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.

Christine Piotrowski,Andrea Sinz,Rocco Moretti,Christian H Ihling,Jens Meiler,Olaf Jahn,Thomas Liepold,Noa Lipstein,André Haedicke

doi:10.3390/cells9010136

Abstract

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.

Highlights

Ca2+ acts as a key regulator of multiple cellular processes, such as cell proliferation, differentiation, apoptosis, and neurotransmitter release [1,2]
As bMunc13-2 is a >220 kDa multi-domain protein that is difficult to express, two different constructs and a synthetic peptide, all derived from its N-terminal region, were employed as proxies for the intact protein to study the CaM–bMunc13-2 interaction
CaM is applied in large excess, we found a predominant occurrence of 1:1 complexes irrespective of the detection method applied, which led us to conclude that the predicted binding site is capable of CaM binding only if the high-affinity binding site is compromised

Summary

Introduction

Ca2+ acts as a key regulator of multiple cellular processes, such as cell proliferation, differentiation, apoptosis, and neurotransmitter release [1,2]. Changes in Ca2+ concentrations are sensed by various proteins that either bind to Ca2+ itself or interact with Ca2+ binding proteins. A prominent Ca2+ binding protein is calmodulin (CaM), a small 17-kDa acidic protein that is highly conserved in all eukaryotes and has an identical amino acid sequence in vertebrates [3,4]. CaM is a 148-amino acid protein arranged in two globular domains, each containing two Ca2+ binding EF-hands that are connected by a flexible linker [5,6,7]. CaM evolved to enable promiscuous target binding, requiring few specific interactions to interact with its more than 300 known target proteins [8,9,10].

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Conclusion