Biology subjects

Krokengen, O. C.

Publications and source records attributed to Krokengen, O. C..

6 recordsLinked to original sources

bioRxiv2024

On the synergy between myelin proteins P0, MBP, and P2 in peripheral nerve major dense line formation

Myelin is a proteolipid membrane multilayer held together by a set of proteins. The proper formation and function of the myelin sheath relies on the coordinated action of several key myelin proteins. Research exploring how proteins from the peripheral myelin cytoplasmic apposition - myelin basic protein (MBP), the cytoplasmic tail of myelin protein zero (P0ct), and peripheral myelin protein 2 (P2) - interact with each other and with myelin-like membranes was conducted using various techniques, such as small-angle X-ray diffraction (SAXD), differential scanning calorimetry (DSC), surface plasmon resonance (SPR), as well as electron and live epifluorescence microscopy. DSC revealed changes in lipid interactions depending on the protein combination, with MBP and P0ct binding more tightly to lipid membranes than P2, resulting in altered membrane fluidity and stability. These results were supported by SPR, which indicated that the myelin proteins may compete for membrane surface binding. Analysis of the Bragg peaks induced by the myelin proteins in lipidic environments showed both lamellar and non-lamellar phases in protein-lipid complexes. The results indicate both synergy and competition between the three main proteins residing in the PNS myelin major dense line. Furthermore, the observed direct effects of myelin proteins on lipid membrane properties may be relevant to their function in myelinating cells.

biochemistry↗

bioRxiv2024

The cytoplasmic tail of myelin protein zero induces morphological changes in lipid membranes

The major myelin protein expressed by the peripheral nervous system Schwann cells is protein zero (P0), representing 50% of the total protein content in myelin. This 30-kDa integral membrane protein consists of an immunoglobulin (Ig)-like domain, a transmembrane helix, and a 69-residue C-terminal cytoplasmic tail (P0ct). The basic residues in P0ct contribute to the tight packing of the myelin lipid bilayers, and alterations in the tail affect how P0 functions as an adhesion molecule necessary for the stability of compact myelin. Several neurodegenerative neuropathies are related to P0, including the more common Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), but also rare cases of motor and sensory polyneuropathy. We find that high P0ct concentrations affect the membrane properties of bicelles and induce a lamellar-to-inverted hexagonal phase transition, which causes the bicelles to fuse into long, protein-containing filament-like structures. These structures likely reflect the formation of semi-crystalline lipid domains of potential relevance for myelination. Not only is P0ct important for stacking lipid membranes, but time-lapse fluorescence microscopy shows that it might affect membrane properties during myelination. We further describe recombinant production and low-resolution structural characterization of full-length human P0. Our findings shed light on P0ct effects on membrane properties, and with successful purification of full-length P0, we have new tools to study in vitro the role P0 has in myelin formation and maintenance.

biochemistry↗

bioRxiv2023

Structural characterization of two nanobodies targeting the ligand-binding pocket of human Arc

The activity-regulated cytoskeleton-associated protein (Arc) is a complex regulator of synaptic plasticity in glutamatergic neurons. Understanding its molecular function is key to elucidate the neurobiology of memory and learning, stress regulation, and multiple neurological and psychiatric diseases. The recent development of anti-Arc nanobodies has promoted the characterization of the molecular structure and function of Arc. This study aimed to validate two anti-Arc nanobodies, E5 and H11, as selective modulators of the human Arc N-lobe (Arc-NL), a domain that mediates several molecular functions of Arc through its peptide ligand binding site. The structural characteristics of recombinant Arc-NL-nanobody complexes were solved at atomic resolution using X-ray crystallography. The structures revealed that both anti-Arc nanobodies specifically bind to the multi-peptide binding site of Arc-NL. Isothermal titration calorimetry showed that the Arc-NL-nanobody interactions occur at nanomolar affinity, and that the nanobodies can displace a TARP{gamma}2-derived peptide from the Arc-NL binding site. Thus, both anti-Arc-NL nanobodies can be used in vitro as competitive inhibitors of endogenous Arc ligands. Differences in the CDR3 loops between the two nanobodies indicate that the spectrum of short linear motifs recognized by the Arc-NL should be expanded. We provide a robust biochemical background to support the use of anti-Arc nanobodies in attempts to target Arc-dependent synaptic plasticity. Function-blocking anti-Arc nanobodies could eventually help unravel the complex neurobiology of synaptic plasticity and help diagnose and treat Arc-associated synaptic disorders.

biochemistry↗

bioRxiv2022

The intrinsically disordered protein glue of myelin: Linking AlphaFold2 predictions to experimental data

Numerous human proteins are either partially or fully classified as intrinsically disordered proteins (IDPs). Due to their properties, high-resolution structural information about IDPs is generally lacking. On the other hand, IDPs are known to adopt local ordered structures upon interactions with ligands, which could be e.g. other proteins or lipid membrane surfaces. While recent developments in protein structure prediction have been revolutionary, their impact on IDP research at high resolution remains limited. We took a specific example of two myelin-specific IDPs, the myelin basic protein (MBP) and the cytoplasmic domain of myelin protein zero (P0ct). Both of these IDPs are known to be crucial for normal nervous system development and function, and while they are disordered in solution, upon membrane binding, they partially fold into helices, being embedded into the lipid membrane. We carried out AlphaFold2 predictions of both proteins and analysed the models in light of previously published data related to solution structure and molecular interactions. We observe that the predicted models have helical segments that closely correspond to the characterised membrane-binding sites on both proteins. We furthermore analyse the fits of the models to SAXS data from the same IDPs. Artificial intelligence-based models of IDPs appear to be able to provide detailed information on the ligand-bound state of these proteins, instead of the form dominating free in solution. We further discuss the implications of the predictions for normal mammalian nervous system myelination and their relevance to understanding disease aspects of these IDPs.

biophysics↗

bioRxiv2020

Determinants for forming a supramolecular myelin-like proteolipid lattice

Myelin protein P2 is a peripheral membrane protein of the fatty acid binding protein family. It functions in the formation and maintenance of the peripheral nerve myelin sheath, and several P2 mutations causing human Charot-Marie-Tooth neuropathy have been reported. Here, electron cryomicroscopy of myelin-like proteolipid multilayers revealed a three-dimensionally ordered lattice of P2 molecules between stacked lipid bilayers, visualizing its possible assembly at the myelin major dense line. A single layer of P2 is inserted between two bilayers in a tight intermembrane space of [~]3 nm, implying direct interactions between P2 and two membrane surfaces. Further details on lateral protein organization were revealed through X-ray diffraction from bicelles stacked by P2. Surface mutagenesis of P2 coupled to structural and functional experiments revealed a role for both the portal region and the opposite face of P2 in membrane interactions. Atomistic molecular dynamics simulations of P2 on myelin-like and model membrane surfaces suggested that Arg88 is an important residue for P2-membrane interactions, in addition to the helical lid domain on the opposite face of the molecule. Negatively charged myelin lipid headgroups anchor P2 stably on the bilayer surface. Membrane binding may be accompanied by opening of the P2 {beta} barrel structure and ligand exchange with the apposing lipid 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 towards deciphering the 3-dimensional assembly of a mature myelin sheath at the molecular level.

biochemistry↗

bioRxiv2019

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-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.

biophysics↗