title: Presynaptic Calmodulin targets: lessons from structural proteomics.
abstract: Calmodulin (CaM) is a highly conserved Ca2+-binding protein that is exceptionally abundant in the brain. In the presynaptic compartment of neurons, CaM transduces changes in Ca2+concentration into the regulation of synaptic transmission dynamics. Areas covered: We review selected literature including published CaM interactor screens and outline established and candidate presynaptic CaM targets. We present a workflow of biochemical and structural proteomic methods that were used to identify and characterize the interactions between CaM and Munc13 proteins. Finally, we outline the potential of ion mobility-mass spectrometry (IM-MS) for conformational screening and of protein-protein cross-linking for the structural characterization of CaM complexes. Expert commentary: Cross-linking/MS and native MS can be applied with considerable throughput to protein mixtures under near-physiological conditions, and thus effectively complement high-resolution structural biology techniques. Experimental distance constraints are applicable best when obtained by combining different cross-linking strategies, i.e. by using cross-linkers with different spacer length and reactivity, and by using the incorporation of unnatural photo-reactive amino acids. Insights from structural proteomics can be used to generate CaM-insensitive mutants of CaM targets for functional studies in vitro or ideally in vivo.
authors.affiliation: a Department of Molecular Neurobiology , Max-Planck-Institute of Experimental Medicine , Göttingen , Germany.
authors.forename: Noa
authors.initials: N
authors.lastname: Lipstein
authors.affiliation: b Institute of Chemistry and Biochemistry , Free University Berlin, Berlin & Fritz Haber Institute of the Max-Planck-Society , Berlin , Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Göth
authors.affiliation: c Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy , Martin Luther University Halle-Wittenberg , Halle (Saale) , Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Piotrowski
authors.affiliation: b Institute of Chemistry and Biochemistry , Free University Berlin, Berlin & Fritz Haber Institute of the Max-Planck-Society , Berlin , Germany.
authors.forename: Kevin
authors.initials: K
authors.lastname: Pagel
authors.affiliation: c Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy , Martin Luther University Halle-Wittenberg , Halle (Saale) , Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
authors.affiliation: d Proteomics Group , Max-Planck-Institute of Experimental Medicine , Göttingen , Germany.
authors.forename: Olaf
authors.initials: O
authors.lastname: Jahn
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics*; D006801:Humans; D013058:Mass Spectrometry; D009474:Neurons / Q000378:metabolism; D017729:Presynaptic Terminals / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics; D009435:Synaptic Transmission / Q000235:genetics*
chemical_list: D002147:Calmodulin; D002118:Calcium
keywords: Calmodulin; Munc13; chemical cross-linking; ion mobility; mass spectrometry; photoaffinity labeling; structural proteomics; synapse; synaptic vesicle cycle