Conformation of bovine myelin basic protein purified with bound lipids

The basic protein of myelin (called MBP) is an extrinsic protein of the myelin membrane. Its structure and function are still unknown. MBP has been extensively studied in its water-soluble form, but it is also known in a detergent-soluble form, which is purified with endogenous myelin lipids and should correspond to the native form of the protein in the membrane. In order to acquire insight into the structure of MBP, we have carried out circular dichroism (CD) experiments on the protein both in the lipid-free and in the lipid-bound form. Our data clearly show that lipid-free MBP is mainly disordered with only a small amount having α-helix and β-sheet motifs. On the other hand, the lipid-bound form of MBP appears to have a consistent amount of ordered secondary structure. Theoretical predictions, made using different computational methods, substantially confirm the tendency of the protein to assume an ordered secondary structure in accordance with our CD results. Received: 13 November 1998 / Accepted: 1 February 1999     

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.     

Small Angle X-Ray Scattering from Lipid-Bound Myelin Basic Protein in Solution

The structure of myelin basic protein (MBP), purified from the myelin sheath in both lipid-free (LF-MBP) and lipid-bound (LB-MBP) forms, was investigated in solution by small angle x-ray scattering. The water-soluble LF-MBP, extracted at pH < 3.0 from defatted brain, is the classical preparation of MBP, commonly regarded as an intrinsically unfolded protein. LB-MBP is a lipoprotein-detergent complex extracted from myelin with its native lipidic environment at pH > 7.0. Under all conditions, the scattering from the two protein forms was different, indicating different molecular shapes. For the LB-MBP, well-defined scattering curves were obtained, suggesting that the protein had a unique, compact (but not globular) structure. Furthermore, these data were compatible with earlier results from molecular modeling calculations on the MBP structure which have been refined by us. In contrast, the LF-MBP data were in accordance with the expected open-coil conformation. The results represent the first direct structural information from x-ray scattering measurements on MBP in its native lipidic environment in solution.     

The secondary structure of myelin basic protein extracted by deoxycholate.

Because of the implication of myelin basic protein in some neurological diseases its in vivo structure is of particular interest. The protein is usually isolated using organic solvents and acid solutions and has previously been shown to contain little alpha-helical or beta-structure; but it is not known how the extraction methods influence the structure. Following recent observations that deoxycholate generally causes minimal structural perturbation when used to dissolve membrane proteins, this detergent has been used to extract the basic protein from bovine myelin. The protein contained in deoxycholate washes of myelin has been purified by gel chromatography and its secondary structure examined by circular dichroism spectroscopy. This protein and conventionally prepared bovine and human basic protein to which 1% deoxycholate has been added appear to have the same structure: they contain 8-14% more helical structure than the chloroform/methanol-extracted protein in pH 4.8 acetate buffer or in pH 9.15 Tris buffer. This conformational change is unaffected by addition of 0.25 M NaCl. The helical content will approach the upper limit if, as is expected, these ordered segments are short. It is suggested that basic protein may adopt this more ordered structure in myelin and possess activity not apparent in its water-soluble unordered conformation. Retention of its encephalitogenic activity following severe treatment may result from an ability to rapidly refold to the original conformation rather than from this activity being inherent in the unordered form.     

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.     

The interaction of zinc with membrane-associated 18.5 kDa myelin basic protein: an attenuated total reflectance-Fourier transform infrared spectroscopic study

Myelin basic protein (MBP) is an essential structural protein required for tight compaction of the myelin sheath of the central nervous system, and belongs to the family of intrinsically disordered proteins. It contains a high proportion of polar and charged amino acids, and has an adaptive conformation depending on its environment and binding surfaces (membranes) or partners (other proteins or small ligands including divalent cations). Zinc is an important stabilizing component of myelin and its concentration is substantially higher than that of any other trace element in the brain. In this study, we investigate the effect of zinc on different variants of 18.5 kDa MBP, including new recombinant forms lacking hexahistidine tags which would interfere with the binding of the cation. Isothermal titration calorimetry showed the dissociation constant to be in the micromolar range for all variants. Circular dichroism spectroscopy showed that there was minimal effect of zinc on the secondary structure on MBP in aqueous solution. When MBP was reconstituted with myelin-mimetic membranes, attenuated total reflectance-Fourier transform infrared spectroscopy revealed that there was a rearrangement of secondary structure components upon addition of zinc that was subtly different for each variant, indicative of a synergistic protein–membrane–cation interaction.     

Effects of the osmolyte trimethylamine-N-oxide on conformation,self-association,and two-dimensional crystallization of myelin basic protein

The osmolyte trimethylamine-N-oxide (TMAO) is a naturally in vivo occurring "chemical chaperone" that has been shown to stabilise the folding of numerous proteins. Myelin basic protein (MBP) is a molecule that has not yet been suitably crystallized either in three dimensions for X-ray crystallography or in two dimensions for electron crystallography. Here, we describe lipid monolayer crystallization experiments of two species of recombinant murine MBP in the presence of TMAO. One protein was unmodified, whereas the other contained six Arg/Lys-->Gln substitutions to mimic the effects of deimination (i.e., the enzymatic modification of Arg to citrulline), which reduces the net positive charge. Planar arrays of both proteins were formed on binary lipid monolayers containing a nickel-chelating lipid and a phosphoinositide. In the presence of TMAO, the diffraction spots of these arrays became sharper and more distinct than in its absence, indicating some improvement of crystallinity. The osmolyte also induced the formation of epitaxial growth of protein arrays, especially with the mutant protein. However, none of these assemblies was sufficiently ordered to extract high-resolution structural information. Circular dichroic spectroscopy showed that MBP gained no increase in ordered secondary structure in the presence of TMAO in bulk solution, whereas it did in the presence of lipids. Dynamic light-scattering experiments confirmed that the MBP preparations were monomodal under the optimal crystallization conditions determined by electron microscopy trials. The salt and osmolyte concentrations used were shown to result in a largely unassociated population of MBP. The amino acid composition of MBP overwhelmingly favours a disordered state, and a neural-network-based scheme predicted large segments that would be unlikely to adopt a regular conformation. Thus, this protein has an inherently disordered nature, which mitigates strongly against its crystallization for high-resolution structure determination.     

Backbone dynamics of the 18.5 kDa isoform of myelin basic protein reveals transient alpha-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.     

Shark Myelin Basic Protein: Amino Acid Sequence, Secondary Structure, and Self-Association

Myelin basic protein (MBP) from the Whaler shark (Carcharhinus obscurus) has been purified from acid extracts of a chloroform/methanol pellet from whole brains. The amino acid sequence of the majority of the protein has been determined and compared with the sequences of other MBPs. The shark protein has only 44% homology with the bovine protein, but, in common with other MBPs, it has basic residues distributed throughout the sequence and no extensive segments that are predicted to have an ordered secondary structure in solution. Shark MBP lacks the triproline sequence previously postulated to form a hairpin bend in the molecule. The region containing the putative consensus sequence for encephalitogenicity in the guinea pig contains several substitutions, thus accounting for the lack of activity of the shark protein. Studies of the secondary structure and self-association have shown that shark MBP possesses solution properties similar to those of the bovine protein, despite the extensive differences in primary structure.     

The Basic Protein of CNS Myelin: Its Structure and Ligand Binding

Consideration of the evidence presented in this review leads to the following conclusions: (a) Isolated MBP in aqueous solution has little ordered secondary or tertiary structure. (b) In this state, the protein can associate with a wide range of hydrophobic and amphiphilic compounds, these interactions involving limited sections of the protein. (c) The strength of binding to bilayers and the accompanying conformational changes in the protein are greatest for systems containing acidic lipids, presumably because of the involvement of ionic interactions. (d) When bound to bilayers of acidic lipids, MBP will have substantially more ordered secondary structure than it manifests in aqueous solution, and it is likely to be oligomeric (possibly hexameric). (e) MBP does affect the organization of lipid aggregates. It influences strongly the separation of bilayers in multilayers of purified lipids, and at present this must be viewed as its prime role within myelin. The greatest impediment to our understanding of MBP is the lack of an assayable biological activity. In contrast to the situation with enzymes, for example, we have no functional test for changes in protein structure or changes accompanying interactions with other molecules. Current evidence suggests that the protein has a structural role within myelin and that its own three-dimensional structure is strongly dependent on the molecules with which it is associated. If this picture is correct, studies of the isolated protein or of the protein in reconstituted lipid systems may yield, at best, a rough guide to the structure within its biological environment. Further clarification of the structure and function of MBP may have to await development of more powerful techniques for studying proteins bound to large molecular aggregates, such as lipid bilayers. The paucity of generally applicable methods is reflected in the fact that even low resolution structures are known for only a handful of intrinsic membrane proteins, and even more limited information exists for proteins associated with membrane surfaces. However, the increasing use of a combination of electron microscopy and diffraction on two-dimensional arrays of proteins formed on lipid bilayers (Henderson et al., 1990) offers the hope that it may not be too long before it will be possible to study at moderate resolution the three-dimensional structure of MBP bound to a lipid membrane.     

Structure and function of the proline-rich region of myelin basic protein

Myelin basic protein (MBP)--the major extrinsic membrane protein of central nervous system myelin--from several species contains a rarely encountered highly conserved triproline segment as residues 99-101 of its 170-residue sequence. Cis peptide bonds are known to arise at X-Pro junctions in proteins and may be of functional significance in protein folding, chain reversal, and/or maintenance of tertiary structure. We have examined the conformation of this proline-rich region using principally 13C nuclear magnetic resonance spectroscopy (125 MHz) both in intact bovine MBP and in several MBP fragment peptides which we synthesized, including octapeptide 97-104 (Arg-Thr-Pro-Pro-Pro-Ser-Gln-Gly). Results suggested an all-trans conformation in aqueous solution for the triproline segment in MBP hexapeptide (99-104), heptapeptide (98-104), and octapeptide. Comparison with the 13C spectrum of intact MBP (125 MHz) suggested that the proline-rich region, as well as all other X-Pro MBP peptide junctures, was also essentially all trans in aqueous solution. Although experiments in which octapeptide 97-104 was bound to a lipid preparation (4:1 dipalmitoylphosphatidylcholine/dimyristoylphosphatidic acid) demonstrated that cis-proline bonds do arise (to the extent of ca. 5%) in the membrane environment, a role of linear chain propagation is suggested for the triproline segment of myelin basic protein.     

Protein kinase C in rat brain myelin

It has been suggested that phosphorylation of myelin basic protein (MBP) in CNS is catalyzed by protein kinase C (PKC). In order to demonstrate that PKC in the myelin phosphorylates MBP, PKC was partially purified from rat CNS myelin by solubilization with Triton X-100 followed by a DEAE-cellulose column. MBP and histone III-S were phosphorylated in the presence of Ca²⁺ and phospholipid by rat myelin PKC. High voltage electrophoresis revealed that the phosphoamino acids in MBP by this kinase was serine residue, which is known to be the amino acid phosphorylated by PKC. The activity of PKC extracted from myelin was inhibited by the addition of psychosine to the incubation mixture. To confirm the presence of PKC molecule and to identify the isoform of PKC in the myelin, the solubilized myelin fraction was applied on SDS-PAGE, transferred to a nitrocellulose sheet and stained with anti-PKC monoclonal antibodies. Rat CNS myelin contained the PKC of about 80 kDa (intact PKC), and no proteolytic fragments were observed. PKC isozymes in myelin were type II and III. A developmental study from 14 to 42 postnatal days showed that PKC activity in CNS myelin seemed to parallel the deposition of myelin protein.     

Spectroscopic assessment of secondary and tertiary structure in myelin basic protein

Myelin basic protein conformation and hydrophobicity, along with the protein's behavior in the presence of the fluorescent probe 6-(p-toluidino)-2-naphthalenesulfonate, have been studied by using Fourier transform infrared (FT-IR) and Raman spectroscopy. The FT-IR and Raman spectra provided compelling evidence for the presence of a small amount of beta structure, ca. 25%, in the aqueous solution and solid-state forms of myelin basic protein. The enhanced fluorescence and shift in the emission maximum of 6-(p-toluidino)-2-naphthalenesulfonate when bound to myelin basic protein are consistent with the presence of at least one hydrophobic region in the molecule. Loss of the fluorescence enhancement in the presence of denaturing agents indicates that native myelin basic protein has a folded structure in solution. All of the results provide support for conformational predictions derived from the application of Edmundson wheels to the primary structure.     

Peptide binding inhibits aggregation of soluble MHC class II in solution

Affinity-purified major histocompatability complex (MHC) class II molecules are known to bind antigenic peptide in vitro. This peptide-bound MHC class II is known to undergo a change in structure upon stable binding of antigenic peptide. Previous results from our, and other laboratories, have suggested a relationship between MHC class II structure and peptide association that enables class II to enter into a stable conformation upon peptide binding. In this report we describe that stable binding of high-affinity antigenic peptide to MHC class II molecule results in transition of aggregated purified MHC class II proteins to a stable heterodimeric state. Such transition was demonstrated by using purified human HLA-DR2 class II molecule and high-affinity myelin basic protein (MBP) 83-102)Y83 peptide. Highly aggregated purified DR2 (high molecular weight; HMW) was first separated from heterodimer (low molecular weight: LMW) in the presence of 50-fold molar excess of MBP(83-102)Y83 peptide. We then show that the aggregated HMW preparation can be successfully converted into a stable dimer by further incubation with MBP(83-102)Y83 and changing various binding parameters such as pH, temperature, reducing agent, and peptide concentrations. Under optimized conditions, the highly aggregated inactive DR2 molecules can be completely loaded with the antigenic peptide. The transformed heterodimers with bound peptide prepared by this method are biologically active, as shown by their ability to induce the production of gamma-interferon by SS8T-transformed human T cells. These results suggest that in solution, MHC class II molecules may be aggregated in the absence of bound peptide. Such aggregated MHC class II molecules can be converted to stable and biologically active heterodimers in the presence of high-affinity antigenic peptide.     

Specificity of zinc binding to myelin basic protein

Z²⁺ appears to stabilize the myelin sheath but the mechanism of this effect is unknown. In a previous report we have shown that zinc binds to CNS myelin basic protein (MBP) in the presence of phosphate and this results in MBP aggregation. For this paper we used a solid phase zinc blotting assay to identify which myelin proteins bind zinc. MBP and a 58 kDa band were found to be the major targets of⁶⁵Zn binding. Moreover, using fluorescence, light scattering and electron microscopy we investigated the binding of zinc and other cations to purified MBP in solution. Among the cations tested for their ability to interfere with the binding of zinc, the most effective were cadmium, mercury and copper, but only cadmium and mercury increased the scattering intensity, whereas MBP aggregation was not inhibited by copper ions. Thus, the effect of zinc on the formation of MBP clusters seems to be specific.     

Racemization of Individual Aspartate Residues in Human Myelin Basic Protein

Human myelin basic protein (MBP), a long-lived brain protein, undergoes gradual racemization of its amino acids, primarily aspartic acid and serine. Purified protein was treated at neutral pH with trypsin to yield peptides that were separated by HPLC using a C18 column. Twenty-nine peptides were isolated and analyzed for amino acid composition and aspartate racemization. Each aspartate and asparagine in the protein was racemized to a different extent, ranging from 2.2 to 17.1% D isomer. When the racemization was examined in terms of the beta-structure model of MBP, a correlation was observed in which six aspartate/asparagine residues assumed to be associated with myelin membrane lipids showed little racemization (2.2-4.9% D isomer), whereas five other aspartate residues were more highly racemized (9.9-17.1% D isomer). Although the observed aspartate racemization may be related to steric hindrance by neighboring residues and/or the protein secondary structure, interaction of aspartates with membrane lipids may also be a major factor. The data are compatible with a model in which each MBP molecule interacts with adjacent cytoplasmic layers of myelin membrane through a beta-sheet on one surface and loops and helices on the other surface, thereby stabilizing the myelin multilamellar structure.     

Force measurements on myelin basic protein adsorbed to mica and lipid bilayer surfaces done with the atomic force microscope.

The mechanical and adhesion properties of myelin basic protein (MBP) are important for its function, namely the compaction of the myelin sheath. To get more information about these properties we used atomic force microscopy to study tip-sample interaction of mica and mixed dioleoylphosphatidylserine (DOPS) (20%)/egg phosphatidylcholine (EPC) (80%) lipid bilayer surfaces in the absence and presence of bovine MBP. On mica or DOPS/EPC bilayers a short-range repulsive force (decay length 1.0-1.3 nm) was observed during the approach. The presence of MBP always led to an attractive force between tip and sample. When retracting the tip again, force curves on mica and on lipid layers were different. While attached to the mica surface, the MBP molecules exhibited elastic stretching behavior that agreed with the worm-like chain model, yielding a persistence length of 0.5 +/- 0.25 nm and an average contour length of 53 +/- 19 nm. MBP attached to a lipid bilayer did not show elastic stretching behavior. This shows that the protein adopts a different conformation when in contact with lipids. The lipid bilayer is strongly modified by MBP attachment, indicating formation of MBP-lipid complexes and possibly disruption of the original bilayer structure.     

Phosphorylation of myelin basic protein and peptides by ganglioside-stimulated protein kinase

Rabbit myelin basic protein (MBP) was phosphorylated by a ganglioside-stimulated protein kinase to a stoichiometry of 1.4 and 2.1 mol phosphate/mol MBP in the presence and absence of GTlb, respectively. Two-dimensional peptide mapping analyses revealed that two of the sites of phosphorylation were distinct from those catalyzed by cAMP-dependent protein kinase or protein kinase C. Phosphorylation of one of these sites by ganglioside-stimulated protein kinase was inhibited by GTlb, suggesting that the inhibitory effect of gangliosides on MBP phosphorylation may be substrate-directed. Although ganglioside-stimulated protein kinase did not phosphorylate MBP at a domain containing residues 82-117, a synthetic peptide Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys corresponding to residues 111-120 was phosphorylated by the kinase in a ganglioside-stimulated manner. These findings suggest that the conformation of MBP may be important in determining its phosphorylatability.     

A novel metalloproteinase associated with brain myelin membranes. Isolation and characterization

Brain myelin membrane preparations contain a metalloproteinase activity which degrades myelin basic protein (MBP). The activity was associated with lentil lectin-binding glycoproteins solubilized from myelin and could be detected in the presence of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS). The metalloproteinase represented about 5% of this glycoprotein fraction and was isolated from it by chromatography on DEAE-Sephacel, CM-Sepharose, and Superose 6. The proteinase had an apparent relative molecular weight (Mr) of approximately 58,000 both by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr value was unaffected by the presence of reducing agents but was diminished to about 52,000 by treating the proteinase with endoglycosidase F. The purified proteinase cleaved many bonds in MBP but did not generate trichloroacetic acid-soluble products. Two major polypeptides, putatively MBP1-73 and MBP74-170, were prominent in digests of MBP by either the purified enzyme or myelin membranes. The proteinase was active between pH 7 and 9 and was inhibited by phenanthroline and dithiothreitol but not phosphoramidon or inhibitors of serine or cysteine proteinases. Histones, but not azocasein, also served as substrates for the proteinase. From its enzymic and molecular characteristics the myelin-derived metalloproteinase appears distinct from previously described enzymes.     

Differential racemization of aspartate and serine in human myelin basic protein

L-Aspartate and L-serine were found to undergo amino acid racemization in brain myelin basic protein (MBP) of aging humans. The observed racemization was different in each chromatographically purified MBP isoform. Pepsin digestion of MBP produced three peptides, each of which exhibited different degrees of racemization of the same amino acids. MBP isolated by the same method from rat and guinea pig brain showed little accumulation of D-amino acids. Total MBP isolated from SDS-polyacrylamide disc gel electrophoresis of total human myelin proteins (delipidated myelin) was racemized to the same extent as purified MBP, indicating that the racemization observed was not an artifact of the isolation procedure. Myelin proteolipid protein from the same gel was racemized approximately half as much as MBP. The age and environment of the aspartates and serines in myelin proteins may strongly affect their observed racemizations.