AbstractAmyloids play key pathological roles in a score of neurodegenerative diseases, but they are also essential to several physiological processes. Perhaps the most fascinating functional amyloid is formed by the Cytoplasmic Polyadenylation Element Binding protein (CPEB) protein family as its structural conversion is essential for memory consolidation in different animals. In vitro, CPEB amyloid formation is driven by the intrinsically disordered region (IDR) present in neuronal-specific isoforms. However, the underlying conformational transitions from the monomeric state to the amyloid state remain poorly understood. Here, we characterize the residue level conformational preferences and ps-ns dynamics for human CPEB3 (hCPEB3) by high field, heteronuclear NMR spectroscopy. At 426 residues, this is the second longest IDR characterized to date in such detail. We find that the residues 1–29: M1QDDLLMDKSKTQPQPQQQQRQQQQQPQP29, adopt an α-helical+disordered Q-rich motif. Similar helix + Q/N-rich motifs are observed in CPEB homologs as well as other RNA-binding proteins like TDP-43 that form pathological amyloids. Residues 86-93: P83QQPPPP93, and residues 166-175: P166PPPAPAPQP175 form polyproline-II (PPII) helices, and we propose NMR chemical shift-based criteria for identifying this conformation. We advance that the presence of helix and amyloid breaker residues, such as proline, in hCPEB3 and its absence in TDP-43 may be a key difference between the functional and pathological amyloids in higher eukaryotes. While the (VG)5 repeat motif (residues 272-282) appears to be completely disordered, residues S221-A235 form a highly populated, rather rigid α-helix and two nearby segments adopt partially populated a-helices. Residues 345–355 also form a partially populated α-helix. These residues comprise the nuclear export signal and border a putative phosphoTyr site which may mediate STAT5B binding. Thus, nascent hCPEB3 protein is not fully disordered like a blank sheet of paper; instead, it contains creases which guide the engraving of our memories. Based on these findings and previous results, a working model for hCPEB3 structural transitions in human memory consolidation is advanced.HighlightsHuman CPEB3’s aggregation-prone disordered region is studied by NMR.In the monomeric state, the polyQ stretch is disordered and flexible.5 α-helices, 2 PPII helices and a rigid nonpolar stretch are identified.Association of the first 4 α-helices could promote functional amyloid formation.The PPII helices could negatively regulate this process.The last α-helix forms the NES and putatively regulates nucleocytoplasmic transport via STAT5B.Short SummaryAmyloids are implicated in neurodegenerative diseases, but they are also essential to several physiological processes, including memory consolidation, neuronal-specific isoforms of the Cytoplasmic Polyadenylation Element Binding (CPEB) protein family. Here, we characterize the atomic level conformation and ps-ns dynamics of the 426-residue intrinsically disordered region (IDR) of human CPEB3 (hCPEB3), which has been associated with episodic memory in humans, by NMR spectroscopy. The first 29 residues: M1QDDLLMDKSKTQPQPQQQQRQQQQQPQP29, adopt a helical+disordered motif, which is key for in vitro amyloid formation. Residues 86-93: P83QQPPPP93, and 166-175: P166PPPAPAPQP175 form polyproline II (PPII) helices. We advance that the presence of amyloid breaker residues, such as proline, is a key difference between functional versus pathological amyloids. While the (VG)5 repeat motif is completely disordered, residues 200-250 adopt three partially populated α-helices. Residues 345 – 355, which comprise the nuclear localization signal, form a modestly populated α-helix and border a phosphoTyr which may mediate STAT5B binding. These findings allow us to advance a model for hCPEB3 structural transitions in memory consolidation in humans.Abstract FigureTable of Contents Graphic
Characterization of the bipartite degron that regulates ubiquitin-independent degradation of thymidylate synthase