An unusual C(2)-domain in the active-zone protein piccolo: implications for Ca(2+) regulation of neurotransmitter release - PubMed (original) (raw)
An unusual C(2)-domain in the active-zone protein piccolo: implications for Ca(2+) regulation of neurotransmitter release
S H Gerber et al. EMBO J. 2001.
Abstract
Ca(2+) regulation of neurotransmitter release is thought to require multiple Ca(2+) sensors with distinct affinities. However, no low-affinity Ca(2+) sensor has been identified at the synapse. We now show that piccolo/aczonin, a recently described active-zone protein with C-terminal C(2)A- and C(2)B-domains, constitutes a presynaptic low-affinity Ca(2+) sensor. Ca(2+) binds to piccolo by virtue of its C(2)A-domain via an unusual mechanism that involves a large conformational change. The distinct Ca(2+)-binding properties of the piccolo C(2)A- domain are mediated by an evolutionarily conserved sequence at the bottom of the C(2)A-domain, which may fold back towards the Ca(2+)-binding sites on the top. Point mutations in this bottom sequence inactivate it, transforming low-affinity Ca(2+) binding (100-200 microM in the presence of phospholipids) into high-affinity Ca(2+) binding (12-14 microM). The unusual Ca(2+)-binding mode of the piccolo C(2)A-domain reveals that C(2)-domains are mechanistically more versatile than previously envisaged. The low Ca(2+) affinity of the piccolo C(2)A-domain suggests that piccolo could function in short-term synaptic plasticity when Ca(2+) concentrations accumulate during repetitive stimulation.
Figures
Fig. 1. Structure of piccolo/aczonin. (A) Domain structures of the short and long splice variants of piccolo/aczonin, and design of the recombinant GST fusion proteins used for the current experiments. Only the C-termini of two alternatively spliced variants of piccolo are shown, with residue numbers (corresponding to the rat short and the mouse long form; Wang et al., 1999; Fenster et al., 2000) indicated above the domains. (B) Sequence alignments of the C2-domains of mouse piccolo (mPic.), rat synaptotagmins I (rSI) and VII (rSVII) and rat rabphilin (rRb). On the left, ‘A’ and ‘B’ denote C2A- and C2B-domains, respectively. Residues present in at least 50% of the sequences are highlighted with a structure-based color code: yellow, top loops; blue, β-strands; green, bottom loops; red, bottom α-helix specific for C2B-domains of synaptotagmins and rabphilin. Aspartates and serines in the top loops involved in Ca2+ binding are highlighted in black and marked by arrowheads. (C) Structure of the Ca2+-binding sites of the synaptotagmin I C2A-domain (Ubach et al., 1998), and model for the Ca2+-binding sites of the piccolo C2A-domain based on the sequence homology between these domains (see B). Only the Ca2+ ligands are drawn. The positions of other residues are indicated by small solid circles along the curves that symbolize the two sequence loops that form the Ca2+-binding region (loops 1 and 3). The three Ca2+-binding sites are indicated as solid circles labeled Ca1, Ca2 and Ca3.
Fig. 2. Analysis of the Ca2+-binding properties of the piccolo C2A-domain by NMR spectroscopy. (A) 1H-15N HSQC spectra of the isolated C2A-domain from piccolo recorded in the absence (black contours) and presence (red contours) of 30 mM Ca2+. Spectra are superimposed to demonstrate widespread Ca2+-dependent cross-peak shifts. (B) Expansions illustrating the progressive changes in the cross-peaks caused by increasing concentrations of Ca2+. The cross-peaks shown are identified in (A) by arrows in the 1H-15N HSQC spectra. Cross-peak positions are displayed at 0.0, 1.0, 2.5, 4.0 and 30.0 mM Ca2+ from top to bottom. Note that in contrast to the gradual cross-peak movements observed for the synaptotagmin C2A-domain (Shao et al., 1996), the Ca2+-free cross-peaks disappear concomitant with the appearance of Ca2+-bound cross-peaks. (C) 1H-15N HSQC spectra of the mutant C2A-domain from piccolo with the double aspartate-to-alanine substitutions in the Ca2+-binding loops (D4668A/D4674A; Figure 1) in the absence (black contours) or presence (red contours) of 30 mM Ca2+. Note the lack of Ca2+-dependent cross-peak shifts in the mutant.
Fig. 3. Ca2+-dependent phospholipid binding by the C2A-domain of piccolo. Purified GST fusion proteins from rat piccolo containing the PDZ-domain (GST–Pic-PDZ) (A), the C2A-domain (GST–Pic-C2A) (B) or the mutant C2A-domain (GST–Pic-C2AD4668A/D4674A substituting two Ca2+-binding aspartates for alanines) (C) were immobilized on glutathione beads. 3H-labeled liposomes composed of 100% PC, 30% PS/70% PC, 50% PI/50% PC, or 50% PE/50% PC were used in binding experiments with the three GST fusion proteins in the presence or absence of 1 mM free Ca2+ (see Materials and methods). Note the expanded scale of the graphs of the GST–PDZ domain and the mutant C2-domain to illustrate the small amount of background binding. Data are mean ± SEM from a representative experiment performed in triplicate.
Fig. 4. Divalent cation specificity of phospholipid binding to the C2A-domains of synaptotagmin I and piccolo. GST alone (as a control), and the GST fusion proteins of the C2A-domain of synaptotagmin I (GST–SytI-C2A) and of piccolo (GST–Pic-C2A) were used in phospholipid-binding assays in the presence of the indicated concentrations of Ca2+, Ba2+, Sr2+ and Mg2+ using liposomes composed of 50% PI/50% PC or 100% PC. Data are mean ± SEM from a representative experiment performed in triplicate.
Fig. 5. Effect of phospholipid composition and NaCl concentration on Ca2+-dependent liposome binding to the C2A-domains of synaptotagmin I and piccolo. Immobilized GST fusion proteins of the C2A-domains of synaptotagmin I (GST–SytI-C2A) and piccolo (GST–Pic-C2A) were used in phospholipid-binding assays in the presence of increasing concentrations of NaCl as indicated, with or without 1 mM Ca2+. Liposomes were composed of 100% PC, 30% PS/70% PC, 50% PE/50% PC, 50% PI/50% PC, 15% PIP/85% PC, or 10% PIP2/90% PC. Note differences in scale between graphs. Data are mean ± SEM from a representative experiment performed in triplicate.
Fig. 6. Ca2+ concentration dependence of phospholipid binding to the C2A-domain of synaptotagmin I (SytI), piccolo (Pic) and the combined PDZ- and C2A-domains of piccolo. Phospholipid-binding reactions with the immobilized GST fusion proteins of the indicated C2-domains were carried out in Ca2+/EGTA buffers to clamp the free Ca2+ concentration. For each experiment, a GST-only control was performed for high Ca2+ concentrations to exclude artifactual Ca2+-dependent lipid precipitation (open symbol in each panel). Titrations were carried out with liposomes composed of 30% PC/70% PC (PS; top panels), or 50% PI/50% PC (PI; bottom panels). The Ca2+ concentration corresponding to half-maximal binding (EC50) and the apparent calculated cooperativity (Hillcoeff) are shown for each experiment in the corresponding panels. Data are mean ± SEM from a representative experiment performed in triplicate; results from multiple experiments are summarized in Table I.
Fig. 7. Evolutionary conservation of the unique bottom sequence in the piccolo C2A-domain, and localization of the sequence in a model of the C2A-domain. (A) Alignment of the primary structures of mouse, rat, human and chicken piccolo C2A-domains in the region containing the piccolo-specific bottom sequence (bold type) that was mutated in the experiments described in Figures 8 and 9 and Table I. Amino acid substitutions are underlined and shown in italic. (B) Model of the piccolo C2A-domain with three bound Ca2+ ions on top (orange spheres) and the unusual sequence that is specific for the C2A-domain on the bottom (shown in yellow). The model proposes that the unique bottom sequence folds back towards the top Ca2+-binding loops of the C2A-domain. The bottom sequence could either directly influence Ca2+ binding to the top loops, or stabilize the Ca2+-free conformation of the overall C2A-domain and thereby indirectly influence Ca2+ binding. The model is based on the structure of the synaptotagmin C2A-domain where the 11 residue loop has been manually inserted in an arbitrary conformation (prepared with the MOLSCRIPT program; Kraulis, 1991).
Fig. 8. NMR spectra of wild-type and mutant piccolo C2A-domains. (A and B) Superpositions of 1H-15N HSQC spectra of the wild-type piccolo C2A-domain (black contours) and the V4690S/V4691S mutant (red contours) in the absence (A) and presence (B) of saturating Ca2+ concentrations (30 mM for wild type and 10 mM for the mutant). (C) Expansions of 1H-15N HSQC spectra of wild-type piccolo C2A-domain, the V4690S/V4691S mutant (VV) and the V4688S/M4689S mutant (VM) acquired at the Ca2+ concentrations described on the left (in millimolar). The expansions illustrate the disappearance of a cross-peak from the Ca2+-free form and the concomitant appearance of the cross-peak from the Ca2+-bound form (identified by arrows in A and B) with increasing Ca2+ concentrations. (D) Ca2+ dependence of the intensity of the cross-peak from the Ca2+-bound form appearing in (C). The intensities were normalized to the intensity of the cross-peak at Ca2+ saturation. Solid lines represent curve fitting of the data to a standard Hill equation; the resulting Ca2+-binding parameters are summarized in Table I. For clarity, the plot only shows data obtained up to 5 mM Ca2+, but the fits were obtained using data up to 30 mM Ca2+ for wild type and up to 10 mM Ca2+ for the mutants (see Table I).
Fig. 9. Effect of increasing NaCl concentrations on Ca2+-dependent liposome binding to wild-type (WT) and mutant piccolo C2A-domains. Ca2+-dependent phospholipid-binding measurements were carried out with immobilized GST fusion proteins of the wild-type and mutant piccolo C2A-domains as described for Figures 3 and 5 using liposomes composed of 30% PS/70% PC. Ca2+-binding affinities of these mutants determined in multiple independent experiments are summarized in Table I. To facilitate comparisons, results were normalized for the amount of binding observed in 0.1 M NaCl. Data are mean ± SEM from a representative experiment performed in triplicate.
References
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