Structural transitions in Orb2 prion-like domain relevant for functional aggregation in memory consolidation

Abstract

The recent structural elucidation of ex vivo Drosophila Orb2 fibrils revealed a novel amyloid formed by interdigitated Gln and His residue side chains belonging to the prion-like domain. However, atomic-level details on the conformational transitions associated with memory consolidation remain unknown. Here, we have characterized the nascent conformation and dynamics of the prion-like domain (PLD) of Orb2A using a nonconventional liquid-state NMR spectroscopy strategy based on 13C detection to afford an essentially complete set of 13Cα, 13Cβ, 1Hα and backbone 13CO and 15N assignments. At pH 4, where His residues are protonated, the PLD is disordered and flexible, except for a partially populated α-helix spanning residues 55-60, and binds RNA oligos, but not divalent cations. At pH 7, in contrast, His residues are predominantly neutral and the Q/H segments adopt minor populations of helical structure, show decreased mobility and start to self-associate. At pH 7, the His residues do not bind RNA or Ca++, but do bind Zn++, which promotes further association. These findings represent a remarkable case of structural plasticity, based on which an updated model for Orb2A functional amyloidogenesis is suggested.

Preferred conformations in the disordered region of human CPEB3, a functional amyloid linked to memory consolidation

“…Indeed, inhibition of Orb2 amyloid formation targeting the N-terminal IDR specifically impairs memory consolidation, but not short-term memory in Drosophila [6][10]. Due to numerous histidine (H) residues in the Orb2 Q/H-rich amyloid core comprised in the in the N-terminal IDR, pH may regulate this structure’s stability as suggested by CryoEM analysis [5] and characterization by NMR spectroscopy [11] In mammals, the N-terminal IDR of the neuronal-specific isoform of CPEB3 is crucial for amyloid formation and memory consolidation [12] [13]. The regulation of functional amyloid formation in mammalian CPEB3 appears to be even more sophisticated due to multiple mechanisms involving post-translational modifications [14], and feedback loops to maintain hCPEB3 expression levels [13].…”

Insight into Polyproline II Helical Bundle Stability and Folding from NMR Spectroscopic Characterization of the Snow Flea Antifreeze Protein Denatured State

“…Reduced, denatured sfAFP might retain some partially structured segments as has often been observed in intrinsically disordered (26) or chemically denatured proteins (15) (27). Since 1 HN chemical shift dispersion arises mainly from >C=Oooo 1 H-N H-bond formation, the loss of dispersion observed for all the conditions studied here indicates that the interhelical H-bond network has broken down following reduction.…”

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

“…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 ). The M9I segment was not observed in the endogenous Orb2 cryo-EM structure ( 32 ), but this may be expected given that the Orb2B isoform is predominantly expressed.…”