Conformational choreography of a molecular switch region in myelin basic protein—Molecular dynamics shows induced folding and secondary structure type conversion upon threonyl phosphorylation in both aqueous and membrane-associated environments

AbstractThe 18.5kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5kDa myelin basic protein (residues Glu80–Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80–Val91 and extended poly-proline type II from Thr92–Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of β-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the α-helix and the accessibility of the proline-rich ligand and can stabilize a β-bend. A bend in this region of 18.5kDa myelin basic protein suggests that the N- and C-termini of the proteins can interact with different leaflets of the myelin membrane and explain how a single protein can bring them close together

Regulation of cell proliferation by nucleocytoplasmic dynamics of postnatal and embryonic exon-II-containing MBP isoforms

AbstractThe only known structural protein required for formation of myelin, produced by oligodendrocytes in the central nervous system, is myelin basic protein (MBP). This peripheral membrane protein has different developmentally-regulated isoforms, generated by alternative splicing. The isoforms are targeted to distinct subcellular locations, which is governed by the presence or absence of exon-II, although their functional expression is often less clear. Here, we investigated the role of exon-II-containing MBP isoforms and their link with cell proliferation. Live-cell imaging and FRAP analysis revealed a dynamic nucleocytoplasmic translocation of the exon-II-containing postnatal 21.5-kDa MBP isoform upon mitogenic modulation. Its nuclear export was blocked upon treatment with leptomycin B, an inhibitor of nuclear protein export. Next to the postnatal MBP isoforms, embryonic exon-II-containing MBP (e-MBP) is expressed in primary (immature) oligodendrocytes. The e-MBP isoform is exclusively present in OLN-93 cells, a rat-derived oligodendrocyte progenitor cell line, and interestingly, also in several non-CNS cell lines. As seen for postnatal MBPs, a similar nucleocytoplasmic translocation upon mitogenic modulation was observed for e-MBP. Thus, upon serum deprivation, e-MBP was excluded from the nucleus, whereas re-addition of serum re-established its nuclear localization, with a concomitant increase in proliferation. Knockdown of MBP by shRNA confirmed a role for e-MBP in OLN-93 proliferation, whereas the absence of e-MBP similarly reduced the proliferative capacity of non-CNS cell lines. Thus, exon-II-containing MBP isoforms may regulate cell proliferation via a mechanism that relies on their dynamic nuclear import and export, which is not restricted to the oligodendrocyte lineage

Recognition Pliability Is Coupled to Structural Heterogeneity: A Calmodulin Intrinsically Disordered Binding Region Complex

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Docking and molecular dynamics simulations of the Fyn-SH3 domain with free and phospholipid bilayer-associated 18.5-kDa myelin basic protein (MBP) – Insights into a non-canonical and fuzzy interaction

The molecular details of the association between the human Fyn-SH3 domain, and the fragment of 18.5-kDa myelin basic protein (MBP) spanning residues S38–S107 (denoted as xα2-peptide, murine sequence numbering), were studied in silico via docking and molecular dynamics over 50-ns trajectories. The results show that interaction between the two proteins is energetically favorable and heavily-dependent on the MBP proline-rich region (P93-P98) in both aqueous and membrane environments. In aqueous conditions, the xα2-peptide/Fyn-SH3 complex adopts a “sandwich”-like structure. In the membrane context, the xα2-peptide interacts with the Fyn-SH3 domain via the proline-rich region and the β-sheets of Fyn-SH3, with the latter wrapping around the proline-rich region in a form of a clip. Moreover, the simulations corroborate prior experimental evidence of the importance of upstream segments beyond the canonical SH3-ligand. This study thus provides a more-detailed glimpse into the context-dependent interaction dynamics and importance of the β-sheets in Fyn-SH3 and proline-rich region of MBP

Molecular dynamics investigation of myelin basic protein stability on lipid membranes

Simulation of proteins and membranes composed of synthetic lipids on computer clusters provides molecular information that complements experimental data. This paper describes molecular dynamics (MD) approaches to study the properties of biological membranes and proteins using the freely available GROMACS package on the C-terminal α-helical peptide of myelin basic protein (MBP). We simulated a mixed membrane – consisting of 2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPC/DMPE), and a pure DMPC membrane, composed of 188 and 248 lipids, respectively – for 200 ns at 309K. The DMPC membrane was approximately three times more fluid compared to the DMPC/DMPE system, with the diffusion coefficients (D) being 0.0207x10-5 cm2/s and 0.0068x10-5 cm2/s, respectively. In addition, we simulated the 14-residue peptide representing the C-terminal α-helical region of murine MBP, with sequence NH2-A141YDAQGTLSKIFKL154-COOH, in both membrane systems for 200 ns. The negatively-charged N-terminal end of the peptide penetrated further into the DMPC bilayer than into the mixed DMPC/DMPE bilayer. Reduced peptide accessibility to a formal positive charge of the DMPC amine ‘N’ atom surrounded by methyl and methylene groups may be the cause [1]. The peptide lost its α-helical structure in DMPC/DMPE but not in the DMPC bilayer. These findings show that membrane composition affects MBP’s interaction with it, a phenomenon that provides insights into myelin structure – and that may eventually be relevant to understanding the pathogenesis of multiple sclerosis (MS)

Backbone Dynamics of the 18.5 kDa Isoform of Myelin Basic Protein Reveals Transient α-Helices and a Calmodulin-Binding Site

The 18.5 kDa isoform of myelin basic protein (MBP) is the predominant form in adult human central nervous system myelin. It is an intrinsically disordered protein that functions both in membrane adhesion, and as a linker connecting the oligodendrocyte membrane to the underlying cytoskeleton; its specific interactions with calmodulin and SH3-domain containing proteins suggest further multifunctionality in signaling. Here, we have used multidimensional heteronuclear nuclear magnetic resonance spectroscopy to study the conformational dependence on environment of the protein in aqueous solution (100 mM KCl) and in a membrane-mimetic solvent (30% TFE-d2), particularly to analyze its secondary structure using chemical shift indexing, and to investigate its backbone dynamics using 15N spin relaxation measurements. Collectively, the data revealed three major segments of the protein with a propensity toward α-helicity that was stabilized by membrane-mimetic conditions: T33-D46, V83-T92, and T142-L154 (murine 18.5 kDa sequence numbering). All of these regions corresponded with bioinformatics predictions of ordered secondary structure. The V83-T92 region comprises a primary immunodominant epitope that had previously been shown by site-directed spin labeling and electron paramagnetic resonance spectroscopy to be α-helical in membrane-reconstituted systems. The T142-L154 segment overlapped with a predicted calmodulin-binding site. Chemical shift perturbation experiments using labeled MBP and unlabeled calmodulin demonstrated a dramatic conformational change in MBP upon association of the two proteins, and were consistent with the C-terminal segment of MBP being the primary binding site for calmodulin