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

Publication year: 2020
Authors: Piotrowski C. 1, Moretti R. 2, Ihling C. 1,Haedicke A. 3†, Liepold T. 4, Lipstein N. 5, Meiler J. 2, Jahn O. 4,*, Sinz A. 1,*
Affiliations:
1 - Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, D-06120 Halle/Saale, Germany
2 - Center for Structural Biology, Department of Chemistry, Vanderbilt University, Nashville, TN 37221, USA
3 - Biophysical Chemistry, Institute of Chemistry, Martin Luther University Halle-Wittenberg, D-06120 Halle/Saale, Germany
4 - Proteomics Group, Max Planck Institute of Experimental Medicine, D-37075 Göttingen, Germany
5 - Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, D-37075 Göttingen, Germany
Present address: Serumwerk Bernburg AG, D-06406 Bernburg, Germany
Published in: Cells, 2020, Vol.9 (1) , p.136
doi: 10.3390/cells9010136

Exploring the interactions between the Ca2+ 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 N-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.


MP-SPR keywords: affinity, avidin kit, binding, interaction, protein