Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C2A domain in asynchronous neurotransmitter release

Abstract

Synaptotagmins (Syts) act as Ca²⁺ sensors in neurotransmitter release by virtue of Ca²⁺-binding to their two C₂ domains, but their mechanisms of action remain unclear. Puzzlingly, Ca²⁺-binding to the C₂B domain appears to dominate Syt1 function in synchronous release, whereas Ca²⁺-binding to the C₂A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C₂A domain and C₂AB region, and analyses of intrinsic Ca²⁺-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca²⁺ saturating conditions, we show that the Syt7 C₂A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of l-α-phosphatidylinositol 4,5-diphosphate (PIP₂). For Syt1, the two Ca²⁺-saturated C₂ domains have similar affinities for membranes lacking PIP₂, but the C₂B domain dominates binding to PIP₂-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C₂A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C₂ domains in neurotransmitter release.

Keywords: X-ray crystallography; membrane binding; neurotransmitter release; synaptotagmin-1; synaptotagmin-7.

Fig. 1.

Crystal structure of the Ca²⁺-bound Syt7 C₂A domain. (A) Ribbon diagram of the crystal structure of the Ca²⁺-bound Syt7 C₂A domain. The three bound Ca²⁺ ions are shown as yellow spheres. The two loops involved in Ca²⁺ binding are indicated (loop 1 and loop 3), and the N and C termini are labeled N and C, respectively. (B) Superposition of the crystal structure of the Ca²⁺-bound Syt7 C₂A domain (cyan) with the solution structure of Ca²⁺-free Syt7 C₂A domain (red) (PDB ID code 2D8K). (C) Superposition of the crystal structure of the Ca²⁺-bound Syt7 C₂A domain (cyan) with the solution structure of Ca²⁺-bound Syt1 C₂A domain (orange) (PDB ID code 1BYN). (D) Diagram illustrating the Ca²⁺-binding sites of the Syt7 C₂A domain. The three bound Ca²⁺ ions are labeled Ca1, Ca2, and Ca3. The Ca²⁺ ligands are shown as stick models and labeled. (E) Diagram of the Ca²⁺-binding sites of Syt7 C₂A domain summarizing all of the residues in loops 1 and 3 (black letters). The residues that are different in the Syt1 C₂A domain are indicated in parenthesis in red letters.

Fig. 2.

Crystal structure of the Ca²⁺-bound Syt7 C₂AB fragment. (A) Ribbon diagram of the crystal structure of the Ca²⁺-bound Syt7 C₂AB fragment, with the C₂A domain in cyan and the C₂B domain in blue. The bound Ca²⁺ ions are shown as yellow spheres. The loops involved in Ca²⁺ binding are indicated (loop 1 and loop 3), and the N and C termini of both domains are labeled N and C, respectively. The sequence linking the two C₂ domains, for which there is insufficient electronic density to build the structure, is illustrated by a dotted line. (B) Superposition of the crystal structure of the Ca²⁺-bound Syt7 C₂AB (cyan and blue) with the crystal structures of Ca²⁺-free Syt1 C₂AB (wheat; PDB ID code 2R83), Syt1 C₂AB bound to the SNARE complex (red; PDB ID code 5CCG) and Ca²⁺-free Syt3 C₂AB (green; PDB ID code 1DQV). Only the C₂A domain of each structure was used for the superposition to illustrate the diversity of relative orientations between the two C₂ domains in these structures. The position of all of the C₂A domains is indicated by C₂A and those of the different C₂B domains are also labeled; Syt1 (SC) C₂B domain refers to Syt1 C₂AB bound to the SNARE complex.

Fig. S1.

There are no stable interactions between the C₂A and C₂B domains of Syt7 in solution. Superpositions of ¹H-¹⁵N HSQC spectra of the Syt7 C₂AB fragment (black contours), the Syt7 C₂A domain (green contours) or C₂B domain (red contours) in the presence of 1 mM EDTA (A) or 2 mM Ca²⁺ (B).

Fig. 3.

ITC analysis of intrinsic Ca²⁺ binding to the Syt7 C₂ domains. Illustrative ITC data obtained by titration of Ca²⁺ versus the isolated Syt7 C₂A (A) or C₂B (B) domain. The curves represent the fits of the data to a three- (A) or four- (B) sequential-binding-site model.

Fig. 4.

The Syt7 C₂A domain dominates binding of Syt7 to membranes and clusters liposomes. (A) Titrations of 20 nM Syt7 C₂A domain, C₂B domain and C₂A*B fragment (the * in C₂A*B denotes that the fluorescent probe is attached to C260 of the C₂A domain) with liposomes lacking PIP₂ in the presence of 1 mM Ca²⁺. Binding was monitored from the FRET developed between a donor BODIPY-FL probe attached to the proteins and a rhodamine acceptor present in the liposomes. Each point represents the average of at least three measurements performed with different liposome preparations. (B) The same titrations shown in A but changing the x axis to better show the points of the titrations obtained at low liposome concentrations. (C) Liposome titrations of 20 nM WT Syt7 C₂AB fragment and mutant fragments where the Ca²⁺-binding sites of the C₂A domain (C₂aB*) or C₂B domain (C₂A*b) were disrupted with D225A,D227A,D233A or D357A,D359A mutations. All of the data in A–C were fit to a Hill function. (D–F) Distribution of particle size measured by DLS on samples containing liposomes alone (D) or liposomes in the presence of the Syt7 C₂A domain (E) or Syt7 C₂B domain (F). In E and F, the diagrams show superpositions of data obtained in the absence (green bars) or presence (red bars) of Ca²⁺.

Fig. 5.

The Syt1 C₂ domains have similar membrane affinities and cooperate in membrane binding. (A) Titrations of 100 nM Syt1 C₂A domain, C₂B domain and C₂AB* fragment (the * in C₂AB* denotes that the fluorescent probe is attached to the C₂B domain) with liposomes lacking PIP₂ in the presence of 1 mM Ca²⁺. Binding was monitored from the FRET developed between a donor BODIPY-FL probe attached to the proteins and a rhodamine acceptor present in the liposomes. Each point represents the average of at least three measurements performed with different liposome preparations. (B) The same titrations shown in A but changing the x axis to better show the points of the titrations obtained at low liposome concentrations. (C) Liposome titrations of 100 nM WT Syt1 C₂AB* fragment and mutant fragments where the Ca²⁺-binding sites of the C₂A domain (C₂aB*) or C₂B domain (C₂A*b) were disrupted with D178A,D230A,D232A or D309A,D363A,D365A mutations. All of the data were fit to a Hill function.

Fig. 6.

PIP₂ enhances the membrane affinity of the Syt1 and Syt7 C₂B domains. Titrations of 100 nM Syt1 C₂A domain (A) or C₂B domain (B), or 20 nM Syt7 C₂A domain (C) and C₂B domain (D) with liposomes lacking (black circles) or containing (red circles) PIP₂ in the presence of 1 mM Ca²⁺. Binding was monitored from the FRET developed between a donor BODIPY-FL probe attached to the proteins and a rhodamine acceptor present in the liposomes. Each point represents the average of at least three measurements performed with different liposome preparations. All of the data were fit to a Hill function.

Fig. 7.

Subtle residue substitutions underlie in part the stronger membrane binding affinity of the Syt7 C₂A domain compared with the Syt1 C₂A domain. (A) Ribbon diagrams of the Syt7 C₂A domain (cyan) and Syt1 C₂A domain (orange) showing the Cα and side chain atoms of residues that are different in their Ca²⁺-binding regions and were mutated to analyze the basis for their different membrane affinities (oxygens are in red, nitrogens in blue, sulfur atoms in yellow, and carbon atoms in cyan for Syt7 and in orange for Syt1). (B and C) Titrations of 20 nM WT and mutant Syt7 C₂A domains with liposomes in the presence of 1 mM Ca²⁺. Binding was monitored from the FRET developed between a donor BODIPY-FL probe attached to the proteins and a rhodamine acceptor present in the liposomes. Each point represents the average of at least three measurements performed with different liposome preparations. (D) Analogous liposome titrations of 100 nM WT and mutant Syt1 C₂A domains. (E) Analogous liposome titrations of 100 nM WT and mutant Syt1 and Syt7 C₂A domains. Note that the data obtained for WT and M173F,K236R Syt1 C₂A domains (orange and gray circles, respectively) are the same as those shown in D, but the scale of the x axis is different to allow comparison with the data obtained for the WT and mutant Syt7 C₂A domains. All of the data were fit to a Hill function.

Fig. S2.

Placing the fluorescent probe on the C₂A or C₂B domain does not alter phospholipid binding to the Syt1 or Syt7 C₂AB fragment. (A) Ribbon diagrams of the structures of the Syt7 and Syt1 C₂A or C₂B domains, as indicated. The structure of the Syt7 C₂A domain is described here. Those of the Syt7 C₂B domain, the Syt1 C₂A domain, and the Syt1 C₂B domain correspond to PDB ID codes 3N5A, 1BYN, and 1K5W, respectively. The bound Ca²⁺ ions are shown as yellow spheres. The two loops involved in Ca²⁺ binding are indicated (loop 1 and loop 3), and the N and C termini are labeled N and C, respectively. The native cysteine side chains of the Syt7 C₂A and C₂B domains, as well as the side chains that were mutated to cysteine in the Syt1 C₂A and C₂B domains to attach a fluorescent probe, are shown as stick models. (B) Titrations of 20 nM Syt7 C₂A*B and C₂AB* fragments (where the * indicates the domain where the fluorescent probe was attached) with liposomes in the presence of 1 mM Ca²⁺. Binding was monitored from the FRET developed between a donor BODIPY-FL probe attached to the proteins and a rhodamine acceptor present in the liposomes. Each point represents the average of at least three measurements performed with different liposome preparations. (C) Analogous liposome titrations of 100 nM Syt1 C₂A*B and C₂AB* fragments. All of the data were fit to a Hill function. (D and E) Superpositions of the data obtained in analogous titrations of 20 nM Syt7 C₂A domain (D) or Syt7 C₂B domain (E), illustrating the reproducibility of the results. Each symbol represents one dataset obtained with a different liposome preparation. The apparent K_Ds derived for the six datasets shown in D are 1.49, 2.35, 2.62, 2.42, 2.37, and 3.17 μM, and those derived for the four datasets shown in E are 34.1, 31.4, 38.1, and 36.4 μM. The Syt7 C₂B domain data provide an example with small variability among different titrations while the Syt7 C₂A domain data illustrate an example with the largest variability, which is reflected in a larger SD in relative terms (Table 1). Even in this case, the data exhibit excellent consistency that allows accurate comparison with the binding curves obtained for other proteins.

Fig. S3.

The Syt7 C₂A domain binds to PIP₂-containing liposomes with higher affinity than the Syt7 C₂B domain. Titrations of 20 nM Syt7 C₂A domain (red circles) or C₂B domain (blue circles) with liposomes containing PIP₂ in the presence of 1 mM Ca²⁺. Binding was monitored from the FRET developed between a donor BODIPY-FL probe attached to the proteins and a rhodamine acceptor present in the liposomes. Each point represents the average of at least three measurements. All of the data were fit to a Hill function. The data are the same shown in Fig. 6 C and D with liposomes containing PIP₂.