Structured Functional Domains of Myelin Basic Protein: Cross-Talk Between Actin Polymerization and Ca 2+-Dependent Calmodulin Interaction

Abstract

The 18.5 kDa myelin basic protein (MBP), the most abundant isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the sheath. A hydrophobic moment analysis of MBP's amino acid sequence reveals three regions with high propensity to form strongly amphipathic alpha-helices. These regions, located in the central, N- and C- terminal parts of the protein, have been shown to play a role in the interactions of MBP with other partners, such as SH3-domain binding proteins, actin, Ca2+-activated calmodulin (Ca2+-CaM), and with myelin-mimetic membrane bilayers. Here, we have further characterized the structure-function relationship of these three domains. We constructed three recombinant peptides derived from the 18.5 kDa murine MBP: A22-K56, S72-S107, and S133-S159 (denoted alpha1, alpha2, and alpha3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, solid-state NMR (ssNMR) spectroscopy, isothermal titration calorimetry, electron microscopy, and fluorimetry) to characterize the interactions of these peptides with actin and calmodulin. Our results show that all three peptides can adopt alpha-helical structure inherently. Although both alpha1 and alpha3 peptides showed strong binding with Ca2+-CaM, only alpha1 exhibited actin polymerization and bundling activity. Calmodulin depolymerized actin that was polymerized by alpha1. Comparing fingerprints of Ala, Pro, and Ser (using ssNMR) in a reconstituted alpha1-actin complex, we showed that this peptide might adopt a better-defined structural state but still exhibits structural polymorphism. The results of this study proved that in addition to the primary calmodulin-binding site located in the C-terminal domain of MBP, there is another N-terminal binding domain for Ca2+-CaM. This secondary binding domain appeared to be essential for CaM-induced actin depolymerization. Acknowledgements: Funded by the CIHR (GH), NSERC (GH), and by Fellowships/Studentships from the MSSC (VVB, GSTS).