Structural Insights into the Calmodulin−Munc13 Interaction Obtained by Cross-Linking and Mass Spectrometry

Abstract

Munc13 proteins are essential regulators of synaptic vesicle priming and play a key role in adaptive synaptic plasticity phenomena. We recently identified and characterized the Ca(2+)-dependent interaction of Munc13 and calmodulin (CaM) as the molecular mechanism linking changes in residual Ca(2+) concentrations to presynaptic vesicle priming and short-term plasticity. Here, we used peptidic photoprobes covering the established CaM-binding motif of Munc13 for photoaffinity labeling (PAL) of CaM, followed by structural characterization of the covalent photoadducts. Our innovative analytical workflow based on isotopically labeled CaM and mass spectrometry revealed that, in the bound state, the hydrophobic anchor residue of the CaM-binding motif in Munc13s contacts two distinct methionine residues in the C-terminal domain of CaM. To address the orientation of the peptide during binding, we obtained additional distance constraints from the mass spectrometric analysis of chemically cross-linked CaM-Munc13 peptide adducts. The constraints from both complementary cross-linking approaches were integrated into low-resolution three-dimensional structure models of the CaM-Munc13 peptide complexes. Our experimental data are best compatible with the structure of the complex formed by CaM and a CaM-binding peptide derived from neuronal NO synthase and show that Munc13-1 and ubMunc13-2 bind to CaM in an antiparallel orientation through a 1-5-8 motif. The structural information about the CaM-Munc13 peptide complexes will facilitate the design of Munc13 variants with altered CaM affinity and thereby advance the detailed functional analysis of the role of Munc13 proteins in synaptic transmission and plasticity.

Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

“…The order of subunits in the top ring is αγζθηϵβδ or CCT1, CCT3, CCT6, CCT8, CCT7, CCT5, CCT2, CCT4 (13687524) that we prefer to write as AGZQHEBD. limited to a small sampling of the conformation space (23,27) or even just to a single model (25). The merits of combinatorial modeling are evident: The top ranking model is chosen without bias, and its significance over the alternatives is clearly quantified.…”

“…For simpler systems involving cross-linking of a pair of proteins, multiple docking models were derived and then filtered by the cross-link sets (23,27). However, studies of more complex systems with multiple proteins only reported a single model in very coarse terms (24,25).…”

Nonconserved Ca2+/Calmodulin Binding Sites in Munc13s Differentially Control Synaptic Short-Term Plasticity

“…Labeling of CaM with Munc13 photoprobes and mapping of the photoadducts by MS was performed according to our recently introduced workflow based on isotopically labeled [ 15 N]CaM (14). To identify and sequence cross-linked peptides, PAL reaction mixtures were subjected to in-solution digestion with trypsin and subsequently analyzed by offline liquid chromatography (LC)-MALDI-MS on a syringe pump-based capillary (300 m inner diameter) LC system coupled to a Bruker Ultraflex MALDI-TOF/TOF mass spectrometer (14). To complement mass spectrometric sequencing, a ligand release assay by on-target CNBr cleavage was used for confirmation of cross-linked sites as described previously (14,23).…”

“…To identify and sequence cross-linked peptides, PAL reaction mixtures were subjected to in-solution digestion with trypsin and subsequently analyzed by offline liquid chromatography (LC)-MALDI-MS on a syringe pump-based capillary (300 m inner diameter) LC system coupled to a Bruker Ultraflex MALDI-TOF/TOF mass spectrometer (14). To complement mass spectrometric sequencing, a ligand release assay by on-target CNBr cleavage was used for confirmation of cross-linked sites as described previously (14,23). In this assay, photoincorporation into the Met side chain is indicated by a newly appearing signal corresponding to the methylthiocyanate derivative (mass shift of ϩ73.00 mass units) of the respective benzophenone-containing tryptic peptide derived from the photoprobe.…”

“…For the photoreaction, the respective Munc13 peptide (10 M) was mixed with SBC-labeled CaM before irradiating the mixture with UV light (365 nm). Analysis of the cross-linking reactions by SDS-PAGE and linear MALDI-TOF-MS was performed as described previously (14). Crosslinked products were separated from nonreacted CaM by SDS-PAGE, and gel bands of interest were subjected to in-gel digestion with trypsin (45) for 2 h at 37°C in the presence of the trypsin enhancer ProteaseMAX surfactant (Promega) according to the manufacturer's protocol.…”