Droplet and fibril formation of the functional amyloid Orb2

Abstract

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Calmodulin binds the N-terminus of the functional amyloid Orb2A inhibiting fibril formation

“…We then wondered if Orb2A 1-88 was still forming fibrils but using a different domain as its cross-β core. For example via its Q/H-rich domain that was the core of ex vivo Orb2 fibrils or of the recombinant Orb2ΔRBD fragments that forms fibrils via phase separation [ 5 , 6 ]. To determine whether or not Orb2A 1-88 was aggregating in the Q/H-rich domain, we again used EPR to track the change in amplitude over time, but this time the MTLS label was at residue 34 (34R1) i.e.…”

“…In this construct, Q/H-rich fibril core of Orb2 was not observed. On the other hand, Orb2AΔRBD fibrils that were made via phase separation showed the presence of the Q/H-rich fibril core rather than the N-terminal core [ 6 ], suggesting that these cores might be mutually exclusive. Similarly, mutually exclusive fibril cores were recently reported for TDP-43 [ 40 ].…”

“…Both are present in the synapse, though Orb2B is found abundantly throughout the cytoplasm whereas Orb2A concentrations are low [3]. Although both Orb2A and Orb2B can form cross-β (amyloid) fibrils in vitro, Orb2A is required for Orb2 aggregation in vivo and both isoforms are found together in in vivo aggregates [3][4][5][6]. This aggregation is necessary for long-term memory [3].…”

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

“…In vitro, Orb2A is reported to form parallel in-register β-sheets over time (31,33,39), with one ssNMR study indicating that the M9I segment, but not the Q/H-rich region, forms the ordered fiber core (33). Orb2A can also undergo liquid-liquid phase separation and subsequent fiber formation, although analysis of immobile residues in that study did not clearly point to the presence of M9I segment (40). In a solution-state NMR study of the Orb2A PLD, the Q/H-rich region adopted varying degrees of α-helical secondary structure whereas the rest of the protein, including M9I, remained disordered (41), and in the presence of lipids the N-terminus was found to form an α-helix and Orb2A fibrillation was inhibited (42).…”

General Principles Underpinning Amyloid Structure

“…In agreement with this, amyloids formed by diverse polypeptides exhibit striking similarities in their mechanisms of self-assembly and pathogenesis. These include: (i) a capacity for seeding and prion-like spreading (Jarrett and Lansbury, 1993 ); (ii) a tendency for pathogenic amyloids to have a highly stable core, whereas many functional amyloids exhibit adaptations to reduce core stability (Sawaya et al, 2021 ); (iii) a nucleated polymerization mechanism of formation (Jarrett and Lansbury, 1992 ; Come et al, 1993 ); (iv) a tendency to nucleate in oligomeric or droplet-like intermediates that are often rich in β-structure (e.g., Serio et al, 2000 ; Bitan et al, 2001 ; Chimon et al, 2007 ; Thakur et al, 2009 ; Lee et al, 2011 ; Molliex et al, 2015 ; Shammas et al, 2015 ; Iljina et al, 2016 ; Ambadipudi et al, 2017 ; Yang et al, 2018 ; Ray et al, 2020 ; Ashami et al, 2021 ); (v) the toxicity of diverse amyloid-related oligomers, and some amyloid fibrils (e.g., Lambert et al, 1998 ; Tucker et al, 1998 ; Rochet et al, 2000 ; Mukai et al, 2005 ; Quist et al, 2005 ; Xue et al, 2009 ; Milanesi et al, 2012 ; Kollmer et al, 2016 ; Schützmann et al, 2021 ); (vi) the capacity of both mature amyloids and oligomers to disrupt lipid membranes (e.g., Rhee et al, 1998 ; Quist et al, 2005 ; Kayed et al, 2009 ; Xue et al, 2009 ; Jang et al, 2010 ; Milanesi et al, 2012 ; Kollmer et al, 2016 ; Flagmeier et al, 2020 ); (vii) and the ability of amyloids to induce further aggregation and toxicity by secondary nucleation (Ruschak and Miranker, 2007 ; Andersen et al, 2009 ; Mizuno et al, 2011 ; Cohen et al, 2013 ; Gaspar et al, 2017 ; Frankel et al, 2019 ). Just as the structural similarities between amyloid fibrils point to shared principles of self-assembly, their behavioral similarities point to shared structure-activity principles.…”