👤 George Harauz

Myelin basic protein (MBP), specifically the 18.5 kDa isoform, is a peripheral membrane protein and a major component of mammalian central nervous system myelin. It is an intrinsically disordered and multifunctional protein that binds cytoskeletal and other cytosolic proteins to a membrane surface and thereby acquires ordered structure. These associations are modulated by post-translational modifications of MBP, as well as by interactions of MBP with Ca(2+)-calmodulin (CaM). Enzymatic deimination of usually six arginine residues to citrulline results in a decrease in the net positive charge of the protein from 19 to ≤13. This deiminated form is found in greater amounts in normal children and in adult patients with the demyelinating disease multiple sclerosis. In this paper, we examine the secondary structure of a calmodulin-binding domain, residues A141-L154, when associated with a lipid bilayer in recombinant murine 18.5 kDa forms rmC1 (unmodified) and rmC8 (pseudodeiminated). We demonstrate here by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy that the Y142-L154 segment in membrane-associated rmC1 forms an amphipathic α-helix, with high accessibility to O(2) and low accessibility to NiEDDA. In membrane-associated rmC8, this segment assumed a structure distorted from an α-helix. Spin-labeled residues in rmC1 in solution were more immobilized on binding Ca(2+)-CaM than those in rmC8. Furthermore, rmC8 was dissociated more readily from a lipid bilayer by Ca(2+)-CaM than was rmC1. These results confirm both a predicted induced ordering upon membrane association in a specific segment of 18.5 kDa MBP, and that this segment is a CaM-binding site, with both interactions weakened by deimination of residues outside of this segment. The deiminated form would be more susceptible to regulation of its membrane binding functions by Ca(2+)-CaM than the unmodified form. Show less

The 18.5 kDa myelin basic protein (MBP), the most abundant splice isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the myelin sheath in the central nervous system. Protein deimination and phosphorylation are two key posttranslational modifications whose balance determines local myelin microdomain stability and function. It has previously been shown that MBP in solution causes both polymerization of G-actin to F-actin and bundling of the microfilaments, and binds them to a negatively charged membrane. However, the binding parameters, and the roles of different possible interacting domains of membrane-associated MBP, have not yet been investigated. Here, we compared the interaction of unmodified (rmC1) and pseudodeiminated (rmC8) recombinant murine MBP (full-length charge variants), and of two terminal deletion variants (rmDeltaC and rmDeltaN), with actin in the presence of DPC (dodecylphosphocholine) to mimic a membrane environment. Our results show that although both charge variants polymerized and bundled actin, the maximal polymerization/bundling due to rmC1 occurred at a lower molar ratio compared to rmC8. In the presence of DPC, rmC1 appeared to be more active than rmC8 in its ability to polymerize and bundle actin, and the binding affinity of both charge variants to G-actin became higher. Moreover, of the two deletion variants studied in the presence of DPC, the one lacking the C-terminal domain (rmDeltaC) was more active compared to the variant lacking the N-terminal domain (rmDeltaN) but exhibited weaker binding to actin. Thus, whereas the N-terminal domain of MBP can be more important for the MBP's actin polymerization activity and membrane-association, the C-terminal domain can regulate its interaction with actin. Show less

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and with cytoskeletal and other proteins. A central segment of MBP is highly conserved in mammals and consists of a membrane surface-associated amphipathic alpha-helix, immediately followed by a proline-rich segment that we hypothesize is an SH3 ligand. We show by circular dichroic spectroscopy that this proline-rich segment forms a polyproline type II helix in vitro under physiological conditions and that phosphorylation at a constituent threonyl residue has a stabilizing effect on its conformation. Using SH3 domain microarrays, we observe that the unmodified recombinant murine 18.5 kDa MBP isoform (rmC1 component) binds the following SH3 domains: Yes1 > PSD95 > cortactin = PexD = Abl = Fyn = c-Src = Itk in order of decreasing affinity. A quasi-deiminated form of the protein (rmC8) binds the SH3 domains Yes1 > Fyn > cortactin = c-Src > PexD = Abl. Phosphorylation of rmC1 at 1-2 threonines within the proline-rich segment by mitogen-activated protein kinase in vitro has no effect on the binding specificity to the SH3 domains on the array. An SH3 domain of chicken Fyn is also demonstrated to bind to lipid membrane-associated C1, phosphorylated C1, and rmC8. Molecular docking simulations of the interaction of the putative SH3 ligand of classic MBP with the human Fyn SH3 domain indicate that the strength of the interaction is of the same order of magnitude as with calmodulin and that the molecular recognition and association is mediated by some weak CH...pi interactions between the ligand prolyl residues and the aromatic ones of the SH3 binding site. One such interaction is well-conserved and involves the stacking of an MBP-peptide prolyl and an SH3 domain tryptophanyl residue, as in most other SH3-ligand complexes. Lysyl and arginyl residues in the peptide canonically interact via salt bridges and cation-pi interactions with negatively charged and aromatic residues in the SH3 domain binding site. Posttranslational modifications (phosphorylation or methylation) of the ligand cause noticeable shifts in the conformation of the flexible peptide and its side chains but do not predict any major inhibition of the binding beyond somewhat less favorable interactions for peptides with phosphorylated seryl or threonyl residues. Show less

The degradation of myelin in the CNS is the hallmark of multiple sclerosis. Reduction in the net positive charge of myelin basic protein (MBP), through deimination, correlates strongly with disease severity and may mediate myelin instability and loss of compaction. Using Cys scanning, spin labeling, EPR spectroscopy, and site-specific proteolysis, we show that in the membrane-bound state the primary immunodominant epitope, V83-T92, of the less cationic recombinant murine MBP C8 mimic (rmC8) forms a more highly surface-exposed and shorter amphipathic alpha-helix than in the unmodified form, recombinant murine MBP C1 mimic (rmC1), analogous to the most cationic and abundant isomer of MBP in normal myelin. Moreover, cathepsin D digested lipid-associated rmC8 3-fold faster than rmC1, and cleavage at F86-F87 occurred more readily in rmC8 than rmC1. These findings suggest a mechanism for initial loss of myelin stability and the autoimmune pathogenesis of multiple sclerosis. Show less

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. Show less

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. Show less