Characterization of the Munc13 - CaM Interaction

Abstract

The Munc13 proteins are the key mediators of synaptic vesicle priming, an essential step in Ca2+-regulated neurotransmitter release that renders docked vesicles fusion-competent prior to exocytosis. They have emerged as important regulators of adaptive synaptic mechanisms such as presynaptic short-term plasticity, a process by which the release of neurotransmitter is dynamically adapted to a changing demand. Indeed, Munc13-1 and ubMunc13-2 contain a conserved calmodulin (CaM) binding site and the Ca2+-dependent interaction of these Munc13 isoforms with CaM constitutes a molecular mechanism that transduces residual Ca2+ signaling to the synaptic exocytotic machinery. This study aimed to (i) establish whether such regulation through CaM exists in the other Munc13 isoforms, bMunc13-2 and Munc13-3, and (ii) provide structural insights into the Munc13-CaM interaction. Bioinformatic tools were used to identify potential CaM recognition motifs in the non-conserved sequences of bMunc13-2 and Munc13-3. Munc13-derived model peptides covering the potential CaM binding sites were used in photoaffinity labeling (PAL) experiments with CaM, followed by mass spectrometric characterization of the covalent photoadducts. Analysis of these peptide-protein interactions demonstrated that all four Munc13 isoforms bind CaM in a stoichiometric and Ca2+-dependent manner and that only slightly elevated intracellular Ca2+ concentrations are sufficient to trigger these interactions. These results support the conclusion that convergent evolution has generated structurally distinct but functionally similar Ca2+/CaM binding sites in Munc13-1/ubMunc13-2, bMunc13-2, and Munc13-3, all of which can contribute to presynaptic short-term plasticity. A novel PAL-based analytical strategy using isotopically labeled CaM and mass spectrometry was established for the structural characterization of the covalent Munc13-CaM photoadducts. It revealed that, in the bound state, the hydrophobic anchor residue of the CaM-binding motif in Munc13 contacts two distinct Met residues in the C-terminal domain of CaM. These contact sites provided a valuable basis for molecular modeling of the Munc13-CaM complex through integration with constraints obtained by chemical cross-linking methods. The PAL analysis, carried out under physiological solvent and concentration conditions, also emerged as an important complement to high-resolution NMR studies on the Munc13/CaM interaction, as it yielded biochemical support for a novel 1-26 CaM binding motif in Munc13-1 and ubMunc13-2. The structural data on the Munc13-CaM complex offer new options for future studies towards a deeper understanding of the role of Munc13 proteins in synaptic vesicle priming and short-term synaptic plasticity.