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. Both anti-Arc nanobodies bind specifically 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 TARPγ2-derived peptide from the binding site. Thus, both anti-Arc-NL nanobodies could be used 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 allow to develop diagnostic and treatment tools.

The intrinsically disordered protein glue of the myelin major dense line: Linking AlphaFold2 predictions to experimental data.

Numerous human proteins are classified as intrinsically disordered proteins (IDPs). Due to their physicochemical 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 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 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 experimental data related to protein structure and molecular interactions. We observe that the predicted models have helical segments that closely correspond to the membrane-binding sites on both proteins. We furthermore analyse the fits of the models to synchrotron-based X-ray scattering and circular dichroism data from the same IDPs. The models are likely to represent the membrane-bound state of both MBP and P0ct, rather than the conformation in solution. Artificial intelligence-based models of IDPs appear to provide information on the ligand-bound state of these proteins, instead of the conformers dominating free in solution. We further discuss the implications of the predictions for mammalian nervous system myelination and their relevance to understanding disease aspects of these IDPs.

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 ß-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.

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. 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.

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

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 ß 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.