Membrane Association Landscape of Myelin Basic Protein Portrays Formation of the Myelin Major Dense Line

Arne Raasakka,Salla Ruskamo,Petri Kursula,Anne S Ulrich,Anne Baumann,Jussi Tuusa,Matti Myllykoski,Robert Barker,Anne Martel,Jochen Bürck,Henning Stahlberg,Julia Kowal

doi:10.1038/s41598-017-05364-3

Abstract

Compact myelin comprises most of the dry weight of myelin, and its insulative nature is the basis for saltatory conduction of nerve impulses. The major dense line (MDL) is a 3-nm compartment between two cytoplasmic leaflets of stacked myelin membranes, mostly occupied by a myelin basic protein (MBP) phase. MBP is an abundant myelin protein involved in demyelinating diseases, such as multiple sclerosis. The association of MBP with lipid membranes has been studied for decades, but the MBP-driven formation of the MDL remains elusive at the biomolecular level. We employed complementary biophysical methods, including atomic force microscopy, cryo-electron microscopy, and neutron scattering, to investigate the formation of membrane stacks all the way from MBP binding onto a single membrane leaflet to the organisation of a stable MDL. Our results support the formation of an amorphous protein phase of MBP between two membrane bilayers and provide a molecular model for MDL formation during myelination, which is of importance when understanding myelin assembly and demyelinating conditions.

Highlights

Myelin basic protein (MBP) is one of the crucial factors in CM membrane stacking in the CNS, its 18.5-kDa isoform being most abundant[3]
To overcome problems arising from construct design or contaminants and heterogeneity in tissue extracts, we used untagged recombinant murine myelin basic protein (MBP) corresponding to the major 18.5-kDa isoform, to other recent studies18, 22–24. recombinant tag-free MBP (rMBP) appeared as a single band in denaturing gel electrophoresis (SDS-PAGE) and monomeric and monodisperse in size-exclusion chromatography (SEC) and dynamic light scattering (DLS), with a hydrodynamic radius (Rh) of 3.5 nm (Supplementary Fig. S1)
Using a recombinant form of MBP accurately mimicking the major 18.5-kDa isoform, we have provided a full physicochemical picture of MBP-induced stacking of lipid bilayers

Summary

Introduction

Myelin basic protein (MBP) is one of the crucial factors in CM membrane stacking in the CNS, its 18.5-kDa isoform being most abundant[3]. The high positive net charge of MBP is related to the intrinsically disordered conformation of MBP in solution; on the other hand, it allows MBP to interact with the phospholipid-rich cytoplasmic face of myelin membranes. This close interaction results in charge neutralisation, folding, and partial membrane insertion[5]. MBP has been suggested to form an ordered, self-assembled protein meshwork of either anti-parallel or stacked MBP molecules with confined degrees of freedom between myelin cytoplasmic leaflets[9, 12, 13]. Our results illustrate a step-wise formation of the MDL at the biomolecular scale

Methods

Results

Conclusion