Structured Functional Domains of Myelin Basic Protein: Cross Talk between Actin Polymerization and Ca2+-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. Solution NMR spectroscopy and a hydrophobic moment analysis of MBP's amino-acid sequence have previously revealed three regions with high propensity to form strongly amphipathic α-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 cytoskeletal proteins, Src homology 3-domain-containing proteins, Ca(2+)-activated calmodulin (Ca(2+)-CaM), and 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) (which are denoted α1, α2, and α3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, isothermal titration calorimetry, transmission electron microscopy, fluorimetry, and solution NMR spectroscopy and chemical shift index analysis) to characterize the interactions of these peptides with actin and Ca(2+)-CaM. Our results show that all three peptides can adopt α-helical structure inherently even in aqueous solution. Both α1- and α3-peptides showed strong binding with Ca(2+)-CaM, and both adopted an α-helical conformation upon interaction, but the binding of the α3-peptide appeared to be more dynamic. Only the α1-peptide exhibited actin polymerization and bundling activity, and the addition of Ca(2+)-CaM resulted in depolymerization of actin that had been polymerized by α1. The results of this study proved that there is an N-terminal binding domain in MBP for Ca(2+)-CaM (in addition to the primary site located in the C-terminus), and that it is sufficient for CaM-induced actin depolymerization. These three domains of MBP represent molecular recognition fragments with multiple roles in both membrane- and protein-association.

The Effects of Threonine Phosphorylation on the Stability and Dynamics of the Central Molecular Switch Region of 18.5-kDa Myelin Basic Protein

“…Separate CD experiments conducted on this peptide in aqueous conditions [23] (see also Figure S3), indicated that although the central α-helical structure is much less well defined, there could be some residual secondary structure present under these conditions. This result was consistent with solution NMR data obtained on the entire 18.5-kDa protein [27].…”

“…The central (P82-I90) segment that is contained within the α 2 -peptides used in this study is an unequivocal α-helical molecular recognition fragment (α-MoRF). This region is disordered in water, but becomes α-helical upon interaction with phospholipids and other surfactants, or in the presence of membrane-mimetic solvents such as TFE ([23], [27], [51], [54], and references therein).…”

“…Unlabeled as well as fully 13 C- 15 N-labelled recombinant murine MBP 36-residue α 2 -peptide (S72–S107, murine 18.5-kDa sequence numbering – see Figure 1) was expressed as a SUMO-fusion, and purified by chromatography as described previously [23], [49]. This construct represented the (i) unmodified peptide variant.…”

“…The latter include cytoskeletal proteins such as actin and tubulin, as well as signaling proteins such as calcium-activated calmodulin and SH3-domain containing proteins [23]–[25]. The murine 18.5-kDa MBP isoform has 168 amino acids, and is intrinsically disordered, like all members of this protein family.…”

“…The murine 18.5-kDa MBP isoform has 168 amino acids, and is intrinsically disordered, like all members of this protein family. There are, however, three segments of the protein that become α-helical in the presence of lipids or membrane-mimetic solvents such as trifluoroethanol (TFE) [23], [26]–[28]. These three segments that undergo this specific disorder-to-order transition are denoted by us here as the α 1 -segment (murine 18.5-kDa residues T33-D46), α 2 -segment (P82-I90), and α 3 -segment (Y142-L154), respectively (Figure 1).…”