Myelin-derived and putative molecular mimic peptides share structural properties in aqueous and membrane-like environments

Background: Despite intense research, the causes of various neurological diseases remain enigmatic to date. A role for viral or bacterial infection and associated molecular mimicry has frequently been suggested in the etiology of neurological diseases, including demyelinating autoimmune disorders, such as multiple sclerosis. Pathogen mimics of myelin-derived autoimmune peptides have been described in the literature and shown to induce myelin autoimmune responses in animal models. Methods: We carried out a structural study on myelin-derived peptides, and mimics thereof from various pathogens, in aqueous and membrane-like environments, using conventional and synchrotron radiation circular dichroism spectroscopy. A total of 13 peptides from the literature were studied, and 290 circular dichroism spectra were analysed. In addition, peptide structure predictions and vesicle aggregation assays were performed. Results: The results indicate a high level of similarity in the biophysical and folding properties of the peptides from either myelin proteins or proteins from pathogenic viruses or bacteria; essentially all of the studied peptides folded in the presence of lipid vesicles or under other membrane-mimicking conditions, which is a sign of membrane interaction. Many of the peptides presented remarkable similarities in their conformation in different environments. Conclusions: As most of the studied epitope segments in myelin proteins are associated with membrane-binding sites, our results support a view of molecular mimicry, involving lipid membrane interaction propensity and similar conformational properties, possibly playing a role in demyelinating disease. The results suggest mechanisms related to protein amphiphilicity and order-disorder transitions in the recognition of peptide epitopes in autoimmune demyelination.publishedVersio

Cryo-EM, X-ray diffraction, and atomistic simulations reveal determinants for the formation of a supramolecular myelin-like proteolipid lattice

Myelin protein P2 is a peripheral membrane protein of the fatty acid?binding protein family that functions in the formation and maintenance of the peripheral nerve myelin sheath. Several P2 gene mutations cause human Charcot-Marie-Tooth neuropathy, but the mature myelin sheath assembly mechanism is unclear. Here, cryo-EM of myelin-like proteolipid multilayers revealed an ordered three-dimensional (3D) lattice of P2 molecules between stacked lipid bilayers, visualizing supramolecular assembly at the myelin major dense line. The data disclosed that a single P2 layer is inserted between two bilayers in a tight intermembrane space of ?3 nm, implying direct interactions between P2 and two membrane surfaces. X-ray diffraction from P2-stacked bicelle multilayers revealed lateral protein organization, and surface mutagenesis of P2 coupled with structure-function experiments revealed a role for both the portal region of P2 and its opposite face in membrane interactions. Atomistic molecular dynamics simulations of P2 on model membrane surfaces suggested that Arg-88 is critical for P2-membrane interactions, in addition to the helical lid domain. Negatively charged lipid headgroups stably anchored P2 on the myelin-like bilayer surface. Membrane binding may be accompanied by opening of the P2 ?-barrel structure and ligand exchange with the apposing bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane protein from human myelin. This is an important step toward deciphering the 3D assembly of a mature myelin sheath at the molecular level.Peer reviewe

Molecular mechanisms of CharcotMarie-Tooth neuropathy linked to mutations in human myelin protein P2

This article has an erratum: Doi 10.1038/s41598-017-18751-7Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neuropathies. Recently, three CMT1-associated point mutations (I43N, T51P, and I52T) were discovered in the abundant peripheral myelin protein P2. These mutations trigger abnormal myelin structure, leading to reduced nerve conduction velocity, muscle weakness, and distal limb atrophy. P2 is a myelin-specific protein expressed by Schwann cells that binds to fatty acids and membranes, contributing to peripheral myelin lipid homeostasis. We studied the molecular basis of the P2 patient mutations. None of the CMT1-associated mutations alter the overall folding of P2 in the crystal state. P2 disease variants show increased aggregation tendency and remarkably reduced stability, T51P being most severe. In addition, P2 disease mutations affect protein dynamics. Both fatty acid binding by P2 and the kinetics of its membrane interactions are affected by the mutations. Experiments and simulations suggest opening of the beta barrel in T51P, possibly representing a general mechanism in fatty acid-binding proteins. Our findings demonstrate that altered biophysical properties and functional dynamics of P2 may cause myelin defects in CMT1 patients. At the molecular level, a few malformed hydrogen bonds lead to structural instability and misregulation of conformational changes related to ligand exchange and membrane binding.Peer reviewe

Structure and dynamics of a human myelin protein P2 portal region mutant indicate opening of the β barrel in fatty acid binding proteins

Abstract Background Myelin is a multilayered proteolipid sheath wrapped around selected axons in the nervous system. Its constituent proteins play major roles in forming of the highly regular membrane structure. P2 is a myelin-specific protein of the fatty acid binding protein (FABP) superfamily, which is able to stack lipid bilayers together, and it is a target for mutations in the human inherited neuropathy Charcot-Marie-Tooth disease. A conserved residue that has been proposed to participate in membrane and fatty acid binding and conformational changes in FABPs is Phe57. This residue is thought to be a gatekeeper for the opening of the portal region upon ligand entry and egress. Results We performed a structural characterization of the F57A mutant of human P2. The mutant protein was crystallized in three crystal forms, all of which showed changes in the portal region and helix α2. In addition, the behaviour of the mutant protein upon lipid bilayer binding suggested more unfolding than previously observed for wild-type P2. On the other hand, membrane binding rendered F57A heat-stable, similarly to wild-type P2. Atomistic molecular dynamics simulations showed opening of the side of the discontinuous β barrel, giving important indications on the mechanism of portal region opening and ligand entry into FABPs. The results suggest a central role for Phe57 in regulating the opening of the portal region in human P2 and other FABPs, and the F57A mutation disturbs dynamic cross-correlation networks in the portal region of P2. Conclusions Overall, the F57A variant presents similar properties to the P2 patient mutations recently linked to Charcot-Marie-Tooth disease. Our results identify Phe57 as a residue regulating conformational changes that may accompany membrane surface binding and ligand exchange in P2 and other FABPs

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

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-4 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 8 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

Production and crystallization of a panel of structure-based mutants of the human myelin peripheral membrane protein P2.

The myelin sheath is a multilayered membrane that surrounds and insulates axons in the nervous system. One of the proteins specific to the peripheral nerve myelin is P2, a protein that is able to stack lipid bilayers. With the goal of obtaining detailed information on the structure-function relationship of P2, 14 structure-based mutated variants of human P2 were generated and produced. The mutants were designed to potentially affect the binding of lipid bilayers by P2. All mutated variants were also crystallized and preliminary crystallographic data are presented. The structural data from the mutants will be combined with diverse functional assays in order to elucidate the fine details of P2 function at the molecular level

Crystal and solution structure of NDRG1, a membranebinding protein linked to myelination and tumour suppression

N-myc downstream-regulated gene 1 (NDRG1) is a tumour suppressor involved in vesicular trafficking and stress response. NDRG1 participates in peripheral nerve myelination, and mutations in the NDRG1 gene lead to Charcot-Marie-Tooth neuropathy. The 43-kDa NDRG1 is considered as an inactive member of the α/β hydrolase superfamily. In addition to a central α/β hydrolase fold domain, NDRG1 consists of a short N terminus and a C-terminal region with three 10-residue repeats. We determined the crystal structure of the α/β hydrolase domain of human NDRG1 and characterised the structure and dynamics of full-length NDRG1. The structure of the α/β hydrolase domain resembles the canonical α/β hydrolase fold with a central β sheet surrounded by α helices. Small-angle X-ray scattering and CD spectroscopy indicated a variable conformation for the N- and C-terminal regions. NDRG1 binds to various types of lipid vesicles, and the conformation of the C-terminal region is modulated upon lipid interaction. Intriguingly, NDRG1 interacts with metal ions, such as nickel, but is prone to aggregation in their presence. Our results uncover the structural and dynamic features of NDRG1, as well as elucidate its interactions with metals and lipids, and encourage studies to identify a putative hydrolase activity of NDRG1

Additional file 1: Table S1. of Myelin-derived and putative molecular mimic peptides share structural properties in aqueous and membrane-like environments

Details of peptide properties. (PDF 60 kb