporter in C2A is monitored, the [Ca²⁺]_1/2 for binding was 69 ± 1.4 μM (Hill coefficient = 2.3; Fig. 1E). Similar results were obtained when the reporter in C2B was monitored ([Ca²⁺]_1/2 = 53 ± 1.3 μM; Hill coefficient = 3.2; Fig. 1E). These values are in the range of the Ca²⁺ requirements for secretion measured in different cell types (from ≈10 to 200 μM Ca²⁺; refs. 28–30).

To confirm that the increase in fluorescence of C2A-C2B* results from penetration of the reporter into bilayers, we carried out quenching experiments using the membrane-embedded fluorescence quencher, doxyl-PC. Doxyl groups are efficient quenchers of AEDANS fluorescence, but because the quencher is located on the acyl chain of the lipid (Fig. 2B Right), quenching can occur only if the fluorophore penetrates into the bilayer. As shown in Fig. 2A, the reporters in C2A*-C2B and C2A-C2B* were efficiently quenched. The degree of quenching by labels at the 5, 7, and 12 positions of the acyl chain indicated that the reporters penetrated ≈1/6th into the hydrophobic core of the bilayer. These results are shown schematically in Fig. 2B, where both C2A and C2B simultaneously penetrate into the hydrophobic core of lipid bilayers, “pinning” the protein to the bilayer surface.

To rule out the possibility that the lipid binding activity of C2A “drags” C2B into membranes, we generated a construct harboring mutations that abolish C2A–membrane interactions (C2A_M-C2B). We then placed fluorophores in loop 3 of either C2 domain (designated C2A*_M-C2B and C2A_M-C2B*). The mutations in C2A strongly inhibited the increase in fluorescence of the C2A probe that results from insertion into bilayers (Fig. 3A Upper), excluding the model in which C2B “repairs” mutations within C2A. In contrast, these mutations had only slight effects on the increase in fluorescence of the fluorescent reporter in C2B (Fig. 3A Middle and Lower). To confirm that mutations in C2A affect the penetration of C2A, but have little effect on the penetration of C2B, we used membrane-embedded quenchers as described above. The relative degree of quenching of reporters placed in the C2A domain was diminished by Ca²⁺-ligand mutations in C2A (Fig. 3B Upper). In contrast, the Ca²⁺ ligand mutations in C2A had little effect on the strong quenching of the fluorophore on C2B (Fig. 3B Middle and Lower). These experiments clearly demonstrate that the ability of C2B to penetrate lipid bilayers is not by means of a passive process in which C2A “drags” C2B. Rather, when C2A is adjacent to C2B, a cryptic lipid penetration activity within C2B is activated. It is interesting that this activation occurs even by using a C2A domain, which itself is not active (discussed further below).

In light of these findings, we sought to better understand the mechanism by which C2A “activates” a cryptic Ca²⁺-triggered lipid penetration activity within C2B. When isolated C2B is expressed and purified, it fails to penetrate lipid bilayers in the AEDANS fluorescence assay (Fig. 4A). We considered two possibilities for this lack of activity: that C2A is needed for correct folding of an active C2B domain or that C2A interacts with C2B to activate it after the protein is synthesized. We addressed the question by engineering a thrombin cleavage site between C2A and C2B (Fig. 4B; designated C2A-TC-C2B). We expressed and purified a His-6-tagged version of C2A-TC-C2B and showed that the C2B domain can penetrate into lipid bilayers using an AEDANS reporter placed in loop 3 (Fig. 4C Top). We then cleaved C2B from C2A by using thrombin. Because this reaction was highly inefficient (≈3% of the protein was cleaved), we assayed whether the conditions used to carry out cleavage affected the ability of C2A-TC-C2B* to penetrate membranes and found that this activity was not diminished (Fig. 4C Middle). We purified cleaved C2B from the fusion protein and assayed its ability to interact with membranes. Analogous to C2B purified as an isolated domain (Fig. 4A); C2B* liberated from C2A also failed to penetrate membranes (Fig. 4C Bottom). These data indicate that the role of C2A is not to direct folding of C2B, but rather that C2A interacts with C2B by means of a novel mechanism that activates cryptic Ca²⁺-triggered membrane penetration properties within C2B, potentially by means of physical contact between the adjacent C2 domains. A recent fluorescence resonance energy transfer study indicates that C2A and C2B can come into closer proximity, albeit at millimolar divalent metal concentrations (31).

Syt IV does not bind liposomes in response to Ca²⁺ (32). This isoform harbors a naturally occurring serine at position 244, which corresponds to a glutamate residue at position 230 in the C2A domain of syt I. In syt I, this glutamate is a critical Ca²⁺ ligand; neutralization by replacement with a serine abolishes Ca²⁺-triggered lipid binding activity of the isolated C2A (22). “Reversal” of this mutation in the C2A domain of syt IV, by means of an S244D substitution, results in robust Ca²⁺-triggered lipid binding activity (33). However, all of the conserved Ca²⁺ ligands are present in the C2B domain of syt IV. Thus, it is surprising that the C2A domain of syt IV does not activate its C2B domain (Fig. 5). These data indicate that either C2A of IV lacks the “activation” activity for C2B or C2B from IV lacks the ability to be activated by C2A. We addressed this issue by generating chimeras between syts I and IV; as controls, syt I/III chimeras were analyzed in parallel. We first observed that the C2A domain of syt IV failed to activate the lipid binding activity of the C2B domain of syt I. In contrast, the C2A_M domain of syt III (D385,387N) was able to activate C2B from isoform I. We next observed that the D230,232N mutant form of the C2A domain of syt I (C2A_MI), which can activate the C2B domain of syts I and III, fails to activate the C2B domain of syt IV. These data indicate that syt IV harbors more than one loss of function; the C2A domain not only fails to penetrate membranes, it also fails to activate the C2B domain of the protein. Furthermore, the C2B of syt IV lacks the ability to be activated by a C2A domain capable of activating other C2B domains. According to these experiments, syt IV harbors additional differences in both C2 domains that disrupt its ability to interact with membranes, consistent with its putative role as an inhibitory syt (34, 35).

Discussion

C2 domains are widespread conserved motifs; to date, 123 human genes encoding proteins that contain C2 domains have been identified (36). These domains are ≈140 residues in length and fold into compact eight-stranded β-sandwiches with flexible loops that protrude from one end (14, 21). In many, but not all, cases, these loops (1 and 3) mediate the binding of divalent metals, which often seems to regulate the binding of the C2 domain to other molecules including lipids and proteins (reviewed in ref. 14). In some proteins, the function of C2 domains seems to be straightforward. For example, Ca²⁺ triggers the translocation of C2 domain harboring phospholipases to membranes where their catalytic domains can efficiently turn-over substrate. In other cases, e.g., SYT, potential functions for C2 domains are less clear. syt was the first membrane protein identified that harbored C2 domains. Biochemical studies demonstrated that the C2A domain of syt I is an autonomously folding Ca²⁺ and lipid-binding module (6, 7). In the Ca²⁺-bound state, the Ca²⁺-binding loops of C2A rapidly penetrate into lipid bilayers at Ca²⁺ concentrations that trigger exocytosis (9–11). Positively charged amino acids within the loops interact with acidic phospholipid head groups. Neutralization of one of the positively charged residues in Ca²⁺-binding loop 3 has been reported to increase the Ca²⁺ requirements for lipid binding and to reduce evoked secretion at hippocampal synapses (4). Together, these data are consistent with a model in which Ca²⁺-triggered syt–membrane interactions serve as a coupling step in exocytosis. The isolated C2B domain of syt I also “senses” Ca²⁺ (8), but in contrast to C2A, Ca²⁺ does not drive the penetration of isolated C2B into membranes (12, 13).

More recently, it was discovered that the initial syt clone harbored a mutation that disrupts activities within the C2B domain (8), prompting a reevaluation of the role of C2A and C2B in mediating Ca²⁺-triggered interactions with membranes. New data demonstrated that mutations that abolished the Ca²⁺-triggered lipid binding activity of C2A did not abolish the overall Ca²⁺-triggered lipid binding activity of C2A-C2B; yet, isolated C2B fails to efficiently bind liposomes in response to Ca²⁺ (13). Here, we report that C2B penetrates into membranes in response to Ca²⁺, but only when tethered to an adjacent C2A domain. Interestingly, the penetration of C2B does not depend on the lipid-binding activity of C2A. However, severing C2A from C2B, after protein synthesis, disrupted the ability of the Ca²⁺-binding loop of C2B to insert into membranes. These data demonstrate that C2A does not activate C2B by directing folding during protein synthesis. This finding is not surprising because isolated C2B still senses Ca²⁺ and does not seem to be misfolded (8). Thus, C2A actively influences the properties of C2B.

This is a striking example of the evolution of multidomain proteins in which activities/properties arise or are lost by means of the use of domain repeats. It is possible that the C2B domain of syt I lost autonomous activity during evolution, giving rise to this form of cooperativity. This cooperativity may underlie the regulation of both C2 domains by molecules that interact with either C2A or C2B. For Ca²⁺-triggered lipid-binding activity, this is true of syts I and III (13) but not syt IV. We found that the C2 domains of syt IV exhibit a number of differences in comparison to syt I: not only is its C2A domain incapable of binding lipids in response to Ca²⁺, it is incapable of activating the lipid-binding activity within the C2B domain of syt I. Furthermore, the C2B domain of syt IV cannot be activated by the C2A domain of syt I (Fig. 5). These properties of syt IV are consistent with a proposed role as a seizure-induced (17) inhibitory isoform of the protein (34, 35).

A model for the structure of the syt–membrane complex is shown in Fig. 2B. In the absence of Ca²⁺, syt forms a weak precomplex with membranes (data not shown). In this view, Ca²⁺ influx would drive penetration of C2A and C2B into the vesicle or plasma membrane (11) with very rapid kinetics (Fig. 1C). If Ca²⁺-triggered syt–membrane interactions are crucial for exocytosis, neutralization of Ca²⁺ ligands within C2A may result in a mild phenotype because the mutant protein would retain the ability to penetrate bilayers in response to Ca²⁺. Penetration into the plasma membrane could help pull the bilayers together to facilitate soluble N-ethylmaleimide-sensitive fusion protein attachment receptor catalyzed fusion. For example, in at least some fusion events, a dimple in the plasma membrane forms as the target membrane is pulled toward the vesicle membrane (reviewed in ref. 37).