The structure of myelin basic protein determined by high resolution electron microscopy and molecular modelling

Abstract

Multiple sclerosis (MS) is characterised by the active degradation of the myelin sheath, and is thought to be precipitated by an autoimmune response to one or more of the sheath’s protein components. Myelin basic protein (MBP) is generally considered to be the antigen responsible for autoimmunity and is one of the most abundant proteins of the sheath. Knowledge of the tertiary structure of MBP, and its organisation on lipid layers and within the compacted myelin multilayers, is essential to understanding the organisation of the myelin membrane and the mechanisms of development of autoimmunity in MS. MBP has hitherto not proved crystallisable for structural studies by X-ray diffractometry despite much effort in this area. We have initiated high-resolution electron microscopical analyses of MBP. When imaged as individual particles, a recurring motif is that of a “C” shape in projection, of approximately 2 nm in thickness and 8 nm in length (Figure a – field of view; Figure b – class averages after single particle analysis; Figure c – 3D reconstruction). The total length of this particle is less than would be expected if the polypeptide chain were fully extended. Crystallisation experiments on MBP using monolayers of lipid mixtures (phosphatidylcholine, phosphatidylserine, dioleoyl phosphatidylethanolamine, galactocerebroside) yielded microcrystalline structures with a repeating pattern of packed protein molecules of size 5 nm, with an arm width of 2 nm and total length of 10-11 nm (Figure d). Molecular modelling of MBP based on the schemes proposed by Stoner and Martenson, and using coordinates of the β sheet backbone of bacteriochlorophyll A protein and energy minimisation of intervening segments, yielded a compact structure of size and appearance consistent with the electron microscopical data (Figure e). The bend of the C shape appears due to a natural curvature in the β sheet. These results are fully congruous with MBP having a regular secondary structure and domains that are potentially flexible with respect to one another. Energy minimisation experiments were performed on this model structure after specific post-translational modifications such as phosphorylation of specific sites (e.g., Ser7, Thr98) and conversion of arginine residues to citrullines. These results are consistent with biochemical data on accessibility of these sites to modification, and provide a framework for hypothesising scenarios of myelin sheath degradation in MS.