Imma Fernandez

A conformational switch in syntaxin during exocytosis: role of munc18

Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N‐terminal domain, a C‐terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP‐25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18‐1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a ‘closed’ conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an ‘open’ syntaxin conformation. Using site‐directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18‐1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18‐1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin–munc18‐1 complex and the core complex.

Three-dimensional structure of the synaptotagmin 1 C2B-domain: Synaptotagmin 1 as a phospholipid binding machine

Synaptotagmin 1 probably functions as a Ca2+ sensor in neurotransmitter release via its two C2-domains, but no common Ca2+-dependent activity that could underlie a cooperative action between them has been described. The NMR structure of the C2B-domain now reveals a beta sandwich that exhibits striking similarities and differences with the C2A-domain. Whereas the bottom face of the C2B-domain has two additional alpha helices that may be involved in specialized Ca2+-independent functions, the top face binds two Ca2+ ions and is remarkably similar to the C2A-domain. Consistent with these results, but in contrast to previous studies, we find that the C2B-domain binds phospholipids in a Ca2+-dependent manner similarly to the C2A-domain. These results suggest a novel view of synaptotagmin function whereby the two C2-domains cooperate in a common activity, Ca2+-dependent phospholipid binding, to trigger neurotransmitter release.

Three-Dimensional Structure of an Evolutionarily Conserved N-Terminal Domain of Syntaxin 1A

Syntaxin 1A plays a central role in neurotransmitter release through multiple protein-protein interactions. We have used NMR spectroscopy to identify an autonomously folded N-terminal domain in syntaxin 1A and to elucidate its three-dimensional structure. This 120-residue N-terminal domain is conserved in plasma membrane syntaxins but not in other syntaxins, indicating a specific role in exocytosis. The domain contains three long alpha helices that form an up-and-down bundle with a left-handed twist. A striking residue conservation is observed throughout a long groove that is likely to provide a specific surface for protein-protein interactions. A highly acidic region binds to the C2A domain of synaptotagmin I in a Ca2+-dependent interaction that may serve as an electrostatic switch in neurotransmitter release.

Synaptotagmin–Syntaxin Interaction: The C2 Domain as a Ca2+-Dependent Electrostatic Switch

Synaptotagmin I is a synaptic vesicle protein that is thought to act as a Ca2+ sensor in neurotransmitter release. The first C2 domain of synaptotagmin I (C2A domain) contains a bipartite Ca2+-binding motif and interacts in a Ca2+-dependent manner with syntaxin, a central component of the membrane fusion complex. Analysis by nuclear magnetic resonance spectroscopy and site-directed mutagenesis shows that this interaction is mediated by the cooperative action of basic residues surrounding the Ca2+-binding sites of the C2A domain and is driven by a change in the electrostatic potential of the C2A domain induced by Ca2+ binding. A model is proposed whereby synaptotagmin acts as an electrostatic switch in Ca2+-triggered synaptic vesicle exocytosis, promoting a structural rearrangement in the fusion machinery that is effected by its interaction with syntaxin.

3D structure of kaliotoxin: is residue 34 a key for channel selectivity?

Kaliotoxin (KTX) is a natural peptide blocker of voltage‐dependent K+ channels. The 3D structure of a truncated analogue of KTX (Fernández et al. (1994) Biochemistry 33, 14256–14263) was determined by NMR spectroscopy and showed significant differences from structures established for other related scorpion toxins. A recent publication with the structure of the complete toxin (Aiyar et al. (1995) Neuron 15, 1169–1181) did not confirm these differences. In this communication we report NMR data for KTX at pH 3.0, 5.5 and 7.2 and the 3D structure obtained from data at pH=5.5. Complete KTX displays a folding similar to that of other toxins with an α‐helix and a β‐sheet linked by two disulphide bonds. The pKa of His 34 is anomalously low (4.7–5.2 depending on the buffer) owing to its interaction with two Lys residues (including the essential Lys 27), the charged N‐terminus and the side chain of Met 29. Charged residues are placed symmetrically with respect to an axis that approximately coincides with one of the principal components of the moment of inertia of the toxin. His 34, which occupies a well‐defined position between two conserved Cys, is located on the centre of a layer of charged groups. Positively and negatively charged residues are found at the same position in related toxins. It is suggested that electrostatic effects modulate the distances between positive charges in flexible side chains, contributing to the fine tuning of the selectivity toward different channel subclasses and that the approximate coincidence between the moment of inertia and the charge axis facilitate the approach of the toxin to the channel. The very low pKa of His 34 implies that it will be completely unprotonated at physiological pH.

NMR characterization of self-association of a helical peptide using deuterium exchange experiments

Abstract The 15-residue hybrid peptide containing residues 1–7 from cecropin A and residues 2–9 from melittin, CA(1–7)M(2–9), is a potent antibiotic with broader activity than cecropin A but without the cytotoxic character of melittin. In the presence of the helix inducer hexafluoroisopropanol the peptide forms aggregates of amphipathic α-helices. Aggregation causes very slow proton-deuterium exchange in some amide protons in the C-terminal region. This provides a method for estimating the association constant (≈ 106 M−1) as well as the stoichiometry of the aggregates. Exchange could be mediated by helix breathing or could involve complete disruption of the helix. These two mechanisms can be differentiated by comparing the decay of nuclear Overhauser effect cross-peaks involving two amide protons with the decay of each individual proton.