Delineating the Molecular Basis of the Calmodulin‒bMunc13-2 Interaction by Cross-Linking/Mass Spectrometry-Evidence for a Novel CaM Binding Motif in bMunc13-2

• Published in: Cells (Basel, Switzerland) Vol. 9; no. 1; p. 136
• Main Authors: Piotrowski, Christine, Moretti, Rocco, Ihling, Christian H, Haedicke, André, Liepold, Thomas, Lipstein, Noa, Meiler, Jens, Jahn, Olaf, Sinz, Andrea
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 07.01.2020; MDPI
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Amino acids; Animals; Binding Sites; Calcium-binding protein; Calmodulin; Calmodulin - chemistry; Calmodulin - metabolism; Cattle; Chromatography; cross-linking; Cross-Linking Reagents - metabolism; E coli; Experiments; Glycerol; Hydrophobic and Hydrophilic Interactions; Mass Spectrometry; Mass spectroscopy; Models, Molecular; munc13; Mutagenesis; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - metabolism; Peptides; Protein Binding; Protein Domains; Proteins; protein–protein interaction; Rats; Scientific imaging; Swine; Synaptic plasticity; Synaptic transmission; Germany; mass spectrometry; protein‒protein interaction; cross-linking; calmodulin; Munc13
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells9010136

Exploring the interactions between the Ca binding protein calmodulin (CaM) and its target proteins remains a challenging task. Members of the Munc13 protein family play an essential role in short-term synaptic plasticity, modulated via the interaction with CaM at the presynaptic compartment. In this study, we focus on the bMunc13-2 isoform expressed in the brain, as strong changes in synaptic transmission were observed upon its mutagenesis or deletion. The CaM‒bMunc13-2 interaction was previously characterized at the molecular level using short bMunc13-2-derived peptides only, revealing a classical 1‒5‒10 CaM binding motif. Using larger protein constructs, we have now identified for the first time a novel and unique CaM binding site in bMunc13-2 that contains an -terminal extension of a classical 1‒5‒10 CaM binding motif. We characterize this motif using a range of biochemical and biophysical methods and highlight its importance for the CaM‒bMunc13-2 interaction.