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.

Effect of arginine loss in myelin basic protein, as occurs in its deiminated charge isoform, on mediation of actin polymerization and actin binding to a lipid membrane in vitro.

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocyte membranes and is most likely responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also polymerize actin, bundle F-actin filaments, and bind actin filaments to lipid bilayers through electrostatic interactions. MBP consists of a number of posttranslationally modified isoforms of varying charge, including C8, in which six arginines are deiminated to the uncharged residue citrulline. The deiminated form decreases with development, but is increased in patients with the demyelinating disease multiple sclerosis. Here we investigate the effect of decreased net positive charge of MBP on its interaction with actin in vitro by comparing a recombinant murine form, rmC1, of the most highly charged unmodified isoform, C1, and a recombinant analogue of C8 in which six basic residues are converted to glutamine, rmC8. The dissociation constant of the less charged isoform rmC8 for actin was a little greater than that of rmC1, and rmC8 had somewhat reduced ability to polymerize actin and bundle F-actin filaments than rmC1. Moreover, rmC8 was more readily dissociated from actin by Ca(2+)-calmodulin than rmC1, and the ability of the deiminated isoform to bind actin to lipid bilayers was reduced. These results indicate that electrostatic forces are the primary determinant of the interaction of MBP with actin. The spin labeled side chains of a series of rmC1 and rmC8 variants containing single Cys substitutions at seven sites throughout the sequence all became motionally restricted to a similar degree on binding F-actin, indicating that the entire sequence is involved in interacting with actin filaments or is otherwise structurally constrained in actin bundles. Thus, this posttranslational modification of MBP, which occurs early in life and is increased in multiple sclerosis, attenuates the ability of MBP to polymerize and bundle actin, and to bind it to a negatively charged membrane.

Charge effects modulate actin assembly by classic myelin basic protein isoforms.

Myelin basic protein (MBP), a highly cationic structural protein of the myelin sheath, is believed to be associated with the cytoskeleton in vivo and interacts with actin in vitro, but little is known about the regulation of this interaction. The rate and extent of actin polymerization induced by 18.5 kDa MBP charge isomers were correlated to charge reduction by post-translational modifications. Increased ionic strength attenuated the initial rate but not the final extent of polymerization achieved. Reduced pH enhanced the rate and extent of polymerization, presumably via partial protonation of intrinsic histidyl residues. The polymerizing activities of the 21.5, 17, and 14 kDa MBP splice variants were not proportionate to their net charges or charge densities. The presence of at least one region derived from exon II or VI of the "classic" MBP gene was required for effective bundling as assessed by light scattering and transmission electron microscopy.

Myelin basic protein-diverse conformational states of an intrinsically unstructured protein and its roles in myelin assembly and multiple sclerosis.

The 18.5 kDa isoform of myelin basic protein (MBP) is a major component of the myelin sheath in the central nervous system of higher vertebrates, and a member of a larger family of proteins with a multiplicity of forms and post-translational modifications (PTMs). The 18.5 kDa protein is the exemplar of the family, being most abundant in adult myelin, and thus the most-studied. It is peripherally membrane-associated, but has generally been investigated in isolated form. MBP is an 'intrinsically unstructured' protein with a high proportion (approximately 75%) of random coil, but postulated to have core elements of beta-sheet and alpha-helix. We review here the properties of the MBP family, especially of the 18.5 kDa isoform, and discuss how its three-dimensional (3D) structure may be resolved by direct techniques available to us, viz., X-ray and electron crystallography, and solution and solid-state NMR spectrometry. In particular, we emphasise that creating an appropriate environment in which the protein can adopt a physiologically relevant fold is crucial to such endeavours. By solving the 3D structure of 18.5 kDa MBP and the effects of PTMs, we will attain a better understanding of myelin architecture, and of the molecular mechanisms that transpire in demyelinating diseases such as multiple sclerosis.

Myelin basic protein has multiple calmodulin-binding sites.

Myelin basic protein (MBP) has been shown to bind calmodulin (CaM) in a specific Ca(2+)-dependent manner via a primary target sequence at its C-terminus [Protein Sci. 12 (2003) 1507]. Upon deimination of MBP, the nature of the interaction changed significantly, suggesting either a new binding site or different conformers with different affinities for CaM. In order to resolve this issue, we investigated here the CaM-binding properties of N- and C-terminal deletion mutants of MBP using Trp fluorescence spectroscopy and mass spectrometry. We conclude that there is an additional CaM-binding site on MBP in a central segment (we posit murine residues 82-93) that forms an amphipathic alpha-helix.

Interaction of the 18.5-kD isoform of myelin basic protein with Ca2+ -calmodulin: effects of deimination assessed by intrinsic Trp fluorescence spectroscopy, dynamic light scattering, and circular dichroism.

The effects of deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) on its binding to calmodulin (CaM) have been examined. Four species of MBP were investigated: unmodified recombinant murine MBP (rmMBP-Cit(0)), an engineered protein with six quasi-citrullinyl (i.e., glutaminyl) residues per molecule (rmMBP-qCit(6)), human component C1 (hMBP-Cit(0)), and human component C8 (hMBP-Cit(6)), both obtained from a patient with multiple sclerosis (MS). Both rmMBP-Cit(0) and hMBP-Cit(0) bound CaM in a Ca(2+)-dependent manner and primarily in a 1:1 stoichiometry, which was verified by dynamic light scattering. Circular dichroic spectroscopy was unable to detect any changes in secondary structure in MBP upon CaM-binding. Inherent Trp fluorescence spectroscopy and a single-site binding model were used to determine the dissociation constants: K(d) = 144 +/- 76 nM for rmMBP-Cit(0), and K(d) = 42 +/- 15 nM for hMBP-Cit(0). For rmMBP-qCit(6) and hMBP-Cit(6), the changes in fluorescence were suggestive of a two-site interaction, although the dissociation constants could not be accurately determined. These results can be explained by a local conformational change induced in MBP by deimination, exposing a second binding site with a weaker association with CaM, or by the existence of several conformers of deiminated MBP. Titration with the collisional quencher acrylamide, and steady-state and lifetime measurements of the fluorescence at 340 nm, showed both dynamic and static components to the quenching, and differences between the unmodified and deiminated proteins that were also consistent with a local conformational change due to deimination.

Terminal deletion mutants of myelin basic protein: new insights into self-association and phospholipid interactions.

The 18.5kDa isoform of myelin basic protein (MBP) has strong and probably specific interactions with phosphoinositides that are of interest regarding this protein's function, and in effecting its two-dimensional crystallization for structural determination. We have designed and constructed truncation mutants of recombinant 18.5kDa murine myelin basic protein (rmMBP) lacking either the N- or C-terminal third, i.e. rmMBPDeltaN and rmMBPDeltaC, respectively. Both variants rmMBPDeltaC and rmMBPDeltaN generally had a reduced ability to aggregate lipid vesicles, compared to the whole protein, especially at lower protein/lipid ratios. Lipid vesicle cosedimentation showed that both truncated variants exhibited altered binding with phosphatidylinositol (PI). Incubation of these proteins under monolayers comprising PI and a nickel-chelating lipid yielded crystalline arrays of rmMBPDeltaC (but not rmMBPDeltaN) in the absence of high salt or osmolytes, which are required for crystallization of whole protein. This result suggests that the C-terminal segment of MBP is a significant source of conformational heterogeneity, and its removal will facilitate future planar or three-dimensional crystallization attempts. Incubation of rmMBPDeltaN and rmMBPDeltaC under monolayers comprising phosphatidylinositol-4-phosphate and a nickel-chelating lipid yielded tubular structures of opposite chirality, suggesting a synergistic effect of both termini of MBP in organizing myelin lipids.