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

Harauz G (2013) The Effects of Threonine Phosphorylation on the Stability and Dynamics of the Central Molecular Switch Region of 18.5-kDa Myelin Basic Protein. PLoS ONE 8(7): e68175. doi:10.1371/journal.pone.0068175 The Effects of Threonine Phosphorylation on the Stability and Dynamics of the Central Molecular Switch Region of 18.5-kDa Myelin Basic Protein Kenrick A. Vassall 0 1 Kyrylo Bessonov 0 1 Miguel De Avila 0 1 Eugenia Polverini 0 1 George Harauz 0 1 Petri Kursula, University of Oulu, Finland 0 Current address: Department of Electrical Engineering and Computer Science (Institut Montefiore), Universite de Lie`ge , Lie`ge , Belgium 1 1 Department of Molecular and Cellular Biology, University of Guelph , Guelph, Ontario , Canada , 2 Department of Physics, University of Parma , Parma , Italy The classic isoforms of myelin basic protein (MBP) are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence -T92-P93-R94-T95-P96-P97-P98-S99-) containing a putative SH3-ligand, adjacent to a region that forms an amphipathic a-helix (P82-I90) upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP) kinases. Here, we have investigated the structure of the a-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the a-helical region, by utilizing four 36-residue peptides (S72-S107) with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the a-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012). Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic a-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the a-helix upon temperature perturbation and affect the global structure of the peptides through altered electrostatic interactions. The results support the hypothesis that the central conserved segment of MBP constitutes a molecular switch in which the conformation and/or intermolecular interactions are mediated by phosphorylation/dephosphorylation at T92 and T95. - Funding: This work was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant RG121541 to GH; http://www.nserccrsng.gc.ca/). GH is a Tier 1 Canada Research Chair. KAV and MDA were recipients of a Postdoctoral Fellowship and of a Doctoral Studentship, respectively, from the Multiple Sclerosis Society of Canada (http://mssociety.ca). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. In the central nervous system (CNS), myelin arises from oligodendrocytes (OLGs), which proceed through a regulated pathway that assembles the components of the myelin membrane [15]. Myelination commences with differentiation of the bipolar early oligodendrocyte progenitor cell (OPC), and culminates with copious synthesis of classic myelin basic protein (MBP) isoforms and proteolipid protein (PLP), when extensive processes form and extend around an axon. Compact myelin is formed by flattening of the multiple, spirally-wrapped lamellae with the extrusion of cytoplasm, a process modeled in various ways, e.g., the liquid croissant and corkscrew models [69]. The amount of white matter in the brain increases with evolutionary complexity [10]. Myelin continues to be formed until the early twenties in humans [11], and remodeling continues throughout adulthood in the healthy CNS [12]. Multiple sclerosis (MS) is a disease that is characterized by inflammatory demyelination of axons, for which the molecular mechanism has remained unknown over 150 years since its first major clinical documentation [13]. An inside-out model suggests that multiple sclerosis results from a cytodegenerative process aimed at the oligodendrocyte-myelin complex [14 16]: a process of gradual physical demyelination can then lead to an autoimmune response and a cycle of further degeneration, characteristic of the most common relapsing-remitting manifestation of multiple sclerosis. For many reasons, it is essential to attain an understanding of myelin formation and architecture at the molecular level in order to comprehend the causes and pathogenesis of this debilitating disease, as well as fundamental aspects of brain development and modeling. One of the most studied candidate auto-antigens in multiple sclerosis is the classic 18.5-kDa isoform of MBP, which is essential to the stability of central nervous system myelin where it plays numerous roles both in myelin development and homeostasis, acting both to adhere membrane leaflets to each other, and as a hub in protein-protein and protein-membrane interaction networks [1722]. The latter include cytoskeletal proteins such as actin and tubulin, as well as signaling proteins such as calciumactivated 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. There are, however, three segments of the protein that become ahelical in the presence of lipids or membrane-mimetic solvents such as trifluoroethanol (TFE) [23,2628]. These three segments that undergo this specific disorder-to-order transition are denoted by us here as the a1-segment (murine 18.5-kDa residues T33-D46), a2-segment (P82-I90), and a3-segment (Y142-L154), respectively (Figure 1). In addition to being membrane-anchoring motifs, these segments can also moonlight as protein-protein interaction sites [22,29]. In vivo, 18.5-kDa MBP undergoes extensive post-translational modifications [30,31], which play a role in its ability to interact with a wide variety of partners [2022]. One of the most important post-translational modifications in MBP is phosphorylation by mitogen-activated and other protein kinases (revi (...truncated)