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

Abstract

Schwann cells myelinate selected axons in the peripheral nervous system (PNS) and contribute to fast saltatory conduction via the formation of compact myelin, in which water is excluded from between tightly adhered lipid bilayers. Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), are incurable demyelinating conditions that result in pain, decrease in muscle mass, and functional impairment. Many Schwann cell proteins, which are directly involved in the stability of compact myelin or its development, are subject to mutations linked to these neuropathies. The most abundant PNS myelin protein is protein zero (P0); point mutations in this transmembrane protein cause CMT subtype 1B and DSS. P0 tethers apposing lipid bilayers together through its extracellular immunoglobulin-like domain. Additionally, P0 contains a cytoplasmic tail (P0ct), which is membrane-associated and contributes to the physical properties of the lipid membrane. Six CMT- and DSS-associated missense mutations have been reported in P0ct. We generated recombinant disease mutant variants of P0ct and characterized them using biophysical methods. Compared to wild-type P0ct, some mutants have negligible differences in function and folding, while others highlight functionally important amino acids within P0ct. For example, the D224Y variant of P0ct induced tight membrane multilayer stacking. Our results show a putative molecular basis for the hypermyelinating phenotype observed in patients with this particular mutation and provide overall information on the effects of disease-linked mutations in a flexible, membrane-binding protein segment. Using neutron reflectometry, we additionally show that P0ct embeds deep into a lipid bilayer, explaining the observed effects of P0ct on the physical properties of the membrane.

How Does Protein Zero Assemble Compact Myelin?

“…The narrow extracellular space between pairs of compact myelin membranes, the intramyelinic compartment, is compacted by a densely arranged P0 zipper in the PNS, which dictates the stability and width of this compartment [28,[37][38][39][40][41]. While numerous studies have specifically focused on the compaction of the intramyelinic compartment, P0 has also been shown to stabilize membrane stacking in the narrow cytoplasmic compartment [42,43]; this is supported by recent biophysical experiments using model membranes [41,44]. In animal models, the stacking occurs in the absence of cytosolic MBP and P2 [42,45,46], which poses open, neglected questions, as cytoplasmic membrane leaflet adhesion by P0 remains poorly understood.…”

“…Cells 2020, 9, 1832 3 of 20 biophysical characterizations using simplified lipid compositions have been performed on P0ct, which revealed that P0ct alone is an IDP in solution but folds upon irreversible association with negatively charged detergent micelles and lipid vesicles [58][59][60][61]. P0ct embeds deep into the lipid structure and affects the mechanical and thermodynamic properties of its surroundings [41,44]. This association in simplified model systems is influenced by ionic strength and the presence of Ca 2+ [62], and it generally resembles the behavior of MBP under similar conditions, which involves a large gain of structural content [41,[58][59][60][61]63].…”

“…Due to available structural information, the Ig-like domain and its role in defining the spacing of the IPL have been the focus of several studies. In contrast to the literature published on the Ig-like domain and IPL formation, studies focusing specifically on the structure-function relationships of P0ct account for a small fraction of experimental data available for P0 [41,44,[58][59][60][61]. MBP and P0ct share similar physicochemical characteristics, which potentially confers P0ct with MBP-like functions.…”

“…Importantly, based on in vitro studies on how P0ct interacts with model lipid bilayers, P0ct might mediate the packing between membranes that form the cytoplasmic compartment [41,44]. Central results supporting this hypothesis arose from experiments where P0ct irreversibly associated with simplified model membrane systems with a similar affinity as MBP [41,44,63] and aggregated lipid vesicles together, producing membrane spacings typical for the MDL [41,44]. The effect was dependent on the dosage of P0ct, the net charge of the lipids, ionic strength, and the presence of Ca 2+ [41,44,62].…”

Flexible Players within the Sheaths: The Intrinsically Disordered Proteins of Myelin in Health and Disease

“…However, these structures are very close to the myelin membrane and therefore likely to be folded, as experimentally shown for the loops of proteolipid protein [23]. Another example is the C-terminal extension of P0 (P0ct), which is disordered in the absence of lipids, but not under membrane-like conditions [19,[51][52][53][54]. In vivo, the lipidated P0ct directly follows the transmembrane helix of P0 in the cytoplasmic compartment of PNS compact myelin, permanently anchoring it to the phospholipid membrane, whereby it folds and is thus unlikely to function as a canonical IDP [55].…”