Conformational dynamics in the disordered region of human CPEB3 linked to memory consolidation

RamírezdeMingoetal. BMC Biology (2022) 20:129 https://doi.org/10.1186/s12915-022-01310-6 RESEARCH ARTICLE Conformational dynamics inthedisordered region ofhuman CPEB3 linked tomemory consolidation D. Ramírez de Mingo1, D. Pantoja‑Uceda2, R. Hervás3, M. Carrión‑Vázquez1* and D. V. Laurents2* Abstract Background: Current understanding of the molecular basis of memory consolidation points to an important func‑ tion of amyloid formation by neuronal‑specific isoforms of the cytoplasmic polyadenylation element binding (CPEB) protein family. In particular, CPEB is thought to promote memory persistence through formation of self‑sustaining prion‑like amyloid assemblies at synapses, mediated by its intrinsically disordered region (IDR) and leading to perma‑ nent physical alterations at the basis of memory persistence. Although the molecular mechanisms by which amyloid formation takes place in CPEB have been described in invertebrates, the way amyloid formation occurs in the human homolog CPEB3 (hCPEB3) remains unclear. Here, we characterize by NMR spectroscopy the atomic level conformation and ps‑ms dynamics of the 426‑residue IDR of hCPEB3, which has been associated with episodic memory in humans. Results: We show that the 426‑residue N‑terminal region of hCPEB3 is a dynamic, intrinsically disordered region (IDR) which lacks stable folded structures. The first 29 residues, M1QDDLLMDKSKTQPQPQQQQRQQQQPQP29, adopt a helical + disordered motif, and residues 86–93: P83QQPPPP93, and 166–175: P166PPPAPAPQP175 form polyproline II (PPII) helices. The (VG)5 repeat motif is completely disordered, and residues 200–250 adopt three partially populated α‑helices. Residues 345–355, which comprise the nuclear localization signal (NLS), form a modestly populated α‑helix which may mediate STAT5B binding. These findings allow us to suggest a model for nascent hCPEB3 structural transi‑ tions at single residue resolution, advancing that amyloid breaker residues, like proline, are a key difference between functional versus pathological amyloids. Conclusion: Our NMR spectroscopic analysis of hCPEB3 provides insights into the first structural transitions involved in protein–protein and protein‑mRNA interactions. The atomic level understanding of these structural transitions involved in hCPEB3 aggregation is a key first step toward understanding memory persistence in humans, as well as sequence features that differentiate beneficial amyloids from pathological ones. Areas: Biophysics, Structural Biology, Biochemistry & Neurosciences. Keywords: Memory consolidation, Intrinsically disordered proteins, NMR spectroscopy © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background The molecular basis of long-term memory, which endures decades despite being built by ephemeral biomolecules, has long fascinated biochemists [1]. Seminal findings by Si, Lindquist, and Kandel suggested that long-term changes in synaptic efficacy require a self-perpetuating amyloid state in the Aplysia CPEB (ApCPEB) Open Access *Correspondence: [email protected]; [email protected] 1 Instituto Cajal, IC‑CSIC, Avda. Doctor Arce 37, 28002 Madrid, Spain 2 Instituto de Química‑Física Rocasolano, IQFR‑CSIC, Serrano 119, 28006 Madrid, Spain Full list of author information is available at the end of the article Page 2 of 18 RamírezdeMingoetal. BMC Biology (2022) 20:129 [2, 3]. This change in CPEB’s conformation would lead to permanent alterations at the synapse constituting a physical substrate of memory persistence. To attain the aggregated state necessary to stabilize memory, ApCPEB contains an N-terminal IDR that is very rich in glutamine (Q) residues which losses α-helix and gains coiled-coil and β-sheet structure during amyloid formation in vitro [4, 5]. The Drosophila homolog of CPEB, called Orb2, behaves in a similar fashion even though its N-terminal IDR has a lower glutamine residue content and a more tightly regulated amyloid formation [6–9]. 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 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]. Compared to the Aplysia and Drosophila homologs, hCPEB3’s content of glutamine residues in its 426-residues long IDR is lower, and it contains diverse segments which are enriched for certain residues such as Ser, Ala, Pro, Gly + Val, and hydrophobic residues (Table1). Mammalian CPEB3 travels to distinct neuronal regions to carry out multiple functions, where the 426-residue long IDR plays a key role (Fig.1A). Following its synthesis, CPEB3 is SUMOlyated, which has been reported to block CPEB3 aggregation [14]. Upon neuronal stimulation, CPEB3, which is mostly cytoplasmic, travels to the nucleus. This process is mediated by the karyopherin IPO5 through interactions with the NLS in the first RNA recognition motif (RRM) of hCPEB3 [15]. Inside the nucleus, CPEB3 interacts with STAT5B, which normally activates the transcription of genes such as EGFR, triggering signaling cascades thought to promote memory consolidation [16, 17]. CPEB3-STAT5B binding, driven by interactions between the IDR of hCPEB3 and residues 639–700 of STAT5B, downregulates STAT5B-dependent Table 1 Sequence of the disordered N‑terminal region of hCPEB3 Segment 1 spans hCPEB3 residues 1-100; segment 2, residues 51 – 150; segment 3, residues 101 – 200; segment 4, residues 151 – 250; segment 5, residues 201 – 300; segment 6, residues 251 350; segment 7, residues 302 – 400 and finally segment 8, which is composed of residues 352 – 450. Residues of structural or functional interest are in bold font. The putative dimethyl-Arg site (R308) is highlighted in green. The putative phosphoTyr site, Y341 plausibly recognized by STAT5B’s SH2 domain is highlighted in blue. The predicted nuclear export signal, L349-L353, is highlighted in yellow. Putative phosphorylation sites: S284, S290, S297, S298, S400, S407, S408, S411 and S412 are highlighted in red. Finally, residues belonging to the first RRM1 domain are written in italics. Page 3 of 18 RamírezdeMingoetal. BMC Biology (2022) 20:129 transcription [17]. However, the details of this interaction have not yet been addressed. By contrast, the 3D structure of the first CPEB3 RRM domain has been elucidated and revealed a β-hairpin (W471-G485) proposed to play a key role in RNA recognition [18]. This domain, as well as the second RRM domain and zinc finger (ZnF) motif, were reported to bind specifically to the 3′UTR mRNA of the AMPA receptor subunit GluR2 [19]. Together, CPEB3 and its target mRNA eventually exit the nucleus and can join distinct biomolecular condensates such as stress granules and neuronal granules, which provide physiological transport to dendritic spines, or to dendritic P-body-like granules [20], where CPEB3 stores GluR2 mRNA and downregulates its translation [19]. After synaptic activity in the hippocampus, SUMOylation of CPEB3 decreases [14] and CPEB3 converts, mediated by the IDR, from a translation repressor into a self-sustaining activator, promoting the translation Fig. 1 A hCPEB3 is present in multiple cellular compartments. Dendritic stimulation leads to temporary, phosphorylation‑mediated short‑term memory and increased synthesis of the protein CPEB3 (1). Composed of an N‑terminal disordered region (black) which includes a Q‑rich segment aiding functional aggregation (magenta), hCPEB3 also contains RRM domains (cyan) and a ZZ‑Zinc finger domain (turquoise). Upon continued neuro‑stimulation, CPEB3 enters the nucleus through the nuclear pore (light magenta), which is a macromolecular condensate (2). Once in the nucleus, CPEB3 indirectly regulates transcription through STAT5B (3) and binds to certain mRNAs (4, red). This binding suppresses translation. After exiting the nucleus through the nuclear pore, (5) CPEB3 + mRNA may associate with a stress granule (6, rose) during moments of adverse conditions. In the absence of stress (7) or its passing (8), CPEB3 + mRNA will combine with another condensate called neuronal granules (light green) for transport to dendritic spines (9), where CPEB3 + mRNA associate with still another class of condensate called a dendritic P‑body‑like structure (golden) [21]. Further neuronal stimulation (10) causes synapse‑specific deSUMOlyation, CPEB3 aggregation, and translational activation of previously repressed mRNA, leading to morphological changes and fortification of the spine, which is proposed to be the basis of long‑term memory. This is a simplified model based on that of Kandel and coworkers [22]. B CPEB3 domain composition and its N‑terminal intrinsically disordered domain (gray) contains key elements with preferred conformers colored blue for α‑helix, magenta for polar amyloidogenic, black for hydrophobic amyloidogenic, green for PPII helix, purple for the putative phosphoTyr site, and red for highly disordered segments. The two RRM domain are colored cyan and the C‑terminal Zinc Finger is shown in turquoise Page 4 of 18 RamírezdeMingoetal. BMC Biology (2022) 20:129 of AMPA receptors [12]. This leads to structural modifications, including a more robust actin network, which fortify the spine and permanently enhance neurotransmission at a given particular synapse [13]. The hypothesis that CPEB3 functional amyloid formation is key for memory persistence in mammals is supported by the impairment in long-term memory and long-term potentiation in the CPEB3 conditional knockout mice [12]. In our species, the causal role of human CPEB3 (hCPEB3) in memory is corroborated by observations that persons carrying a rare CPEB3 allele, which leads to a decreased production of hCPEB3 protein, have episodic memory impairments [23]. The 426-residue long IDR of hCPEB3 plays a key role mediating memory persistence through this prion-like mechanism. It contains an amyloid-forming region spanning residues 1–200 and a condensate-promoting region formed by residues 250–426 which are linked by an alanine rich segment [24]. The full IDR is followed by two folded RRM which bind RNA and finally a ZZ-type ZnF domain (Fig.1B). Recent sequence and deletion mutational analyses of the IDR have begun to identify subregions key for aggregation, such as the first 30 residues [13]. However, for the hCPEB3 IDR, programs to predict secondary structure tendencies give different outputs, Alpha Fold 2 structural predictions [25] are marked as low to very low confidence (see https:// alpha fold. ebi. ac. uk/ entry/ Q8NE35), and, to date, no high-resolution experimental data on the partial structures or motions have been reported. Here, motivated by the key roles of the IDR in CPEB prion-like aggregation required for memory persistence, we characterize the atomic level conformations and dynamics of the complete IDR of hCPEB3 by NMR spectroscopy. Results hCPEB3’s IDR ischiefly disordered As a first step to experimentally characterize hCPEB3’s IDR, we probed the complete 426-residue IDR of hCPEB3 by biophysical techniques and homonuclear NMR. Its fluorescence emission spectra, recorded at temperatures ranging from 2 to 70°C, show emission maximum > 350nm. This is consistent with its six Trp residues being solvent exposed and not buried in the hydrophobic core of a folded domain (AdditionalFile1. Fig. S1A) [26]. The far UV CD spectra of the hCPEB3 IDR also shows the hallmarks of a disordered protein, namely a minimum near 200nm [27]. No spectral features indicative of α-helix and β-sheet; namely, minima at 208, 218, or 222nm and no maximum at 195nm, are evident (AdditionalFile1. Fig. S1B). The 1D 1H and 2D 1H-1H NOESY spectra show 1H signals clustered into narrow bands near the values observed for short, unstructured peptides (AdditionalFile1. Fig. S1C) [28, 29]. The sequence alignment of several representative vertebrate CPEB3 proteins using the T-Coffee program is shown in AdditionalFile1. Fig. S2. Very similar results were obtained from the Omega Clustal program (not shown). Whereas most IDPs show poor levels of sequence conservation, some stretches rich in hydrophobic residues, such residues M1-T12, W111-F139, and Y341-I357, are highly conserved. By contrast, glutamine rich, alanine rich, and some proline rich segments are present only in mammals. Taking all these data together, the presence of large, stably folded domains in the IDR can be ruled out, but short segments with partly populated secondary structures could still be present. Atomic level characterization reveals partially structured elements inhCPEB’s N‑terminal “disordered” region To discover and characterize possible segments with partial secondary structure, we applied multidimensional heteronuclear NMR. As the full length IDR is too long to characterize by this methodology, we have followed the “divide and conquer” approach implemented by Zweckstetter etal. to characterize tau, a similarly sized IDP implicated in Alzheimer’s disease and other tauopathies [30]. As described in the “Methods” section, and shown in Table1, eight overlapping segments of 100 residues were characterized. Using our powerful 13CO, 15N, 1HN-based assignment strategy, over 99% of the main chain 13CO, 13Cα, 15N, 1HN, and the 13Cβ resonances were assigned for residues 1–450 of hCPEB3. The chemical shifts of the complete IDR of hCPEB3 are reported in the BMRB (entry number 50256), and the original 2D and 3D spectral data have been deposited in the Mendeley data repository. The assigned 2D 1H-15N HSQC and 2D 13CO15N spectra of segment 4 are shown in AdditionalFile1. Fig. S3 and AdditionalFile1. Fig. S4, respectively. The 2D 1H-15N HSQC spectra of segments 1, 3, 4, 5, 6, 7, and 8 are shown in AdditionalFile1. Fig. S5. The similar positions of most 1H-15N signals in neighboring segments additionally suggest a sparsity of long range interactions under these conditions. Likewise, the majority of the crosspeaks of the same residues in adjacent segments also overlap or are close together in the 2D 13CO15N spectra of segments 1, 3, 4, 5, 6, and 8 (AdditionalFile1.Fig. S6). Multiple attempts to express and purify hCPEB3 segment 2, which spans residues 51–150, by recombinant methods were unsuccessful. Nevertheless, all the residues within segment 2 are present and have been characterized structurally in the context of segments 1 and 3. To test if there might be some structure in the neighborhood Page 5 of 18 RamírezdeMingoetal. BMC Biology (2022) 20:129 of residues 91–110 located in the middle of segment 2 and the Cand N-termini of segments 1 and 3, respectively, we studied the conformation of a twenty-residue peptide corresponding to this region by NMR spectroscopy. No significant trends towards structure formation were detected (AdditionalFile1. Fig. S7). Fig. 2 The N‑terminal 25 residues of hCPEB3 adopt a hydrophobic α‑helix followed by a disordered polyQ segment flanked by PQP mini‑breaker motifs. The N‑terminus of hCPEB3 contains an α‑helix‑forming and a disordered amyloidogeneic Q4RQ4 segments, which are separated by PQP mini breaker motifs. A Schematic representation as a gray cylinder of partial (20%) α‑helix formation by the first ten residues of hCPEB3. The disordered conformational ensemble of residues 11–32 is represented curved lines colored purple, blue, cyan, green, orange, red, and black. B 13Cα (blue) and 13CO (black) conformational chemical shifts indicate a 20% population of helix at 25 °C. Uncertainties in the conformational chemical shifts (Δδ) are 0.02 and 0.10 ppm for 13CO and 13Cα, respectively. C {1H}‑15 N NOE and D R1ρ relaxation measurements indicate that this helical conformation is less mobile than the polyQ segment at ns/ps and µs/ms timescales, respectively at 25 °C. Error bars are shown in C and D but are small as the estimated uncertainties are < 0.01 for the hNOE and < 0.1 s−1 for R1ρ. Missing values in C and D are due to overlap of 1H15N peaks or a lack of 1H15N signals in the case of proline residues (see Additional File 1. Fig. S9 for additional values from 13C‑detected relaxation experiments). E Eight representative backbone conformers, colored purple, blue, teal, green, amber, orange, red, and black, of the proline rich segment, H84‑Q94, featuring a PPII helix that spans residues P86‑Q94. All heavy atoms are shown for the purple conformer Page 6 of 18 RamírezdeMingoetal. BMC Biology (2022) 20:129 The 13Cα and 13CO conformational chemical shifts (Δδ) of the hCPEB3 segments 1 and 3–8 are plotted in AdditionalFile1. Fig. S8. These data show five segments, comprising residues 1–10, 202–210, 222–234, 238–246, and 346–356, with significantly high Δδ13Cα and Δδ13CO values. Such values are characteristic of partially populated α-helices and are examined in detail in the following paragraphs. The first residues ofhCPEB3 adopt apartly populated α‑helix which precedes theQ‑rich stretch The conformational chemical shifts point to the formation of partly populated (20%) α-helix in the first ten residues of the protein (Fig. 2A, B). Increased conformational chemical shifts are observed at 5°C, reflecting a higher amount of helical structure upon cooling. Standard 1H-detected 15 N relaxation measurements detect that these residues are the most rigid part of segment 1 (Fig. 2C, D). These results are corroborated by 13C-detected 15 N relaxation experiments (AdditionalFile1 Fig. S9). The α-helix detected for these first residues extends N-terminally into the His/Tev tag. To rule out a possible structure-promoting effect on segment 1, we tried to remove it by proteolytic cleavage with the TEV protease. Multiple attempts failed, which suggests that the helix spanning the last residues of His/Tev tag and the first residues of the hCPEB3 IDR is present and impedes the proteolytic cleavage. Therefore, we characterized a dodecamer peptide whose sequence corresponds to the first twelve residues, M1QDDLLMDKSKT12, of the hCPEB3 IDR. The observation of a series of weak 1HNi—1HNi+1 nuclear Overhauser enhancement (NOE) crosspeaks reveals that this peptide has a slight tendency to form α-helix in aqueous buffer (AdditionalFile1. Fig. S10A). Fluorinated alcohols like trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP) are known to increase the population of helical conformations in peptides which have an α-helix forming tendency, but not in peptides which prefer to adopt β-strands or random coil [31]. In the presence of 20% HFIP, the population of α-helix in this peptide increases strongly, based on the observation of stronger and more numerous NOE crosspeaks as well as 1Hα and 13Cα conformational chemical shifts (AdditionalFile1. Fig. S10B). These findings evince that the first 12 residues of hCPEB3 do tend to adopt an α-helix. Interestingly enough, the polyQ segment, Q16QQQRQQQQ24, does not form an α-helix or a β-strand and appears to be thoroughly disordered and flexible (Fig.2A, B). A construct spanning residues 1–200 of hCPEB3, which contains the Q4RQ4 motif, plays a role in hCPEB3 amyloid formation as indirectly evidenced by the anti-amyloid action of the polyglutamine-binding peptide 1 (QBP1) [24]. This polyQ segment is preceded and followed by Pro-Gln-Pro residue triplets (P13QP15 and P25QP27). Considering the inhibitory effect of proline residues previously observed for polyQ amyloid formation in Huntingtin by Wetzel and co-workers [32], it is likely that these PQP mini-motifs check amyloidogenesis by the polyQ segment. The first 100 residues also contain a predicted SUMOylation site [24] at Lys 47 and ends with a proline-rich segment P86PQQPPPPQEPAAPG100, which is associated with solubility. Whereas recently reported NMR criteria [33] allow us to rule out that this stretch folds into a stable polyproline II (PPII) helical bundle, the steric limitations of polyproline segments mean that residues 86–93 adopt an isolated, partly populated PPII helix (Fig.2E). In fact, the consecutive proline residues show a distinct pattern of conformational chemical shifts; namely + 0.6 ppm, − 1.0, and − 0.3 for 13Cα, 13Cβ, and 13CO, respectively (Table2, AdditionalFile1 Fig. S11). Not observed in isolated proline residues, we advance that they are hallmarks of a PPII helical conformation. Residues 101–200 ofhCPEB3 contains arigid nonpolar segment andaPPII helix Regarding residues 101–200, no strong trends to adopt α-helical or β-structures are detected. Nevertheless, the stretch composed of residues, W111STGTTNAVEDSFFQGITPVNGTMLFQNF139 which contain numerous aliphatic and aromatic residues, shows relatively high rigidity, both on fast ns/ps as well as slower µs/ms timescales (Additional File 1. Fig. S12). This finding is interesting considering that this relatively hydrophobic segment also appears to be essential for hCPEB3 amyloid formation in vitro [24], and very recently, it has been reported to form amyloid in mouse CPEB3 [37]. In addition, the stretch of residues 161–190 Q1 61 HHQ QPP PPA 17 0P APQ PAQ PAQ 18 0P PQAQPPQQR190 has a very high Q/P content, and Pro and Gln are the residues with the highest intrinsic tendencies to adopt PPII helices [38]. The consecutive proline residues, P166PPPAPAPQP175, also display the characteristic PPII pattern of conformational chemical shifts (AdditionalFile1. Fig. S11 ABC) seen for residues 86–93. Although more weakly than long stretches of pure polyproline [39, 40], a synthetic peptide corresponding to residues P166-P175 of hCPEB3 binds to human Profilin 1, a known mediator of interactions with actin (AdditionalFile1. Fig. S11 D). Residues 201–300 contain three α‑helical segments andadisordered (VG)5 segment Significant 13Cα and 13CO chemical shift deviations with respect to values predicted for a statistical coil, Page 7 of 18 RamírezdeMingoetal. BMC Biology (2022) 20:129 for three residue segments spanning A202QRSAAAY 210 GHQPIMTSKP220-S221SSSAVAAAA 230AAAAA) SSASS240SWNTHQSVHAA250 (Fig. 3A). These results indicate that the three segments of underlined residues adopt partially populated α-helices. Based on the magnitude of the conformational chemical shifts, the helical populations are different, being about 30% for the A202-Y210 α-helix, 80% for the S222-A234 α-helix and 20% for the A238-Q246 α-helix at 5°C; whereas these populations decrease at 25°C to approximately 10%, 40%, and 15%, respectively, they are still significant (Fig.3B). The presence of the first and second helices are confirmed by 1HN-1Hα coupling constants (AdditionalFile1 Fig. S13). Moreover, analysis with TALOS + , which predicts secondary structure taking into account 13Cβ, 15N and 1Hα chemical shifts in addition to 13Cα and 13CO, confirms the presence of these three helical segments and structural calculations with CYANA suggest that the three helices do not tend to adopt a preferred alignment relative to each other (data not shown). The helices are not especially rigid on fast ps-ns time scales (Fig.3C) or the slower µs-ms time domain (Fig.3D) at 25°C but do show a heightened stiffness at 5°C (AdditionalFile1. Fig. S12). Helical wheel projections (Additional File 1. Fig. S14) suggest that different interactions contribute stability to these α-helices. Gly 211 and His 212 are positioned to stabilize the A202-Y210 α-helix by a C-capping motif [41]. Whereas Ala has a very high intrinsic helix forming propensity, the propensity of Ser is low [42]. In this segment, however, the Ser residues are positioned at the N-terminus of theα-helices, where adding negative charge via phosphorylation would increase the helical population, considering the well-known stabilizing effects of charge/ macrodipole interactions and N-capping H-bonds [43]. This proposal is supported by NMR spectroscopic characterization of a peptide EAVAAA AAA AKK, with a phosphomimetic N-terminal Glu residue, which reveals a modest increase in helicity as the pH is raised from three, where the Glu is mostly neutral to five, where the Glu is chiefly anionic (AdditionalFile1. Fig. S15). Although this sequence’s insolubility thwarts attempts to more directly test the impact of phosphorylation, we note that these Ser residues are placed at the positions where phosphorylation is expected to increase α-helix stability the most [44]. Moreover, at neutral pH, where phosphoserine carries two negative charges, the stabilization is substantially greater than at pH 4, where it carries one [44]. The last α-helix, A238-Q246, is less populated, but its stability might increase if W242 were to engage in long-range interactions, such as with the hydrophobic or cationic residues of the first α-helix, i.e. MQDDLLMDKSKT. To test this possibility, we studied two polypeptides containing the M1-T12 and A238-Q246 helical segments with and without an N-terminal Dansyl group, connected by a flexible (Gly)4 linker. The results of FRET and 2D NMR spectroscopy evince that this polypeptide adopts a conformational ensemble significantly more compact than a Table 2 Conformational chemical shifts for α‑helices, β‑strands, and PPII helices a From Spera and Bax (1991) [34] b From Wang and Jardetzky (2002) [35] for alanine c Calculated from Wishart and Skyes (1994) [36] d From Treviño etal. (2018) [33] e This study α‑Helix β‑Strand Gly‑rich PPII helical bundle Isolated Pro‑rich PPII helix δΔ 13Cα 3.1a/2.9b − 1.5a/ − 1.8 b − 0.6d + 0.6e δΔ 13Cβ − 0.4a/ − 0.8b + 2.2a/ + 2.7b + 0.3d − 1.0e δΔ 13CO 2.2c/2.2b − 2.2c/ − 2.1b − 0.2d − 0.3e (See figure on next page.) Fig. 3 Residues 201–250 adopt three partial populated α‑helices. A (Top) Schematic representation as gray cylinders of the three partially populated helices present in residues 200–250. (Bottom) One conformer with all three α‑helices is shown; residues are colored: cationic residues (R and K) = blue, aromatics (F, Y, W and H) = purple, anionic (E and D) = red, aliphatic (A, I, L, M) = dark gray, amyloidogenic (N and Q) = magenta, hydroxyl bearing (S and T) = cyan, and proline = green. B 13CO (black) and 13Cα (blue) conformational chemical shifts (Δδ) of residues 201–250 at 25 °C. Note that the second α‑helix which contains nine consecutive Ala residues has a relatively high helical population. Uncertainties in the conformational chemical shifts (Δδ) are 0.02 and 0.10 ppm for 13CO and 13Cα, respectively. C {1H}‑15 N NOE ratios. Values shown in dark blue are of individual 1H15N resonances; those in light blue correspond to overlapped peaks. D R1ρ values reveal the ps/ns and µs/ms time scales. Significantly higher {1H}‑15 N NOE ratios and R1ρ values are observed for these residues at 5 °C (Additional File 1. Fig. S10) Page 8 of 18 RamírezdeMingoetal. BMC Biology (2022) 20:129 Fig. 3 (See legend on previous page.) Page 9 of 18 RamírezdeMingoetal. BMC Biology (2022) 20:129 statistical coil but that the α-helix contents are not significantly altered (AdditionalFile1. Fig. S16). One of the most striking features in hCPEB3’s sequence is a short dipeptide protein motif (Val-Gly)5 spanning residues 271–281, which is reminiscent of longer (AlaGly)N and (Pro-Gly)N and (Arg-Gly)N dipeptide repeat proteins encoded by mutant C9orf72 which have been implicated in ALS [45, 46]. Our in silico analysis identified this segment as having a high potential to form amyloid [24]. In the context of hCPEB3, however, this segment is among the most disordered and flexible of all the zones of the IDR (AdditionalFile1. Fig. S8, S12). Just beyond the (VG)5 segment, there is a stretch of 15 residues, S284PLNPISPLKKPFSS298, whose NMR parameters indicate disorder and flexibility (AdditionalFile1. Fig. S8, S12). Nevertheless, this stretch contains four Ser residues reported to phosphorylated by protein kinase A (PKA) or calcium/calmodulin-dependent protein kinase II [47] (Table1) and therefore might be important for the transition between shortand long-term memory. Residues P303-PKFPRAAP311 are proline rich. Predictions suggest that Arg 308 can be methylated (Table1). This modification, whose impact has not been probed here, was reported to fortify cation–π interactions, reduce interactions with RNA, and destabilize condensates in other proteins [48]. The residues forming theNuclear Export Signal (NES) show amarked tendency toadopt α‑helical structures Significant conformational chemical shifts were also observed for residues L349-L353 which form the NES (Fig.4) indicating the presence of helical structure. Using the 13CO, 15N, 1HN, 13Cα, and 13Cβ chemical shift data as input, a family of conformers was calculated using the programs TALOS + and CYANA for residues P333-P363. This 31-residue segment is rich in aromatic (five) and aliphatic (six) residues, which is unusual for a disordered polypeptide. The resulting structures reveal that residues L346-L349 adopt one turn of α-helix and residues S352-M356 form a short α-helix (Fig.4A). It is notable that this conformer positions five nonpolar residues: L346, L349, L353, M354, and I357 on the same face of the α-helices. Y341, the putative phosphorylation site, is in an extended portion of the backbone and would be accessible for this post-translational modification (PTM). Whereas the conformational ensemble will contain many other structures, based on conformational chemical shifts as illustrated by the Δδ13Cα and Δδ13CO values shown in Fig.4B, the α-helical population is about one third. The presence of rigid conformers is corroborated by relatively high {1H}-15N NOE ratios (Fig.4C) and elevated transverse relaxation ratios (Fig.4D). Beyond the NES α-helix, no segments with preferred secondary structure are detected. The last residues of the segment 8 construct S426-RKVFVGGLPPDIDEDEITASFRRF450 belong to the RRM1 domain. According to the 3D structure [18], residues K428–G432 adopt a β-strand and residues E440–R449 form an α-helix in the context of the complete RRM1 domain. Here, these segments appear to be largely disordered. After a proline-rich zone ending around residue 380, the next fifty residues have a higher content of nonpolar residues and tend to be more rigid (Additional File 1. Fig S12). Residues 400–412 SHGDQALSSGLSS contain five Ser residues reported to be phosphorylated [47] (Table1). Discussion Like its homologs in Aplysia and Drosophila, hCPEB3 resembles the diverse superfamily of RNA-binding proteins that contain RRM and/or ZnF domains as well as intrinsically disordered prion-like regions, such as fused in sarcoma (FUS) or transactive response DNA-binding protein of 43kDa (TDP-43). FUS and TDP-43 are essential proteins, but their anomalous aggregation has been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Thus, comparing the CPEB3 and TDP-43 IDR conformational tendencies and dynamics may reveal why the latter can become pathological. The biophysical analysis of the full length hCPEB3 IDR shows that it lacks stable secondary structure. Disordered regions tend to change their sequence much more rapidly over the course of evolution due to a lack of structural constraints [49]. The strong conservation in vertebrate CPEB3 of the N-terminal α-helix and the 350’s (NES) α-helix and the nearby putative phosphoTyr site suggests physiological importance (AdditionalFile1. Fig. S2). The latter’s hypothetical binding to the SH2 domain of STAT5B might occlude the NES, leading to nuclear retention. Additional segments, like the hydrophobic stretch key for amyloid formation [24] and a cluster of three Trp residues (W242, W252 and W259), are also conserved from mammals to fish. In contrast, the N-terminal Gln-rich segment, the Pro-rich “breaker” regions and the Ala-rich helices are well conserved in mammals but not across all vertebrates. In some lower vertebrates, there is an alternative Q-rich region positioned after the 100’s hydrophobic segment. These elements’ rapid evolution could be related to the development of the mammalian brain. By contrast, the ability to move the poly-Q segment or substitute it for a hydrophobic amyloidogenic segment highlights the cassette or modular nature of PLDs, which was previously established for the Drosophila CPEB homolog [5]. Page 16 of 18 RamírezdeMingoetal. BMC Biology (2022) 20:129 Funding This study was supported by projects SAF2016‑76678‑C2‑1‑R (MC‑V) and SAF2016‑76678‑C2‑2‑R (DVL) from the Spanish Ministry of Economy and Competitivity (MINECO/AEI/FEDER, UE) and PID 2019‑109306RB‑I00/ AEI/10.13039/501100011033 from the Spanish Ministry of Science and Innova‑ tion (DVL). The authors declare no competing interests. Availability of data and materials All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. All data generated or analyzed during this study are included in this published article, its supplementary information files, and publicly available repositories. In particular, NMR chemical shift data are deposited in the BMRB database under file number 50256 [93], and NMR raw spectral data are deposited in the Mendeley data repository at Laurents, Douglas; Pantoja, David (2021) “hCPEB3_NMR_Spectra” Mendeley Data V1, doi:1,017,632/hpyjdp33fx/2, at this link: https:// data. mende ley. com/ datas ets/ hpyjd p33fx/2. Declarations Ethics approval and consent to participate No animal or human participation was involved in this research. Consent for publication All authors given their consent for publication. Competing interests The authors declare that they have no competing interests. Author details 1 Instituto Cajal, IC‑CSIC, Avda. Doctor Arce 37, 28002 Madrid, Spain. 2 Instituto de Química‑Física Rocasolano, IQFR‑CSIC, Serrano 119, 28006 Madrid, Spain. 3 School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China. Received: 14 June 2021 Accepted: 25 April 2022 References 1. Crick F. Memory and molecular turnover. 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