Molecular structure and function of myelin protein P0 in membrane stacking

Abstract

Compact myelin forms the basis of nerve insulation essential for higher vertebrates. Dozens of myelin membrane bilayers undergo tight stacking, and in the peripheral nervous system, this is partially enabled by myelin protein zero (P0). Consisting of an immunoglobulin (Ig)-like extracellular domain, a single transmembrane helix, and a cytoplasmic extension (P0ct), P0 harbours an important task in ensuring the integrity of compact myelin in the extracellular compartment, referred to as the intraperiod line. Several disease mutations resulting in peripheral neuropathies have been identified for P0, reflecting its physiological importance, but the arrangement of P0 within the myelin ultrastructure remains obscure. We performed a biophysical characterization of recombinant P0ct. P0ct contributes to the binding affinity between apposed cytoplasmic myelin membrane leaflets, which not only results in changes of the bilayer properties, but also potentially involves the arrangement of the Ig-like domains in a manner that stabilizes the intraperiod line. Transmission electron cryomicroscopy of native full-length P0 showed that P0 stacks lipid membranes by forming antiparallel dimers between the extracellular Ig-like domains. The zipper-like arrangement of the P0 extracellular domains between two membranes explains the double structure of the myelin intraperiod line. Our results contribute to the understanding of PNS myelin, the role of P0 therein, and the underlying molecular foundation of compact myelin stability in health and disease.

Myelin Basic Protein dynamics from out-of-equilibrium functional state to degraded state in myelin

“…Correspondingly, the fluctuations RSD increase dramatically, going from 1.2 % in the fresh sample to 3.04 % and 8.37 % for the pH=6 and pH=5 samples, respectively. For the pH 6 sample, the little increase of d cyt spacing are consistent with the maintained structure of the myelin proteins P2 and P0, as their R g (1.53 nm and 2.2 nm) (54,55), together with MBP action, which has increased its rigidity, therefore its strength (Figure 3b). On the other hand, for the pH 5 sample the P2 and P0 structures become unfolded because the cyt apposition fluctuates assuming values too small, no longer consistent with the size of structured and folded proteins.…”

Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail

“…1c, Supplementary Table 1). All of the variants showed high apparent molecular weight in SDS-PAGE, which reflects the intrinsically disordered nature of P0ct (Raasakka et al 2019b). The molecular weight and the presence of the mutations were confirmed using mass spectrometry (Table 1).…”

“…P0ct contains a neuritogenic segment, which can be used to induce experimental autoimmune neuritis (EAN) in animal models (de Sèze et al 2016). In vitro , P0ct is disordered in aqueous solution, gaining secondary structure upon binding to negatively charged phospholipids (Luo et al 2007; Raasakka et al 2019b). In its lipid-bound state, P0ct affects the phase behaviour of lipids and promotes the fusion of lipid vesicles.…”

“…In its lipid-bound state, P0ct affects the phase behaviour of lipids and promotes the fusion of lipid vesicles. High-degree molecular order, most likely from stacked lipid bilayers, can be detected via X-ray diffraction of P0ct-bound membranes (Raasakka et al 2019b). This suggests that P0ct harbours a structural role in mature myelin.…”

“…Considering the small size of P0ct and the nature of the disease mutations in it, many of which change its electrostatic charge, impairment in the function of P0ct as a membrane binding/stabilizing segment is a possible functional mechanism. We used methodologies established earlier for myelin basic protein (MBP) (Raasakka et al 2017) and wild-type P0ct (wt-P0ct) (Raasakka et al 2019a; Raasakka et al 2019b) to characterize structure-function relationships of the CMT- and DSS-related P0ct variants. Our results suggest that D224Y is a hypermyelinating gain-of-function mutation, which is in line with the clinically relevant phenotype of abnormally thickened myelin sheaths (Fabrizi et al 2006).…”