Micro-electron diffraction structure of the aggregation-driving N terminus of Drosophila neuronal protein Orb2A reveals amyloid-like β-sheets

Abstract

Amyloid protein aggregation is commonly associated with progressive neurodegenerative diseases, however not all amyloid fibrils are pathogenic. The neuronal cytoplasmic polyadenylation element binding protein is a regulator of synaptic mRNA translation and has been shown to form functional amyloid aggregates that stabilize long-term memory. In adult Drosophila neurons, the cytoplasmic polyadenylation element binding homolog Orb2 is expressed as 2 isoforms, of which the Orb2B isoform is far more abundant, but the rarer Orb2A isoform is required to initiate Orb2 aggregation. The N terminus is a distinctive feature of the Orb2A isoform and is critical for its aggregation. Intriguingly, replacement of phenylalanine in the fifth position of Orb2A with tyrosine (F5Y) in Drosophila impairs stabilization of long-term memory. The structure of endogenous Orb2B fibers was recently determined by cryo-EM, but the structure adopted by fibrillar Orb2A is less certain. Here we use micro-electron diffraction to determine the structure of the first 9 N-terminal residues of Orb2A, at a resolution of 1.05 Å. We find that this segment (which we term M9I) forms an amyloid-like array of parallel in-register β-sheets, which interact through side chain interdigitation of aromatic and hydrophobic residues. Our structure provides an explanation for the decreased aggregation observed for the F5Y mutant and offers a hypothesis for how the addition of a single atom (the tyrosyl oxygen) affects long-term memory. We also propose a structural model of Orb2A that integrates our structure of the M9I segment with the published Orb2B cryo-EM structure.

Keywords: amyloid; cytoplasmic polyadenylation element binding (CPEB) protein; electron microscopy; functional amyloid; intrinsically disordered protein; micro-electron diffraction (micro-ED); orb2; protein aggregation; protein structure.

Figure 1

The hydrophobic nine-residue N-terminal segment of Orb2A (M9I) forms amyloid-like fibers. A, schematic of full-length Orb2A, showing the N-terminal sequence unique to the Orb2A isoform (M9I, blue), followed by a 13-residue linker (white), and the Q/H-rich domain (pink). Both the linker and Q/H-rich domain are common to the Orb2A and Orb2B isoforms. The corresponding residue positions in the Orb2B isoform are in parentheses, and the segment forming the structured core of Orb2B fibers (32)are indicated in bold. B, left: segment sequences for wildtype M9I (blue) and M9I with the F5Y mutation (purple). Right: kinetic thioflavin T (ThT) assay comparing aggregation kinetics of wildtype M9I and M9I-F5Y at 1 mg/ml concentration (∼850 μM). The darker line represents the average reading of 3 independent technical repeats, and the lighter vertical bars below represent 1 standard deviation. C, left: negative-stain TEM analysis of fibers formed by M9I and M9I-F5Y after 20 h of incubation under identical conditions as (panel B) but without added ThT. Middle: X-ray diffraction of aligned dried fibers at 20 h incubation. Right: negative-stain TEM analysis of M9I and M9I-F5Y fibers after 5 days incubation. RRM, RNA-recognition motif.

Figure 2

Structure of M9I segment as determined by micro-electron diffraction. A, representative electron diffraction pattern from micro-ED data collection on M9I microcrystals. Strong reflections at 4.8 Å (white arrow) correspond to inter-β-strand spacings. Inset: electron micrograph of M9I microcrystals on Quantifoil grids; scale bar: 2 μm. B, structural model of M9I shows formation of in-register parallel β-sheets. Two sheets are viewed down the fibril axis, illustrating the water-excluded interface formed between sheets. Strands are related to each other via a 2₁-screw axis perpendicular to the fiber axis (class 4 steric zipper). Red spheres represent ordered water molecules. C, view perpendicular to fibril axis, showing side chain stabilizing interactions along the fibril axis. Asparagine 7 (N7) side chain forms a ladder of hydrogen bonds, and aromatic residues (Y2, F5, F8) stack in a parallel-displaced fashion. F5 and F8 also interact closely in the dry interface. Atomic separations are indicated in red; distances between aromatic side chains were calculated using the benzyl ring centroid point (red stars). Red spheres represent ordered water molecules. D, space-filling model of M9I colored according to the Kyte-Doolittle hydrophobicity scale (magenta-hydrophilic, teal-hydrophobic), showing tight packing of hydrophobic residues in the intersheet interface.

Figure 3

Phenylalanine replacements reduce fibrillation of Orb2A prion-like domain. A, schematic of Orb2A prion-like domain (PLD) constructs (residues 1–80) with either the fifth (F5Y) or eighth (F8Y) phenylalanine replaced by tyrosine. B, ThT assay comparing aggregation of 10 μM wildtype PLD (blue) to the F5Y (purple) or F8Y (green) PLD constructs. Samples were incubated at 25 °C in Hepes-KCl-urea pH 7.4 buffer. The darker line represents the average reading of technical triplicates, and the lighter vertical bars represent 1 standard deviation. C, negative stain TEM imaging of WT, F5Y, and F8Y Orb2A-PLD after 7 days incubation in identical conditions as above, but without ThT added. ThT, thioflavin-T; TEM, transmission electron microscopy.

Figure 4

Proposed structural model of an Orb2A–Orb2B heterocomplex. Aggregation of Orb2A (gray and purple) may initially be driven by steric zipper formation of the hydrophobic N-terminal M9I segment. The downstream Q/H-rich region, connected by a disordered 13-residue linker, would then be oriented in-register and can adopt an identical protofilament structure as that formed by endogenous Orb2B fibers (32). Formation of such an Orb2A protofibril seed could then nucleate Orb2B (green) fibril formation via structurally homotypic seeding of their identical Q/H-rich regions.