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Ionic Strength and Calcium Regulate the Membrane Interactions of Myelin Basic Protein and 2 the Cytoplasmic Domain of Myelin Protein Zero

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Ionic Strength and Calcium Regulate the Membrane Interactions of Myelin Basic Protein and 2 the Cytoplasmic Domain of Myelin Protein Zero bioRxiv preprint doi: https://doi.org/10.1101/529586; this version posted January 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Ionic strength and calcium regulate the membrane interactions of myelin basic protein and 2 the cytoplasmic domain of myelin protein zero 3 4 Arne Raasakka1, Nykola C. Jones2, Søren Vrønning Hoffmann2 & Petri Kursula1,3,† 5 6 7 1 Department of Biomedicine, University of Bergen, Bergen, Norway 8 2 ISA, Department of Physics and Astronomy, Aarhus University, Ny Munkegade 120, 8000 Aarhus C, Denmark 9 3 Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland 10 † 11 Corresponding author. E-mail: [email protected] 12 1 bioRxiv preprint doi: https://doi.org/10.1101/529586; this version posted January 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Abstract 2 The formation of a mature myelin sheath in the vertebrate nervous system requires specific protein- 3 membrane interactions. Several myelin-specific proteins are involved in the stacking of lipid membranes into 4 multilayered structures around neuronal axons, and misregulation of these processes may contribute to 5 chronic demyelinating diseases. Two key proteins functioning in myelin membrane binding and stacking are 6 the myelin basic protein (MBP) and protein zero (P0). Other factors, including Ca2+, are important for the 7 regulation of myelination. Here, we studied the effects of ionic strength and Ca2+ on the direct molecular 8 membrane interactions of MBP and the cytoplasmic domain of P0 (P0ct). While both MBP and P0ct bound 9 and aggregated negatively charged lipid vesicles, while simultaneously folding, both ionic strength and 10 calcium had systematic effects on these interactions. Especially when decreasing membrane net negative 11 charge, the level and kinetics of vesicle aggregation, which is a functional assay for myelin membrane- 12 stacking proteins, were affected by both salt and Ca2+. The results indicate that the effects on lipid membrane 13 surfaces by ions can directly affect myelin protein-membrane interactions at the molecular level, in addition 14 to signalling effects in myelinating glia. 15 16 Keywords 17 Myelin basic protein; myelin protein zero; intrinsically disordered protein; protein folding; lipid binding; 18 calcium; ionic strength 19 20 2 bioRxiv preprint doi: https://doi.org/10.1101/529586; this version posted January 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Introduction 2 Myelin contributes greatly to the efficiency of both the central and peripheral nervous system (CNS and PNS 3 respectively) via axonal insulation and trophic support (1,2). Comprised of tightly stacked lipid membranes, 4 held together by a specific assortment of proteins, compact myelin (CM) – a water-deficient structure – 5 insulates selected axonal segments and enables fast saltatory nerve impulse conduction. While neuronal 6 axons undergo frequent membrane depolarization, and [K+] and [Na+] vary upon action potential firing, the 7 ionic conditions in myelinating glia remain fairly constant, with higher [K+] and [Na+] compared to neurons 8 in general (3). 9 In myelinating cells, [Ca2+] and [Zn2+] (1 mM and 50 µM, respectively) are relatively high compared to 10 many other cell types (3,4). This signifies the importance of the underlying Ca2+ signalling (5–7); due to the 11 narrow bilayer spacing and low water content in CM, intracellular divalent cations must be subjects of 12 attractive and repulsive interactions. These involve negatively charged and zwitterionic phospholipid 13 headgroups, as well as myelin proteins, which are often highly positively charged and directly participate in 14 interactions with other proteins and lipids. Both Ca2+ and Zn2+ have been studied with respect to 15 autoinflammatory diseases and pathogenesis (5,8–10), and intracellular Ca2+ levels and changes therein have 16 been directly linked to the correct development of myelin (11–14). 17 Myelin basic protein (MBP) plays a fundamental role in forming and maintaining stable myelin membrane 18 stacks, defining the boundaries between CM and non-compact myelin (15–18). MBP is involved in multiple 19 sclerosis (MS) (19–21), being an intrinsically disordered protein (IDP) that folds upon lipid binding-induced 20 charge neutralization (16,22,23). MBP is highly positively charged and manifests as a pool of post- 21 translationally citrullinated variants with differing net charges (24). MBP requires negatively charged lipids 22 for membrane adhesion, but correct myelin morphology is also dependent on the presence of other lipids and 23 a controlled ionic content (14,16,25). MBP interacts with divalent cations (26–30), with the underlying 24 mechanisms being very specific; one would expect electrostatic repulsion due to its unusually high positive 25 net charge. MBP binds to calmodulin in a Ca2+-dependent manner (27,31–33), and the pool of different MBP 26 isoforms has been shown to regulate oligodendroglial Ca2+ influx (34,35). Concentration-dependent binding 27 of divalent cations to different MBP variants could, together with altered membrane compositions, influence 28 their in vivo functions, having implications in MS etiology and the stability of CM (6,14,27,36). 29 Myelin protein zero (P0) is a transmembrane protein in PNS myelin, with relevance in human peripheral 30 neuropathies and animal models (37–39). It consists of an extracellular domain with an immunoglobulin-like 31 fold (40), a single transmembrane helix, and a short cytoplasmic segment (P0ct) involved in the regulation of 32 membrane fluidity (41). Similarly to MBP, P0ct is highly positively charged and directly interacts with 33 phospholipids through electrostatics, gaining secondary structure in the process (41,42), which might make it 34 susceptible to effects caused by cationic species. 3 bioRxiv preprint doi: https://doi.org/10.1101/529586; this version posted January 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 In this study, the effects of ionic strength and Ca2+ on the structural and functional properties of recombinant 2 MBP and P0ct binding to lipid membranes were studied using different biophysical techniques. The results 3 shed new light on the interplay between ions and proteins in CM and support the proposed role of Ca2+ as a 4 regulator of the bilayer-MBP interaction (14,36,43). 5 6 4 bioRxiv preprint doi: https://doi.org/10.1101/529586; this version posted January 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Experimental procedures 2 Protein expression and purification 3 Mouse MBP and human P0ct were expressed and purified as described (16,41). The final size-exclusion 4 chromatography after affinity purification and tag removal was performed using Superdex75 16/60 HiLoad 5 and Superdex75 increase 10/300GL columns (GE Healthcare) with 20 mM HEPES, 150 mM NaCl, pH 7.5 6 (HBS) as mobile phase. When required, the buffer was exchanged to water by sequential dialysis. The 7 purified proteins were either used fresh or snap-frozen in liquid N2 and stored at -80 °C for later use. 8 Vesicle preparation 9 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac- 10 glycerol) (Na-salt) (DMPG), and the deuterated d54-DMPC, d54-DMPG, and d7-cholesterol were from Avanti 11 Polar Lipids (Alabaster, Alabama, USA). The detergent n-dodecylphosphocholine (DPC) was from 12 Affymetrix. 13 Lipid stocks were prepared by dissolving the dry lipids in chloroform or chloroform:methanol (9:1 v/v) at 10 14 – 30 mM. All mixtures were prepared from the stocks at desired molar ratios, followed by solvent 15 evaporation under an N2 stream and freeze-drying overnight at -52 °C under vacuum. The dried lipids were 16 either stored air-tight at -20 °C or used fresh for liposome preparation. 17 Liposomes were prepared by agitating dried lipids with either water or HBS at a concentration 10 – 15 mM, 18 followed by gentle mixing at ambient temperature, to ensure that no unsuspended lipids remained in the 19 vessel. MLVs were prepared by freeze-thawing using liquid N2 and a warm water bath, with vigorous 20 vortexing afterwards. Such a cycle was performed 7 times. SUVs were prepared by sonicating fresh MLVs 21 using a probe tip sonicator (Materials Inc. Vibra-Cell VC-130) until clear, while avoiding overheating. All 22 lipid preparations were immediately used for experiments. 23 Vesicle turbidimetry 24 For turbidimetric measurements, samples containing 0.5 mM DMPC:DMPG (1:1) SUVs were prepared with 25 2.5 µM P0ct or MBP in duplicate at 150 µl final volume on a Greiner 655161 96-well plate. Optical density 26 at 450 nm was recorded immediately after thorough mixing at 25 °C using a Tecan Spark 20M plate reader. 27 Synchrotron radiation circular dichroism spectroscopy 28 SRCD data were collected from 0.1–0.4 mg ml-1 protein samples in water or 0.5% SDS on the AU-CD 29 beamline at ASTRID2 (ISA, Aarhus, Denmark).
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