Dataset Information

Backbone dynamics of the 18.5 kDa isoform of myelin basic protein reveals transient alpha-helices and a calmodulin-binding site.

ABSTRACT: 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-d(2)), particularly to analyze its secondary structure using chemical shift indexing, and to investigate its backbone dynamics using (15)N spin relaxation measurements. Collectively, the data revealed three major segments of the protein with a propensity toward alpha-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 alpha-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.

SUBMITTER: Libich DS

PROVIDER: S-EPMC2397351 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Backbone dynamics of the 18.5 kDa isoform of myelin basic protein reveals transient alpha-helices and a calmodulin-binding site.

Libich David S DS Harauz George G

Biophysical journal 20080307 12

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 spec ...[more]

PMID: 18326633

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Project description: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 ?-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 ?-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 ?-helical region, by utilizing four 36-residue peptides (S72-S107) with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the ?-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 ?-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the ?-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.

Project description:The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5-kDa MBP isoform has a proline-rich region comprising amino acids 92-99 (murine sequence -T(92)PRTPPPS(99)-) that contains a minimal SH3 ligand domain. We have previously shown that 18.5-kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBP's SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild-type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N-terminal-tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca(2+) influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein-protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBP's SH3 ligand domain. These results suggest that MBP's SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that phosphorylation of Thr92 and Thr95 can regulate this function.

Dataset Information

Backbone dynamics of the 18.5 kDa isoform of myelin basic protein reveals transient alpha-helices and a calmodulin-binding site.

Publications

Backbone dynamics of the 18.5 kDa isoform of myelin basic protein reveals transient alpha-helices and a calmodulin-binding site.

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