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-d(2)), particularly to analyze its secondary structure using chemical shift indexing, and to investigate its backbone dynamics using (15)N spin relaxation measurements. Collectively, the data revealed three major segments of the protein with a propensity toward alpha-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 alpha-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.
Libich
David S
DS
Department of Molecular and Cellular Biology, and Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada.
Harauz
George
G
D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry* / Q000648:ultrastructure*; D003198:Computer Simulation; D008956:Models, Chemical; D008958:Models, Molecular; D009186:Myelin Sheath / Q000737:chemistry* / Q000648:ultrastructure*; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000648:ultrastructure
D002147:Calmodulin; D020033:Protein Isoforms