Abstract
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.
Highlights
In the central nervous system (CNS), myelin is generated by oligodendrocytes and forms a multilamellar, periodic, lipid-rich structure that surrounds axons to facilitate saltatory conduction [1,2]
An 18.5 kDa myelin basic protein (MBP) segment upstream of the primary Src homology 3 (SH3)-ligand is involved in interaction We reasoned on the basis of our previous experience that constructing a smaller peptide of MBP that could still interact in a mode that is representative of the full-length protein could potentially ameliorate difficulties with aggregation, at the relatively high protein concentrations required for isothermal titration calorimetry (ITC) and NMR spectroscopy [10,11,58]
We first used in cellula imaging to define the minimal segments of MBP required for interaction with Fyn–SH3 as assessed by morphological phenotype of the immortalized N19-cell line, representing an early-developmental oligodendrocyte [10,44,45]
Summary
In the CNS (central nervous system), myelin is generated by oligodendrocytes and forms a multilamellar, periodic, lipid-rich structure that surrounds axons to facilitate saltatory conduction [1,2]. The adhesion of the cytoplasmic leaflets of compact myelin in the mature CNS is primarily carried out by MBP (myelin basic protein), which is a highly positively-charged, developmentally regulated protein family expressed from the gene in the oligodendrocyte lineage (Golli) [3,4]. As previously reviewed [4,6,7], the protein has many other binding partners: cytoskeletal proteins (actin, tubulin), Ca2 + -activated calmodulin, and proteins containing SH3 (Src homology 3)-domains The latter include mainly Fyn kinase [8,9,10,11], and ZO-1 (zonula occludens 1) and cortactin [12,13]. This protein’s multifunctionality in myelin is derived partly from its conformational plasticity as an IDP (intrinsically disordered protein) [6,7,14,15]
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