Induced Secondary Structure and Polymorphism in an Intrinsically Disordered Structural Linker of the CNS: Solid-State NMR and FTIR Spectroscopy of Myelin Basic Protein Bound to Actin

The 18.5 kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that maintains the structural integrity of the myelin sheath of the central nervous system by conjoining the cytoplasmic leaflets of oligodendrocytes and by linking the myelin membrane to the underlying cytoskeleton whose assembly it strongly promotes. It is a multifunctional, intrinsically disordered protein that behaves primarily as a structural stabilizer, but with elements of a transient or induced secondary structure that represent binding sites for calmodulin or SH3-domain-containing proteins, inter alia. In this study we used solid-state NMR (SSNMR) and Fourier transform infrared (FTIR) spectroscopy to study the conformation of 18.5 kDa MBP in association with actin microfilaments and bundles. FTIR spectroscopy of fully ¹³C,¹⁵N-labeled MBP complexed with unlabeled F-actin showed induced folding of both protein partners, viz., some increase in β-sheet content in actin, and increases in both α-helix and β-sheet content in MBP, albeit with considerable extended structure remaining. Solid-state NMR spectroscopy revealed that MBP in MBP-actin assemblies is structurally heterogeneous but gains ordered secondary structure elements (both α-helical and β-sheet), particularly in the terminal fragments and in a central immunodominant epitope. The overall conformational polymorphism of MBP is consistent with its in vivo roles as both a linker (membranes and cytoskeleton) and a putative signaling hub.     

Solution NMR structure of an immunodominant epitope of myelin basic protein. Conformational dependence on environment of an intrinsically unstructured protein

Using solution NMR spectroscopy, three-dimensional structures have been obtained for an 18-residue synthetic polypeptide fragment of 18.5 kDa myelin basic protein (MBP, human residues Q81-T98) under three conditions emulating the protein's natural environment in the myelin membrane to varying degrees: (a) an aqueous solution (100 mM KCl pH 6.5), (b) a mixture of trifluoroethanol (TFE-d2) and water (30 : 70% v/v), and (c) a dispersion of 100 mM dodecylphosphocholine (DPC-d38, 1 : 100 protein/lipid molar ratio) micelles. This polypeptide sequence is highly conserved in MBP from mammals, amphibians, and birds, and comprises a major immunodominant epitope (human residues N83-T92) in the autoimmune disease multiple sclerosis. In the polypeptide fragment, this epitope forms a stable, amphipathic, alpha helix under organic and membrane-mimetic conditions, but has only a partially helical conformation in aqueous solution. These results are consistent with recent molecular dynamics simulations that showed this segment to have a propensity to form a transient alpha helix in aqueous solution, and with electron paramagnetic resonance (EPR) experiments that suggested a alpha-helical structure when bound to a membrane [I. R. Bates, J. B. Feix, J. M. Boggs & G. Harauz (2004) J Biol Chem, 279, 5757-5764]. The high sensitivity of the epitope structure to its environment is characteristic of intrinsically unstructured proteins, like MBP, and reflects its association with diverse ligands such as lipids and other proteins.     

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

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

The proline-rich region of 18.5 kDa myelin basic protein binds to the SH3-domain of Fyn tyrosine kinase with the aid of an upstream segment to form a dynamic complex in?vitro

The intrinsically disordered 18.5 kDa classic isoform of MBP (myelin basic protein) interacts with Fyn kinase during oligodendrocyte development and myelination. It does so primarily via a central proline-rich SH3 (Src homology 3) ligand (T92–R104, murine 18.5 kDa MBP sequence numbering) that is part of a molecular switch due to its high degree of conservation and modification by MAP (mitogen-activated protein) and other kinases, especially at residues T92 and T95. Here, we show using co-transfection experiments of an early developmental oligodendroglial cell line (N19) that an MBP segment upstream of the primary ligand is involved in MBP–Fyn–SH3 association in cellula. Using solution NMR spectroscopy in vitro, we define this segment to comprise MBP residues (T62–L68), and demonstrate further that residues (V83–P93) are the predominant SH3-target, assessed by the degree of chemical shift change upon titration. We show by chemical shift index analysis that there is no formation of local poly-proline type II structure in the proline-rich segment upon binding, and by NOE (nuclear Overhauser effect) and relaxation measurements that MBP remains dynamic even while complexed with Fyn–SH3. The association is a new example first of a non-canonical SH3-domain interaction and second of a fuzzy MBP complex.     

NMR assignment of an intrinsically disordered protein under physiological conditions: the 18.5 kDa isoform of murine myelin basic protein

We report the NMR assignment of 18.5 kDa recombinant murine myelin basic protein (MBP) in 100 mM KCl as a prerequisite to structural analyses of its Ca²⁺-dependent interaction with calmodulin.     

Divalent cations induce a compaction of intrinsically disordered myelin basic protein

Central nervous system myelin is a dynamic entity arising from membrane processes extended from oligodendrocytes, which form a tightly-wrapped multilamellar structure around neurons. In mature myelin, the predominant splice isoform of classic MBP is 18.5 kDa. In solution, MBP is an extended, intrinsically disordered protein with a large effective protein surface for myriad interactions, and possesses transient and/or induced ordered secondary structure elements for molecular association or recognition. Here, we show by nanopore analysis that the divalent cations copper and zinc induce a compaction of the extended protein in vitro, suggestive of a tertiary conformation that may reflect its arrangement in myelin.     

Marburg's Variant of Multiple Sclerosis Correlates with a Less Compact Structure of Myelin Basic Protein

Multiple sclerosis (MS) is an autoimmune disease in which the myelin sheath of the central nervous system is degraded, and the 18.5 kDa isoform of myelin basic protein (MBP) is reduced in cationicity. In a unique case of acute, fulminating MS (Marburg's variant), MBP is considerably less cationic than MBP from both normal, and chronic MS-afflicted individuals. This electron microscopical study has identified that,in vitro,the less cationic Marburg MBP isomer forms a more extended protein-lipid complex than MBP from healthy or chronic MS-afflicted individuals. This correlation implies that chemical modifications to MBPin vivocontribute directly to the structural instability of myelin, and subsequent autoantigenic presentation of this protein, observedin vivoin MS.     

An analysis of the regions of the myelin basic protein that bind to phosphatidylcholine

The interactions of phosphatidylcholine (PC) to regions of the myelin basic protein (MBP) was examined. In solid phase binding assays the nature of the binding of unilamellar vesicles of¹⁴C-labeled phosphatidylcholine to bovine 18.5 kDa MBP, its N- and C-terminal peptide fragments, photooxidized 18.5 kDa MBP and the mouse 14 kDa protein, with an internal deletion of residues 117–157, was studied. The data were analyzed by computer-generated Scatchard plots in which non-specific binding was eliminated. Non-cooperative, low affinity binding of PC vesicles to MBP was observed, and this binding found to be sensitive to pH and ionic changes. At an ionic strength of 0.1 and pH 7.4, the binding of PC to the 14 kDa mouse MBP exhibited a K_d similar to that obtained with both the N-terminal and photooxidized 18.5 kDa bovine MBP. The studies indicated that the sites of PC interaction with MBP are located in the N-terminal region of the protein. The C-terminal region appeared to modulate the strength of the interaction slightly. Under similar conditions, lysozyme did not bind PC liposomes, and histone bound them nonspecifically.     

Myelin management by the 18.5‐kDa and 21.5‐kDa classic myelin basic protein isoforms

The classic myelin basic protein (MBP) splice isoforms range in nominal molecular mass from 14 to 21.5 kDa, and arise from the gene in the oligodendrocyte lineage (Golli) in maturing oligodendrocytes. The 18.5‐kDa isoform that predominates in adult myelin adheres the cytosolic surfaces of oligodendrocyte membranes together, and forms a two‐dimensional molecular sieve restricting protein diffusion into compact myelin. However, this protein has additional roles including cytoskeletal assembly and membrane extension, binding to SH3‐domains, participation in Fyn‐mediated signaling pathways, sequestration of phosphoinositides, and maintenance of calcium homeostasis. Of the diverse post‐translational modifications of this isoform, phosphorylation is the most dynamic, and modulates 18.5‐kDa MBP's protein‐membrane and protein‐protein interactions, indicative of a rich repertoire of functions. In developing and mature myelin, phosphorylation can result in microdomain or even nuclear targeting of the protein, supporting the conclusion that 18.5‐kDa MBP has significant roles beyond membrane adhesion. The full‐length, early‐developmental 21.5‐kDa splice isoform is predominantly karyophilic due to a non‐traditional P‐Y nuclear localization signal, with effects such as promotion of oligodendrocyte proliferation. We discuss in vitro and recent in vivo evidence for multifunctionality of these classic basic proteins of myelin, and argue for a systematic evaluation of the temporal and spatial distributions of these protein isoforms, and their modified variants, during oligodendrocyte differentiation.     

Role of phosphorylation in conformational adaptability of bovine myelin basic protein.

Controlled thrombic digestion of a preparation of components 2 + 3 isolated from the 18.5 kDa bovine myelin basic protein (MBP) yielded a polypeptide that was monophosphorylated on threonine 97 (component 3pT97). This is the first posttranslationally phosphorylated MBP isolated in pure form. We studied the effect of this single phosphate on the conformational adaptability of 18.5 kDa bovine MBP by comparing the circular dichroism (CD) spectrum of component 3pT97 with the spectra of highly purified nonphosphorylated components 1 and 2. The CD spectra of nonphosphorylated component 1 and component 2 [monodeamidated form(s) of component 1] were indistinguishable, while component 3pt97 exhibited a different spectrum. The singly phosphorylated MBP component exhibited 13% more ordered conformations than that adopted by nonphosphorylated MBP in dilute aqueous solutions. This was estimated from the CD spectra, and apparently involved about 17 additional amino acid residues in beta-structure(s).     

Solid-State NMR Spectroscopy of Membrane-Associated Myelin Basic Protein—Conformation and Dynamics of an Immunodominant Epitope

Myelin basic protein (MBP) maintains the tight multilamellar compaction of the myelin sheath in the central nervous system through peripheral binding of adjacent lipid bilayers of oligodendrocytes. Myelin instability in multiple sclerosis (MS) is associated with the loss of positive charge in MBP as a result of posttranslational enzymatic deimination. A highly-conserved central membrane-binding fragment (murine N81-PVVHFFKNIVTPRTPPP-S99, identical to human N83-S101) represents a primary immunodominant epitope in MS. Previous low-resolution electron paramagnetic resonance measurements on the V83-T92 fragment, with Cys-mutations and spin-labeling that scanned the epitope, were consistent with it being a membrane-associated amphipathic α-helix. Pseudodeimination at several sites throughout the protein, all distal to the central segment, disrupted the α-helix at its amino-terminus and exposed it to proteases, representing a potential mechanism in the autoimmune pathogenesis of MS. Here, we have used magic-angle spinning solid-state NMR spectroscopy to characterize more precisely the molecular conformation and dynamics of this central immunodominant epitope of MBP in a lipid milieu, without Cys-substitution. Our solid-state NMR measurements have revealed that the α-helix present within the immunodominant epitope is shorter than originally modeled, and is independent of the pseudodeimination, highlighting the importance of the local hydrophobic effects in helix formation and stability. The main effect of pseudodeimination is to cause the cytoplasmic exposure of the fragment, potentially making it more accessible to proteolysis. These results are the first, to our knowledge, to provide atomic-level detail of a membrane-anchoring segment of MBP, and direct evidence of decreased MBP-membrane interaction after posttranslational modification.     

And Yet it is Modified—Holding a Candle to the Dark Matter of White Matter

The multilamellar membrane myelin sheath of the CNS, that enwraps axons to facilitate saltatory conduction in higher vertebrates, is held together by myelin basic protein (MBP). Yet this generalization masks how enigmatic MBP is, much like cosmological “dark matter.” First, the casual use of the singular form for “protein” distracts that there are multiple, developmentally regulated “classic” splice isoforms ranging from 14 to 21.5 kDa, each with extensive PTMs. Second, the static image of MBP adhering two cytoplasmic leaflets of the oligodendrocyte membrane together in close apposition, suggests it to be inaccessible to modifying enzymes. And yet it is modified (to paraphrase Galileo's phrase on the earth's motion). In this issue of Proteomics, Sarg et al. apply an integrated CE–MS approach to investigate the PTMs of 18.5 kDa MBP from mouse brains of different ages. They identify new sites and types of modification, as well as confirming previously known PTMs. Innovative tools for unraveling the intricacies of the myelin basic proteome and how it organizes CNS myelin (much like basic histones organize chromatin), will help us understand white matter development and plasticity in health, during ageing, and in demyelinating diseases such as multiple sclerosis.     

Possible Hydrophobic Region in Myelin Basic Protein Consisting of an Orthogonally Packed β-Sheet

Theoretical analysis was carried out to determine how the approximately 20% of beta-structure observed in the 18.5 kilodalton (kDa) myelin basic protein (MBP) could be organized into a relatively stable beta-sheet. The beta-sheet is presumed to consist of the five most hydrophobic segments of polypeptide chain, which have beta-structure potential. These correspond approximately to sequences 15-21, 37-45, 84-92, 106-112, and 148-154 (rabbit MBP sequence numbering) and constitute beta-strands a, b, c, d, and e, respectively. A number of constraints are imposed upon the sheet; e.g., it should have the same topology in all MBP forms (21.5, 18.5, 17, and 14 kDa); strand e should lie at the sheet edge; strands b, c, and d should be ordered sequentially; the sheet formed by strands a, b, c, and d should be antiparallel; a maximum of the nonpolar surface area should be removed from the aqueous milieu; and charged side chains should be solvent-accessible. On the basis of these constraints it is possible to propose six orthogonally packed beta-sheets having different topologies. If strand e is restricted to an antiparallel alignment, the number of different sheets is reduced to four. Each of these sheets can form a relatively compact hydrophobic globular region. Two of the strands (a and e) can undergo transitions to alpha-helix without disrupting the structure of the remaining sheet bcd or producing major topologic rearrangements of the polypeptide chain.     

Interactions of the 18.5 kDA isoform of myelin basic protein with a2+-calmodulin: In vitro studies using gel shift assay

The interactions of the 18.5 kDa isoform of myelin basic protein (MBP) with calmodulin (CaM) in vitro have been investigated using glutaraldehyde or dithiobis[succinimidylpropionate] (DSP) cross-linking, and SDS-polyacrylamide gel electrophoresis. The following forms of MBP were used: the natural bovine C1 charge isomer (bMBP/C1) and a recombinant murine product (rmMBP), and their fragments generated by digestion with cathepsin D (EC 3.4.23.5). In physiological buffers (10 mM HEPES-NaOH, pH 7.4, 5 mM CaCl₂, 0.0035% glutaraldehyde; or 50 mM HEPES-NaOH, pH 7.4, 100 mM NaCl, 1 mM CaCl₂, 0.0035% DSP), MBP and CaM interacted primarily in a 1:1 molar ratio, consistent with previous studies that used 6 M urea, i.e. denaturing conditions. Moreover, the appearance of higher-order bands (not previously observed) suggested that the mechanism of interaction of the two proteins involved a series of relatively complex equilibria, resulting in 2:1 ratios of MBP to CaM. This observation would explain the cooperativity of association inferred from fluorescence studies [13]. Our results demonstrated further that the interaction involved the C-terminal domain of MBP, again in a primarily 1:1 molar ratio with CaM, consistent with our identification of a CaM-binding motif at the C-terminus.     

Assembly of tubulin by classic myelin basic protein isoforms and regulation by post-translational modification

Myelin basic protein (MBP), a highly cationic protein that maintains the structure of the myelin sheath, associates with tubulin in vivo. The in vitro assembly of tubulin by MBP was examined here using several assays. The unmodified C1 component of 18.5 kDa bovine MBP (bC1) assembled tubulin into microtubules in a dose-dependent manner via filamentous intermediates, and was able simultaneously to promote the formation of microtubule bundles. The critical tubulin concentration in the presence of bC1 was 0.69 +/- 0.05 microM. The effects of post-translational modifications (such as deamidation and phosphorylation) were assayed by comparing the bC1-bC6 components of 18.5 kDa bovine MBP; an increasing level of modification enhanced the ability of MBP to assemble tubulin. The effects of charge reduction via deimination were examined using recombinant murine isoforms emulating the unmodified C1 and deiminated C8 isoforms of 18.5 kDa MBP; both rmC1 and rmC8 exhibited a comparable ability to assemble tubulin. The effects of alternate exon recombination of the classic MBP variants were tested using the recombinant murine 21.5, 17.22, and 14 kDa isoforms. The isoforms containing regions derived from exon II of the classic MBP gene, 21.5 and 17.22 kDa MBP, showed no substantial difference in the extent of tubulin polymerization and bundling when compared to those of 18.5 kDa MBP. The 14 kDa isoform and two terminal deletion mutants of rmC1 were able to induce microtubule polymerization, but not bundling, to the same degree as the longer proteins. Finally, bC1 was shown to disrupt and aggregate planar sheets of crystalline tubulin stabilized by paclitaxel, establishing that these structures are not suitable substrates for the formation of MBP cocrystals.     

Detection of conformationally changed MBP using intramolecular FRET

The principal objective of this study was to explore protein conformational changes using fluorescence resonance energy transfer (FRET) technology. Maltose binding protein (MBP) was adopted as a target model, due to its well-characterized structure and ligand specificity. To the best of our knowledge, this is the first report to provide information regarding the biological distance between the two lobes of MBP upon maltose binding. For the FRET pair, ECFP and EYFP were used as the donor and the acceptor, and were linked genetically to the C-terminal and N-terminal regions of MBP (ECFP:MBP:EYFP), respectively. After the FRET reaction, maltose-treated MBP was shown to exhibit a considerable energy transfer (FRET efficiency (E) = ∼0.11, Distance (D) = ∼6.93 nm) at the ensemble level, which was regarded as reflective of the increase in donor quenching and the upshift in acceptor emission intensity, thereby suggesting that the donor and the acceptor had been brought close together as the result of structural alterations in MBP. However, upon glucose treatment, no FRET phenomenon was detected, thereby implying the specificity of interaction between MBP and maltose. The in vitro FRET results were also confirmed via the acceptor photobleaching method. Therefore, our data showed that maltose-stimulated conformational changes of MBP could be measured by FRET, thereby providing biological information, including the FRET efficiency and the intramolecular distance.     

Is myelin basic protein crystallizable?

Myelin basic protein (MBP) is the predominant extrinsic protein in both central and peripheral nervous system myelins. It is thought to be involved in the stabilizing interactions between myelin membranes, and it may play an important role in demyelinating diseases such as multiple sclerosis. In spite of the fact that this abundant protein has been known for almost three decades, its three-dimensional crystal structure has not yet been determined. In this study we report on our extensive attempts to crystallize the major 18.5 kDa isoform of MBP. We used MBP having different degrees of purity, ranging from crude MBP (that was acid or salt extracted from isolated myelin), to highest purity single isoform. We used conventional strategies in our search for a suitable composition or a crystallization medium. We applied both full and incomplete factorial searches for crystallization conditions. We analyzed the available data on proteins which have previously resisted crystallization, and applied this information to our own experiments. Nevertheless, despite our efforts which included 4600 different conditions, we were unable to induce crystallization of MBP. Previous work on MBP indicates that when it is removed from its native environment in the myelin membrane and put in crystallization media, the protein adopts a random coil conformation and persists as a population of structurally non-identical molecules. This thermodynamically preferred state presumably hinders crystallization, because the most fundamental factor of protein crystallization-homogeneity of tertiary structure-is lacking. We conclude that as long as its random coil flexibility is not suppressed, 18.5 kDa MBP and possibly also its isoforms will remain preeminent examples of proteins that cannot be crystallized.     

Triton X-100 extractions of central nervous system myelin indicate a possible role for the minor myelin proteins in the stability of lamellae

Isolated CNS myelin membranes were extracted with Triton X-100 under conditions previously established for the isolation of cytoskeletal proteins. Treated myelin retained much of its characteristic lamellar structure despite the removal of most of the major myelin basic protein (18.5 kDa) and the proteolipid protein, which together normally constitute 60% of the total myelin protein. The SDS-PAGE profile of this extract residue demonstrated an enrichment in proteins of Mr 30 to 60 kilodaltons (the Wolfgram group). The major myelin proteins were identified by antibodies on Western immunoblots, as were the 23-cyclic nucleotide 3-phosphodiesterase (CNP), actin, tubulin, myelin-associated glycoprotein (MGP) and the 21.5 kDa MBP. The overall behavior of CNP, the 21.5 kDa MBP, MGP and tubulin towards Triton extraction is reminiscent of the behavior of other membrane-skeletal complexes, supporting the idea that these and other minor myelin proteins might be part of heteromolecular complexes with interactions spanning several lamellae of the myelin sheath.     

Extensive degradation of myelin basic protein isoforms by calpain following traumatic brain injury

Axonal injury is one of the key features of traumatic brain injury (TBI), yet little is known about the integrity of the myelin sheath. We report that the 21.5 and 18.5-kDa myelin basic protein (MBP) isoforms degrade into N-terminal fragments (of 10 and 8 kDa) in the ipsilateral hippocampus and cortex between 2 h and 3 days after controlled cortical impact (in a rat model of TBI), but exhibit no degradation contralaterally. Using N-terminal microsequencing and mass spectrometry, we identified a novel in vivo MBP cleavage site between Phe114 and Lys115. A MBP C-terminal fragment-specific antibody was then raised and shown to specifically detect MBP fragments in affected brain regions following TBI. In vitro naive brain lysate and purified MBP digestion showed that MBP is sensitive to calpain, producing the characteristic MBP fragments observed in TBI. We hypothesize that TBI-mediated axonal injury causes secondary structural damage to the adjacent myelin membrane, instigating MBP degradation. This could initiate myelin sheath instability and demyelination, which might further promote axonal vulnerability.