Cytoplasmic domain of human myelin protein zero likely folded as beta-structure in compact myelin

Myelin protein zero (P0 or P0 glycoprotein), the major integral membrane protein in peripheral nervous system myelin, plays a key role in myelin membrane compaction and stability. While the structure of P0 extracellular domain was determined by crystallography, the paucity of any structural data on the highly positive-charged P0 cytoplasmic domain (P0-cyt) has greatly limited our understanding of the mechanism of P0 function. Here, using circular dichroism and intrinsic fluorescence spectroscopy, we attempted to elucidate the structure of human P0-cyt (hP0-cyt) in membrane mimetic environments composed of detergents or lipid vesicles. We found that the secondary structure of P0-cyt was polymorphic—at the lipid/protein ratio corresponding to that of mature peripheral myelin (~50:1), hP0-cyt mainly adopted a β-conformation, whereas when the proportion of lipid increased, the structure underwent a β→α transition. By contrast, the secondary structure of the major isoform of myelin basic protein, another myelin protein with a very large positive charge, remained unchanged across a wide range of lipid/protein ratios. We propose that when hP0-cyt is bound at sufficient concentration to lamellar lipid bilayers such as myelin, it folds into a β-conformation; before this threshold lipid/protein ratio is reached, the domain is α-helical. We suggest that the cytoplasmic apposition (major dense line) in compact myelin may be stabilized via the hydrogen-bonding of β-strands formed as a result of local P0-P0 aggregation.     

Peripheral myelin of Xenopus laevis: role of electrostatic and hydrophobic interactions in membrane compaction

P0 glycoprotein is the major structural protein of peripheral nerve myelin where it is thought to modulate inter-membrane adhesion at both the extracellular apposition, which is labile upon changes in pH and ionic strength, and the cytoplasmic apposition, which is resistant to such changes. Most studies on P0 have focused on structure-function correlates in higher vertebrates. Here, we focused on its role in the structure and interactions of frog (Xenopus laevis) myelin, where it exists primarily in a dimeric form. As part of our study, we deduced the full sequence of X. laevis P0 (xP0) from its cDNA. The xP0 sequence was found to be similar to P0 sequences of higher vertebrates, suggesting that a common mechanism of PNS myelin compaction via P0 interaction might have emerged through evolution. As previously reported for mouse PNS myelin, a similar change of extracellular apposition in frog PNS myelin as a function of pH and ionic strength was observed, which can be explained by a conformational change of P0 due to protonation-deprotonation of His52 at P0's putative adhesive interface. On the other hand, the cytoplasmic apposition in frog PNS myelin, like that in the mouse, remained unchanged at different pH and ionic strength. The contribution of hydrophobic interactions to stabilizing the cytoplasmic apposition was tested by incubating sciatic nerves with detergents. Dramatic expansion at the cytoplasmic apposition was observed for both frog and mouse, indicating a common hydrophobic nature at this apposition. Urea also expanded the cytoplasmic apposition of frog myelin likely owing to denaturation of P0. Removal of the fatty acids that attached to the single Cys residue in the cytoplasmic domain of P0 did not change PNS myelin structure of either frog or mouse, suggesting that the P0-attached fatty acyl chain does not play a significant role in PNS myelin compaction and stability. These results help clarify the present understanding of P0's adhesion role and the role of its acylation in compact PNS myelin.     

Myelin Structure and Composition in Zebrafish

To establish a standard for genotype/phenotype studies on the myelin of zebrafish (Danio rerio), an organism increasingly popular as a model system for vertebrates, we have initiated a detailed characterization of the structure and biochemical composition of its myelinated central and peripheral nervous system (CNS; PNS) tissues. Myelin periods, determined by X-ray diffraction from whole, unfixed optic and lateral line nerves, were approximately 153 and approximately 162 Angstrom, respectively. In contrast with the lability of PNS myelin in higher vertebrates, zebrafish lateral line nerve myelin exhibited structural stability when exposed to substantial changes in pH and ionic strength. Neither optic nor lateral line nerves showed swelling at the cytoplasmic apposition in CaCl(2)-containing Ringer's solution, in contrast with nerves from other teleost and elasmobranch fishes. Zebrafish optic nerve showed greater stability against changes in NaCl and CaCl(2) than lateral line nerve. The nerves from zebrafish having mutations in the gene for myelin basic protein (mbpAla2Thr and mbpAsp25Val) showed similar myelin periods as the wildtype (WT), but gave approximately 20% less compact myelin. Analysis of proteins by SDS-PAGE and Western blotting identified in both CNS and PNS of WT zebrafish two orthologues of myelin P0 glycoprotein that have been characterized extensively in trout--intermediate protein 1 (24 kDa) and intermediate protein 2 (28 kDa). Treatment with endoglycosidase-F demonstrated a carbohydrate moiety of approximately 7 kDa, which is nearly threefold larger than for higher vertebrates. Thin-layer chromatography for lipids revealed a similar composition as for other teleosts. Taken together, these data will serve as a baseline for detecting changes in the structure and/or amount of myelin resulting from mutations in myelin-related genes or from exogenous, potentially cytotoxic compounds that could affect myelin formation or stability.     

Expression and Purification of the Extracellular Domain of Human Myelin Protein Zero

Myelin protein zero (P0), an adhesion protein of the immunoglobulin superfamily, is the major protein of peripheral nervous system myelin in higher vertebrates. Protein zero is required for the formation and maintenance of myelin structure in the internode, likely through homophilic interactions at both the extracellular and the intracellular domains. Mutations and deletions in the P0 gene correlate with hereditary peripheral neuropathies of varying severity. Comparisons between the human and rat isoforms, whose three-dimensional structure has been determined by X-ray crystallography, suggest that these disease-associated genetic alterations lead to structural changes in the protein that alter P0-P0 interactions and hence affect myelin functionality. Knowing the crystal structures of native and altered human P0 isoforms could help to elucidate the structural changes in myelin membrane packing that underlie the altered functionality. Alterations of P0 extracellular domain (P0-ED) are of additional interest as previous X-ray diffraction studies on myelin membrane packing suggest that P0-ED molecules can assume distinct adhesive arrangements. Here, we describe an improved method to express and purify human P0-ED (hP0-ED) suitable for crystallographic analysis. A fusion protein consisting of maltose binding protein fused to hP0-ED was secreted to the periplasm of Escherichia coli to allow an appropriate folding pathway. The fusion protein was extracted via osmotic shock and purified by affinity chromatography. Factor Xa was used to cleave the fusion protein, and a combination of affinity and ion-exchange chromatography was used to further purify hP0-ED. We document several significant improvements to previous protocols, including bacterial growth to approximately 15 OD using orbital shakers and the use of diafiltration, which result in yields of approximately 150 mg highly pure protein per liter of medium.     

Myelin Membrane Structure and Composition Correlated: A Phylogenetic Study

We have correlated myelin membrane structure with biochemical composition in the CNS and PNS of a phylogenetic series of animals, including elasmobranchs, teleosts, amphibians, and mammals. X-ray diffraction patterns were recorded from freshly dissected, unfixed tissue and used to determine the thicknesses of the liquid bilayer and the widths of the spaces between membranes at their cytoplasmic and extracellular appositions. The lipid and protein compositions of myelinated tissue from selected animals were determined by TLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblotting, respectively. We found that (1) there were considerable differences in lipid (particularly glycolipid) composition, but no apparent phylogenetic trends; (2) the lipid composition did not seem to affect either the bilayer thickness, which was relatively constant, or the membrane separation; (3) the CNS of elasmobranch and teleost and the PNS of all four classes contained polypeptides that were recognized by antibodies against myelin P0 glycoprotein; (4) antibodies against proteolipid protein (PLP) were recognized only by amphibian and mammalian CNS; (5) wide extracellular spaces (ranging from 36 to 48 A) always correlated with the presence of P0-immunoreactive protein; (6) the narrowest extracellular spaces (approximately 31 A) were observed only in PLP-containing myelin; (7) the cytoplasmic space in PLP-containing myelin (approximately 31 A) averaged approximately 5 A less than that in P0-containing myelin; (8) even narrower cytoplasmic spaces (approximately 24 A) were measured when both P0 and 11-13-kilodalton basic protein were detected; (9) proteins immunoreactive to antibodies against myelin P2 basic protein were present in elasmobranch and teleost CNS and/or PNS, and in mammalian PNS, but not in amphibian tissues; and (10) among mammalian PNS myelins, the major difference in structure was a variation in membrane separation at the cytoplasmic apposition. These findings demonstrate which features of myelin structure have remained constant and which have become specifically altered as myelin composition changed during evolutionary development.     

Shiverer and Normal Peripheral Myelin Compared: Basic Protein Localization, Membrane Interactions, and Lipid Composition

We have correlated membrane structure and interactions in shiverer sciatic nerve myelin with its biochemical composition. Analysis of x-ray diffraction data from shiverer myelin swollen in water substantiates our previous localization of an electron density deficit in the cytoplasmic half of the membrane. The density loss correlates with the absence of the major myelin basic proteins and indicates that in normal myelin, the basic protein is localized to the cytoplasmic apposition. As in normal peripheral myelin, hypotonic swelling in the shiverer membrane arrays occurs in the extracellular space between membranes; the cytoplasmic surfaces remain closely apposed notwithstanding the absence of basic protein from this region. Surprisingly, we found that the interaction at the extracellular apposition of shiverer membranes is altered. The extracellular space swells to a greater extent than normal when nerves are incubated in distilled water, treated at a reduced ionic strength of 0.06 in the range of pH 4-9, or treated at constant pH (4 or 7) in the range of ionic strengths 0.02-0.20. To examine the biochemical basis of this difference in swelling, we compared the lipid composition of shiverer and normal myelin. We find that sulfatides, hydroxycerebroside, and phosphatidylcholine are 20-30% higher than normal; nonhydroxycerebroside and sphingomyelin are 15-20% lower than normal; and ethanolamine phosphatides, phosphatidylserine, and cholesterol show little or no change. A higher concentration of negatively charged sulfatides at the extracellular surface likely contributes to an increased electrostatic repulsion and greater swelling in shiverer. The cytoplasmic surfaces of the apposed membranes of normal and shiverer myelins did not swell apart appreciably in the pH and ionic strength ranges expected to produce electrostatic repulsion. This stability, then, clearly does not depend on basic protein. We propose that P0 glycoprotein molecules form the stable link between apposed cytoplasmic membrane surfaces in peripheral myelin.     

Effects of ZnCl2 on membrane interactions in myelin of normal and shiverer mice

X-ray diffraction was used to record the effects of metal cations on the structure of peripheral nerve myelin. Acidic saline (pH 5.0) either with or without added metal cations caused myelin to swell by 10-20 A from its native period of 178 A. The X-ray patterns usually showed broad reflections, and higher orders were either weak or unobserved. With added ZnCl2, however, the swollen myelin gave diffraction patterns that retained sharp reflections to approx. 15 A spacing. Alkaline saline (pH 9.7) containing ZnCl2 produced a reduction of the myelin period by approx. 5 A which was at least twice as much as that produced by other metals. To examine the underlying chemical basis for these unique interactions of Zn2+ with myelin, we carried out parallel X-ray experiments on sciatic nerve from the shiverer mutant mouse, which lacks the major myelin basic proteins. Shiverer myelin responded like normal myelin to ZnCl2 in acidic saline; however, in alkaline saline shiverer myelin showed broadened X-ray reflections which indicated disordering of the regularity of the membrane arrays, and additional reflections were recorded which indicated lipid phase separation. This breakdown may come about by the binding of Zn2+ to negatively-charged lipids which could be more exposed due to the absence of myelin basic proteins. Electron density profiles were calculated on the assumption that, except for changes in their packing, the myelin membranes were minimally altered in structure. For both normal and shiverer myelins, treatments under acidic conditions resulted in swelling at the extracellular apposition and a slight narrowing of the cytoplasmic space. This swelling is likely due to adsorption of protons and divalent cations. Interaction between Zn2+ and myelin P0 glycoprotein could preserve an ordered arrangement of the apposed membrane surfaces. Alkaline saline containing ZnCl2 produced compaction at the cytoplasmic apposition in both normal and shiverer myelins possibly through interactions with a portion of P0 glycoprotein which extends into the cytoplasmic space between membranes.     

Lipid/myelin basic protein multilayers. A model for the cytoplasmic space in central nervous system myelin

A multilayered complex forms when a solution of myelin basic protein is added to single-bilayer vesicles formed by sonicating myelin lipids. Vesicles and multilayers have been studied by electron microscopy, biochemical analysis, and X-ray diffraction. Freeze-fracture electron microscopy shows well-separated vesicles before myelin basic protein is added, but afterward there are aggregated, possibly multilayered, vesicles and extensive planar multilayers. The vesicles aggregate and fuse within seconds after the protein is added, and the multilayers form within minutes. No intra-bilayer particles are seen, with or without the protein. Some myelin basic protein, but no lipid, remains in the supernatant after the protein is added and the complex sedimented for X-ray diffraction. A rather variable proportion of the protein is bound. X-ray diffraction patterns show that the vesicles are stable in the absence of myelin basic protein, even under high g-forces. After the protein is added, however, lipid/myelin basic protein multilayers predominate over single-bilayer vesicles. The protein is in every space between lipid bilayers. Thus the vesicles are torn open by strong interaction with myelin basic protein. The inter-bilayer spaces in the multilayers are comparable to the cytoplasmic spaces in central nervous system myelins . The diffraction indicates the same lipid bilayer thickness in vesicles and multilayers, to within 1 A. By comparing electron-density profiles of vesicles and multilayers, most of the myelin basic protein is located in the inter-bilayer space while up to one-third may be inserted between lipid headgroups. When cytochrome c is added in place of myelin basic protein, multilayers also form. In this case the protein is located entirely outside the unchanged bilayer. Comparison of the various profiles emphasizes the close and extensive apposition of myelin basic protein to the lipid bilayer. Numerous bonds may form between myelin basic protein and lipids. Cholesterol may enhance binding by opening gaps between diacyl-lipid headgroups.     

Amino acid variations resulting in functional and nonfunctional zebrafish P2X<Subscript>1</Subscript> and P2X<Subscript>5.1</Subscript> receptors

Several zebrafish P2X receptors (zP2X(1), zP2X(2), and zP2X(5.1)) have been reported to produce little or no current although their mammalian orthologs produce functional homomeric receptors. We isolated new cDNA clones for these P2X receptors that revealed sequence variations in each. The new variants of zP2X(1) and zP2X(5.1) produced substantial currents when expressed by Xenopus oocytes, however the new variant of zP2X(2) was still nonfunctional. zP2X(2) lacks two lysine residues essential for ATP responsiveness in other P2X receptors; however introduction of these two lysines was insufficient to allow this receptor to function as a homotrimer. We also tested whether P2X signaling is required for myogenesis or synaptic communication at the zebrafish neuromuscular junction. We found that embryonic skeletal muscle expressed only one P2X receptor, P2X(5.1). Antisense knockdown of P2X(5.1) eliminated skeletal muscle responsiveness to ATP but did not prevent myogenesis or behaviors that require functional transmission at the neuromuscular junction.     

Accessibility of Galactosyl Ceramides to Probe Reagents in Central Nervous System Myelin

The suitability of isolated central nerve myelin preparations for probe labelling studies was assessed and the accessibility of galactosyl ceramides in myelin to galactose oxidase and sodium periodate was determined. Isolated myelin preparations present a uniform external membrane surface to added probes because lamellae in the myelin sheath separate at their external apposition surfaces exclusively during isolation. The cytoplasmic apposition remains intact in isolated myelin. Cationised ferritin can gain access along external apposition regions of inner lamellae in multilamellar fragments of isolated myelin, indicating that proteins and lipids on the external membrane surface will be accessible to probes. Over 50% of the total galactosyl ceramides of myelin are accessible to galactose oxidase attack; hydroxy fatty acid- and nonhydroxy fatty acid-containing cerebrosides are equally attacked. Sodium periodate attacks over 90% of the galactosyl ceramides in isolated myelin at 20°C and electron micrographs of the periodate-treated myelin reveal changes at the external apposition only. Galactosyl ceramides in vesicles of myelin lipid vesicles are not so readily attacked by periodate. The disposition of galactosyl ceramides in the myelin lamellae is discussed.     

Reconstituted P2/Myelin-Lipid Multilayers

A complex forms when bovine P2 protein is added to single-bilayer vesicles created by sonicating myelin lipids. The complex was studied by biochemical analysis, freeze-fracture (FF) and thin-section electron microscopy (EM), and by X-ray diffraction. Smaller amounts of P2 cause the vesicles to aggregate and fuse whereas larger amounts (greater than or equal to 4 wt%) cause multilayers to form. Binding saturates at 15 wt% P2. FF EM shows that large, flat multilayers form within 15 min of addition of P2. Only smooth fracture faces are seen, as expected for a peripheral membrane protein. X-ray diffraction shows a constant repeating distance in the multilayers: 86.0 +/- 0.7 A between the centers of bilayers in the range 4 wt% less than or equal to P2/(P2 + lipid) less than or equal to 15 wt%. Assuming a 53 A-thick bilayer, the space between bilayers is 33 A wide. This is a wider space than for myelin basic protein (MBP) (20-25 A wide). The respective widths are consistent with a compact, globular structure for P2 and a flattened shape for MBP. Calculated electron-density profiles of the lipids with and without P2 reveal the protein largely in the interbilayer spaces, with a small part possibly inserted into the lipid headgroup layers. The different proportions of P2 in the sciatic nerve of various species are tentatively correlated with the different average widths observed by X-ray diffraction for the cytoplasmic space (major period line) between bilayers in the respective sciatic myelins.     

The cytoplasmic domain of the myelin P0 protein influences the adhesive interactions of its extracellular domain

The extracellular domain of the myelin P0 protein is believed to engage in adhesive interactions and thus hold the myelin membrane compact. We have previously shown that P0 can behave as a homophilic adhesion molecule through interactions of its extracellular domains (Filbin, M. T., F. S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. Nature (Lond.) 344:871-872). To determine if the cytoplasmic domain of P0 must be intact for the extracellular domains to adhere, we compared the adhesive capabilities of P0 proteins truncated at the COOH-terminal to the full-length P0 protein. P0 cDNAs lacking nucleotides coding for the last 52 or 59 amino acids were transfected into CHO cells, and surface expression of the truncated proteins was assessed by immunofluorescence, surface labeling followed by immunoprecipitation, and an ELISA. Cell lines were chosen that expressed at least equivalent amounts of the truncated P0 proteins at the surface as did a cell line expressing the full-length P0. The adhesive properties of these three cell lines were compared. It was found that when a suspension of single cells was allowed to aggregate for a period of 60 min, only the cells expressing the full-length P0 had formed large aggregates, while the cells expressing the truncated P0 molecules were still mostly single cells indistinguishable from the control cells. Furthermore, 25-30% of the full-length P0 was insoluble in NP40, indicative of an interaction with the cytoskeleton, whereas only 5-10% of P0 lacking 52 amino acids and none of P0 lacking 59 amino acids were insoluble. These results suggest that for the extracellular domain of P0 to behave as a homophilic adhesion molecule, its cytoplasmic domain must be intact, and most probably, it is interacting with the cytoskeleton.     

Tyrosine phosphorylation of PNS myelin P(0) occurs in the cytoplasmic domain and is maximal during early development

P(0), the major protein of PNS myelin, is considered to play a critical role in the compaction and stabilization of myelin lamellae. The protein undergoes extensive posttranslational modifications, including phosphorylation at multiple serine moieties in the cytoplasmic region. Recently, we demonstrated that P(0) is phosphorylated on one or more tyrosine residues in rat nerve homogenates after incubation. In this study, we show that P(0) phosphorylated on tyrosine is also present in the intact animal. The proportion of P(0) molecules phosphorylated on tyrosine is highest during the first postnatal week, a period that coincides with the most rapid period of myelin deposition in the PNS. A peptide that constitutes the cytoplasmic domain was isolated from purified P(0) and shown by immunochemical and chemical means to be phosphorylated on the tyrosine corresponding to Y(191) in the intact protein. No evidence was obtained supporting the possibility that P(0) is phosphorylated on other tyrosine residues. The sequence of amino acids surrounding Y(191) resemble known substrate phosphorylation sites for some nonreceptor cytoplasmic tyrosine kinases, as well as tyrosine-based recognition signals associated with clathrin vesicle-mediated cndocytosis.     

Incorporation of P0 Protein into Liposomes: Demonstration of a Two-Domain Structure by Immunochemical and PAGE Analysis

The amphiphilic nature of P0, the major glycoprotein of peripheral nerve myelin, has been suggested previously. In the present study, purified P0 from human peripheral nerve myelin was incorporated into an artificial lipid bilayer consisting of dimyristoyl lecithin and cholesterol. The liposomes were fractionated on a sucrose gradient. The continued expression of P0 antigenicity by the liposomes was shown by specific complement consumption with a multivalent antiserum against P0 or with an IgM monoclonal antibody. Both antibodies recognized P0 expressed on the surface of peripheral nerve myelin and the P0 liposomes. P0 liposomes and peripheral nerve myelin treated with trypsin lost the surface determinant that reacted with the monoclonal antibody. Analysis of the trypsin-treated liposomes and peripheral nerve myelin by polyacrylamide gel electrophoresis revealed molecular weights for this protein of 19,500 and 20,500, respectively. Similar treatment of the P0 in the fluid phase resulted in many smaller fragments. These results indicate that P0 consists of two domains, a hydrophilic domain accessible to trypsin digestion and a hydrophobic domain, which is potentially trypsin-sensitive, but shielded by the lipid bilayer. Binding studies with an anti-P0 monoclonal antibody and polyacrylamide gel analysis of the lipid-shielded P0 fragment in liposomes and peripheral nerve myelin suggest that the orientation of the protein in the liposome is similar to that in peripheral nerve myelin.     

Complete amino acid sequence of PO protein in bovine peripheral nerve myelin

The P0 protein is a major structural glycoprotein of molecular weight 28,000 in peripheral nerve myelin. The complete amino acid sequence of bovine P0 protein was determined. The polypeptide chain consists of 219 amino acid residues and includes a highly hydrophobic domain (residues 125-150) in the middle, which probably represents a transmembrane segment. The amino terminal domain (residues 1-124) is relatively hydrophobic, but contains a negatively charged carbohydrate chain at Asn93. This domain is most likely located on the extracellular side of the membrane and may contribute to formation of the myelin intraperiod line by hydrophobic and electrostatic interactions. On the other hand, the basic carboxyl-terminal domain (residues 151-219) may protrude from the cytoplasmic side of the membrane and is probably involved together with basic proteins in the formation of the major myelin dense line through electrostatic interaction with acidic lipids in the membrane. The few interspecies amino acid variations between the bovine P0 and the rat P0 sequences, deduced from the cDNA (Lemke, G., and Axel, R. (1985) Cell 40, 501-508), indicate that the P0 protein is conserved across species.     

The three-dimensional structure of P2 myelin protein.

The three-dimensional structure of P2 protein from peripheral nervous system myelin has been determined at 2.7 A resolution by X-ray crystallography. The single isomorphous replacement/anomalous map was interpreted using skeletonized electron density on a computer graphics system. An atomic model was built using fragment fitting. The structure forms a compact 10-stranded up-and-down beta-barrel which encapsulates residual electron density that we interpret as a fatty acid molecule. This beta-barrel shows some similarity to, but is different from, the retinol binding protein family of structures. The relationship of the P2 structure to a family of cytoplasmic, lipid binding proteins is described.     

The aromatic domain of the coronavirus class I viral fusion protein induces membrane permeabilization: putative role during viral entry

Coronavirus (CoV) entry is mediated by the viral spike (S) glycoprotein, a class I viral fusion protein. During viral and target cell membrane fusion, the heptad repeat (HR) regions of the S2 subunit assume a trimer-of-hairpins structure, positioning the fusion peptide in close proximity to the C-terminal region of the ectodomain. The formation of this structure appears to drive apposition and subsequent fusion of viral and target cell membranes; however, the exact mechanism is unclear. Here, we characterize an aromatic amino acid rich region within the ectodomain of the S2 subunit that both partitions into lipid membranes and has the capacity to perturb lipid vesicle integrity. Circular dichroism analysis indicated that peptides analogous to the aromatic domains of the severe acute respiratory syndrome (SARS)-CoV, mouse hepatitis virus (MHV) and the human CoV OC43 S2 subunits, did not have a propensity for a defined secondary structure. These peptides strongly partitioned into lipid membranes and induced lipid vesicle permeabilization at peptide/lipid ratios of 1:100 in two independent leakage assays. Thus, partitioning of the peptides into the lipid interface is sufficient to disorganize membrane integrity. Our study of the S2 aromatic domain of three CoVs provides supportive evidence for a functional role of this region. We propose that, when aligned with the fusion peptide and transmembrane domains during membrane apposition, the aromatic domain of the CoV S protein functions to perturb the target cell membrane and provides a continuous track of hydrophobic surface, resulting in lipid-membrane fusion and subsequent viral nucleocapsid entry.     

New observations on the compact myelin proteome

Myelin formation and maintenance depends on the establishment of two structurally and biochemically discernible domains: (a)compact myelin, that is multilamellar stacks of plasma membrane sheets; and (b) cytoplasmic channels that border the compact myelin domains, attach them to the cell body and anchor the myelin sheath to the axonal membrane. To identify proteins involved in the organization of these domains we took advantage of the high lipid content of compact myelin to separate it cleanly from other neural membranes and then used reverse-phase HPLC coupled to Electro-Spray Double Mass Spectrometry('MudPIT') to characterize the proteome of this sample. MudPIT allowed us to sidestep the bias of 2D-PAGE against either highly charged or transmembrane proteins. Thus, of 97 proteins that presented at least two, fully tryptic peptides (a stringent threshold), seven were well known myelin markers, including the mayor CNS myelin proteins: proteolipid protein and myelin basic protein, which are not resolvable by 2D-PAGE. Furthermore, we have confirmed and extended the known compact myelin proteome by 22 proteins and confirmed that CNS and PNS myelinated tracts present Sirtuin 2, a tubulin deacetylase, and Septin7, a small GTPase that is likely to be involved in membrane and cytoplasm partitioning.     

Mutations in the cytoplasmic domain of P0 reveal a role for PKC-mediated phosphorylation in adhesion and myelination.

Mutations in P0 (MPZ), the major myelin protein of the peripheral nervous system, cause the inherited demyelinating neuropathy Charcot-Marie-Tooth disease type 1B. P0 is a member of the immunoglobulin superfamily and functions as a homophilic adhesion molecule. We now show that point mutations in the cytoplasmic domain that modify a PKC target motif (RSTK) or an adjacent serine residue abolish P0 adhesion function and can cause peripheral neuropathy in humans. Consistent with these data, PKCalpha along with the PKC binding protein RACK1 are immunoprecipitated with wild-type P0, and inhibition of PKC activity abolishes P0-mediated adhesion. Point mutations in the RSTK target site that abolish adhesion do not alter the association of PKC with P0; however, deletion of a 14 amino acid region, which includes the RSTK motif, does abolish the association. Thus, the interaction of PKCalpha with the cytoplasmic domain of P0 is independent of specific target residues but is dependent on a nearby sequence. We conclude that PKC-mediated phosphorylation of specific residues within the cytoplasmic domain of P0 is necessary for P0-mediated adhesion, and alteration of this process can cause demyelinating neuropathy in humans.     

Myelin P0-Glycoprotein: Predicted Structure and Interactions of Extracellular Domain

Protein zero (P₀), a transmembrane glycoprotein, accounts for over 50% of the total protein in PNS myelin. The extracellular domain of P₀ (P₀-ED) is similar to the immunoglobulin variable domain, carrying one acceptor sequence for N-linked glycosylation. The x-ray diffraction analysis of PNS myelin has demonstrated reversible transitions that depend on pH and ionic strength, resulting in three distinct structures characterized by widths of about 36 Å, 50 Å (native), and 90 Å between the extracellular surfaces of the membranes. In the current work, we considered the constraints imposed by these x-ray diffraction data on the orientation of P₀-ED, and we propose how this immunoglobulin-like domain could be accommodated in the variable widths of the extracellular space between myelin membranes. The modeling made use of the finding that β-strand predictions for P₀-ED are virtually superimposable with those of the V_H domain of the phosphocholine-binding immunoglobulin M603 of mouse, which has a similar number of residues as P₀-ED and a structure that has been solved crystallographically. The dimensions of P₀-ED from the space-filling model, developed using PC- based molecular modeling software, were found to be 44 Å× 25 Å× 23 Å. On the assumption that neither the shape nor the orientation of P₀-ED changes appreciably, then the different widths at the extracellular apposition would easily accommodate P₀-ED from apposed membranes if the molecules were oriented so that the β- strands were approximately perpendicular to the membrane surface. The apposed P₀-EDs would fully overlap at the closest apposition of the membranes, partially overlap in the native state, and align end to end in the incompletely swollen state. The P₀-ED regions analogous to the complementarity-determining regions of immunoglobulins can account for the recognition of P₀-ED from apposed membranes in the incompletely swollen state. Two of the faces of P₀-ED that show charge complementarity could account for the homophilic interactions of P₀-ED from apposed membranes in the native state. This association can be stabilized further by hydrophobic interactions. The N- linked nonasaccharide after energy minimization fit into a cavity, which was at the base of P₀-ED and which was lined with three positively charged residues. Thus, the carbohydrate may help to maintain the orientation of P₀ at the membrane surface. Our model shows how the single immunoglobulin-like domain of P₀ can account for distinct structural states of myelin membrane packing by homophilic interactions.