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

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. 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 also 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 advanced. Highlights · The Orb2 prion like domain that forms the structures related to memory consolidation is studied by solution NMR. · The amyloidogenic Q/H-rich stretch is disordered and flexible at low pH. · Residues 55-60 form a partly populated α-helix at pH 4. · At pH 7, the Q/H-rich segment also adopts a low population of α-helix and rigidifies. · Zn++ binding induces associative changes in the Orb2 prion-like domain.


Introduction
The comprehension of how the nervous system encodes memory has been an important goal since the beginning of modern neuroscience. At the turn of the twentieth century, memory was proposed to be encoded as alterations in the neurite network (Cajal, 1894) as a persistent chemical change (Semon, 1904). This hypothesis has gained support over the decades (Hebb, 1949;Frey & Morris, 1997;Kandel, 2006;Josselyn & Tonegawa, 2020), and in 2003, Si, Lindquist and Kandel published a seminal paper providing the first evidence for the molecular basis of memory consolidation (Si et al., 2003). In that study, Si and coworkers proposed that functional aggregation of the protein CPEB into ordered structures is key to memory consolidation in Aplysia. Numerous studies have corroborated the key functional role of CPEB aggregation as well as that of its homologs in memory consolidation in Drosophila and mammals (Si & Kandel, 2016).
The Drosophila CPEB homolog, named Orb2, has been investigated in the most detail. Two Orb2 isoforms, called Orb2A and Orb2B, are relevant for long term memory in Drosophila melanogaster (Keleman et al., 2007;Krüttner et al., 2012). Whereas they differ at the N-terminus, both Orb2A and Orb2B contain a prion-like domain (PLD) rich in Gln and His residues, followed by a stretch of Gly and Ser residues (Sup. Fig. 1A). Whereas the Orb2A isoform is rare, it is essential for triggering the aggregation of the more abundant Orb2B and to maintain the memory trace (Krüttner et al., 2012;Majumdar et al., 2012). The first nine residues of Orb2A are unique to this isoform (Sup. Fig. 1A). Rich in hydrophobic side chains, these nine residues have been reported to interact with membrane mimetics , to be crucial for aggregation (Majumdar et al., 2012) and to adopt a bstrand within an Orb2 amyloid structure formed in vitro as characterized by solid-state NMR and EPR (Cervantes et al. 2016).
Both Orb2A and Orb2B contain a 31-residue long Q/H-rich stretch, followed by a short amphiphilic segment (residues N55LSAL59) and a second modest Q/H-stretch (H60HHHQQQQQ68) (Sup. Fig. 1A). Due to its similarity to amyloid-forming polyQ stretches in huntingtin (Scherzinger et al., 1997) and the androgen receptor (Eftekharzadeh et al., 2016), the main Q/H-rich stretch (residues 23-53) was suspected to be key for Orb2 amyloid formation. Indeed, while both Q/H-rich stretches appear rather disordered in Orb2 amyloids formed in vitro (Cervantes et al. 2016), Orb2 aggregation and memory consolidation can be blocked in Drosophila by a peptide which inhibits polyglutamine aggregation (Hervás et al., 2016). Very recently, the elucidation of the structure of physiological Orb2 amyloid isolated directly from Drosophila fly brains has shown that the main Q/H-rich stretch does in fact form the physiologically relevant amyloid structure . This amyloid features three-fold symmetry and side chain to mainchain H-bonds (Sup. Fig. 1B). The participation of numerous His residues in the amyloid core provides a singular mechanism for amyloid destabilization through acidification. Intriguingly, the first nine residues of Orb2A, while relevant for triggering aggregation (Majumdar et al., 2012), do not contribute to the amyloid core, which is composed of the more abundant Orb2B isoform ) (Sup. Fig. 1).
Following the second Q/H-rich stretch, there is a two-hundred residue, presumptively disordered region with an elevated content of Gly, Ser, Pro and Asn (Sup. Fig. 1A, Sup. Fig.   2). The conserved C-terminal half of Orb2 is composed of two RRM domains, which bind Orb2-specfic mRNA targets, and a ZZ-type zinc finger domain (Sup. Fig. 1A). ZZ-type zinc fingers are a special sub-class that bind two Zn ++ ions and generally mediate protein/protein interactions, not nucleic acid binding (Legge et al., 2004). In Orb2, the conserved C-terminal domains are crucial for binding mRNAs containing a uridine-rich 3'UTR (Mastushita-Sakai et al., 2010) and potentially different translation complexes . This binding suppresses the translation of mRNAs coding for factors promoting synapse formation, synapse growth and proteases (Mastushita-Sakai et al., 2010). Upon neuronal stimulation, Orb2 forms amyloid leading to the activation of these mRNAs (Khan et al., 2015). Due to its physiological importance, Orb2 aggregation is finely controlled. Studies have reported key roles of intron retention (Gill et al., 2017), phosphorylation and protein stability (White-Grindley et al., 2014) and chaperones  in tightly regulating Orb2 functional amyloid formation. Besides the mentioned control of Orb2 aggregation via acidification , Siemer and co-workers proposed that the spacing of His residues within the long Q/H-rich stretch would position them on the same face of a hypothetical a-helix . This may contribute to the binding of Ni ++ , Cu ++ and Zn ++ which might impact aggregation . Interestingly, the longer Q/H-rich region of Orb2 may be sequestered into pathological amyloids formed by expanded polyQ segments of huntingtin, providing a novel explanation for memory loss in dementia (Hervás et al., 2016;Joag et al., 2019).
Whereas all these results have provided molecular insight into Orb2's role in memory consolidation, much is still unclear regarding the atomic level details of the amyloid formation process. In particular, it is unknown whether the nascent protein contains elements of partial structure which predispose the formation of the functional amyloid, or alternatively, act as a safety mechanism to prevent premature or excess amyloid formation. The objective of this study is to characterize the atomic level conformation and dynamics of Orb2A PLD and the first residues of the G/S-rich region using solution NMR spectroscopy. Because pH is proposed to impact amyloid formation and stability , we have characterized the conformation of Orb2A both at pH 4, where His residues are positively charged which impedes aggregation, as well as pH 7, where neutral His are compatible with amyloid formation. In addition, the binding of Zn ++ promotes self-association via the Q/Hrich regions. The structural transitions observed may represent nascent structures in the process of aggregation into functional amyloids.

Results
The Orb2A PLD is disordered at pH 4.0, except for a partly populated a-helix spanning residues 55-60. Initial 1D 1 H and 2D 1 H-15 N HSQC spectra recorded in PBS at pH 7.0 revealed broad lines which are not optimal for spectral analysis and assignment. Therefore, following the low pH, low ionic strength strategy of Song and coworkers (Li et al., 2006), which we have recently applied to assign the snow flea antifreeze protein (Treviño et al., 2018) and a RepA winged helix domain (Pantoja-Uceda et al., 2020), spectra of Orb2A PLD were recorded at pH 4.0 in 1 mM acetic acid buffer at 25 ºC. Under these conditions, the signals were sharper and gave excellent quality 2D and 3D spectra. The 1 Ha, 1 HN, 13 Ca, 13 Cb and backbone 15 N and 13 CO chemical shifts recorded at pH 4.0, 25 ºC have been deposited in the BMRB data bank under accession number 50274. The assignments are complete except for the 1 Ha of proline residues and the 13 CO and 13 Cb of terminal Ser 88.
The assigned 2D 1 H-15 N HSQC and 2D 13 C-15 N CON spectra registered at pH 4.0, 25 ºC are shown in Figure 1A and 1B, respectively. The resonances are generally sharp and well defined except for clusters of overlapped Gln and His residues belonging to the amyloidogenic tract (Sup. Fig. 1B). The low 1 HN signal dispersion is a hallmark of disordered proteins and an assessment of the 13 Ca, 13 Cb, 13 CO and 15 N chemical shifts using the TALOS+ program confirmed the overall dearth of structure in the Orb2A PLD under these conditions. With regard to the glutamine side chain amide groups, their 1 H2-15 N signals are also clumped into overlapped peaks (Sup. Fig. 3). Notably, they do not show the striking pattern of disperse signals observed for the glutamine side chain resonances of the androgen receptor, which participate in side chain to backbone hydrogen bonds (Escobedo et al., 2019).
Orb2A PLD contains a single Cys residue. Its 13 Cb chemical shift indicates that it is reduced at pH 4.0 in 1 mM acetic acid buffer in the absence of reducing agent. Regarding the proline residues, all Xaa-Pro peptide bonds are in the trans conformation, which is expected for a disordered chain, as judged by their 13 Cb and 13 CO chemical shifts.
The 13 Ca, 13 Cb and 13 CO Dd values, which reveal partially populated elements of secondary structure, are plotted in Figure 2A. The segment composed of residues 55-60, which follows the amyloidogenic Q/H-rich stretch, adopts a minor population of a-helix.
Based on the conformational chemical shift (Dd) values expected for 100% a-helix of 3.1, -0.4 and 2.2 ppm for 13 Ca, 13 Cb and 13 CO, respectively (Spera & Bax, 1991;Wishart & Skyes 1994), the population of a-helix for these residues is approximately 20%. Otherwise, the Nterminal hydrophobic stretch, the Q/H-rich segments and the C-terminal G-rich element do not contain detectable populations of secondary structure under these conditions. Whereas the Dd 13 CO values of terminal G82GGGGG87 are anomalously high at pH 4 (and also pH 7, see below), which suggests a-helix formation, they are not corroborated by high Dd 13 Ca values, so this may rather reflect a shortcoming in the prediction of secondary structure from 13 CO d values for polyG repeats. As an orthogonal measure of secondary structure, the 3 JHNHA coupling constants were measured and analyzed using the Karplus equation to obtain backbone f angles. Some 3 JHNHA coupling constants appear to be somewhat lower for residues 55-60 ( Figure 2B) which may reflect the minor population of a-helix. Next, the µs/ms dynamics were assessed by R1r relaxation measurements. The obtained values are consistent with high mobility, except for residues 55-60, whose slightly elevated R1r rates reflect a modestly increased rigidity, due to the partial a-helical structure ( Figure 2C).
The population of marginally stable elements of secondary structure in polypeptides is generally enhanced by cooling. To check if this is the case for Orb2A PLD, additional spectra were recorded at 15ºC and 5ºC, pH 4.0. Nevertheless, the analysis of the conformational chemical shifts did not reveal any additional elements of secondary structure, beyond the 55-60 helix already detected at 25ºC ( Figure 2D). Finally, the fast ps/ns dynamics were characterized by { 1 H}-15 N NOE measurements at 5ºC. Low values indicative of flexibility are seen throughout the domain and are only slightly higher for the hydrophobic segment and a-helix spanning residues 55-60 ( Figure 2E). The G/S-rich C-terminal residues are especially mobile. Taking all these results together, we can conclude that at pH 4.0, where the His residues of Orb2A's PLD are chiefly positively charged, the polypeptide chain is disordered and flexible, except for the minor population of a-helix formed by residues 55-60 ( Figure 2F).

The amyloidogenic Q/H-rich segment adopts partly populated helical conformations and rigidifies at neutral pH.
A visible precipitate formed when the pH of the sample was increased from 4.0 to 7.0.
Nevertheless, the concentration of protein remaining in solution was sufficient to record good quality 2D 1 H-15 N and 1 H-13 C HSQC as well as 3D 1 H-detected HNCO, HNCA and CBCAcoNH spectra. The 2D 1 H-15 N HSQC spectrum ( Figure 3A) is significantly broadened. The average 1 HN peak width is 27 ± 4 Hz at pH 7.0 compared to 17 ± 2 Hz at pH 4.0 ( Figure 3B).
This is an indication of conformational interconversion, oligomerization, or both (Charlier et al., 2016). The assignments at pH 7.0 are complete for 13 Ca, 13 Cb, 1 HN and backbone 13 CO and 15 N nuclei save the 13 Cb of S77, the 13 CO of S79, the 13 Cb and 13 CO of S88 as well as the 13 Cb and 13 CO of L15 and other residues preceding proline residues. Moreover, all assignments are missing for H63, H64 and H78 as these residues' 1 HN signals may be adversely affected by exchange broadening. The assigned chemical shift values, like those measured at pH 4.0, have been deposited in the BMRB under assession number 50274.
Remarkably, the assessment of the Dd values shows that at neutral pH, the main Q/Hrich stretch partly adopts a-helical conformations, whose population is approximately 10% ( Figure 3C). Residues S53-P54 separate this helix from that formed by residues 55-60. The shorter Q/H segment also appears to adopt a minor population of a-helix. The nine Nterminal residues do not show a clear tendency to adopt a preferred secondary structure at pH 7. Cys10 remains reduced and Xaa-Pro peptide bonds continue to be mainly trans at pH 7. Exchange measurements at pH 7 yielded { 1 H}-15 N NOE and R1r values which are elevated for most residues (Figure 3D, 3E) relative to values obtained at pH 4.0. These measurements reflect stiffening on ps/ns and µs/ms timescales and is consistent with the formation of preferred conformers and associative processes. However, the G/S-rich segment located C-terminal to the PLD continues to be highly dynamic at pH 7, both on faster ps/ns timescales as well as slower µs/ms timescales.
After a two month incubation at 4 ºC, Orb2A PLD's 1 H-15 N NMR spectra at 25ºC in PBS revealed that despite some changes, most peaks retained their positions; this suggest that slow conformational changes or aggregative processes are minimal (Fig. 4A). Zinc chloride was then added to a final concentration of 2 mM. In the presence of this divalent cation, the peak intensities of His and Gln 1 H-15 N resonances drop practically to the signal/noise limit ( Fig. 4B, C). Strong decreases are also seen for the 1 H-13 Cb and 1 H-13 C Ca signals of His in 2D 1 H-13 C HSQC spectra (Sup. Fig. 4). This strongly suggest that Zn ++ binds to His residues and promotes association processes of nearby Gln residues which are manifested as a loss of signal intensity. The signal intensity of other residues also decreased following Zn ++ addition, but sufficed to record good quality 3D HNCO, HNCA and CBCAcoNH spectra.
The analysis of the these spectra revealed that the conformational ensemble of the molecules remaining visible to NMR is poor in helical structures and slightly enriched in extended conformations (Fig. 4D). Thus, Zn ++ binds the His residues and drives the association of helical conformations of Orb2A PLD into large assemblies invisible to liquid state NMR.

Discussion
The key findings of this study are that the amyloidogenic Q/H-rich region of Orb2's PLD is disordered and flexible at low pH, but adopts a minor population of a-helix at pH 7.0, where the His residues are mostly in the neutral state. The hypothesis that Orb2 aggregation triggers enduring synapse-specific alterations in translation which are key for memory consolidation received strong support by the elucidation of its amyloid structure from adult Drosophila heads . Gln and His side chains are interlocked in this amyloid (Sup. Fig. 1B). The relative instability of Orb2 amyloid at acidic conditions, as shown in Sup. Fig. 15   , which we corroborate here by NMR for Zn ++ (Figure 4). Those researchers also proposed that if this segment were to adopt an a-helical conformation (just as we have shown here), then the His residues, which are mostly spaced i, i+3 and i, i+4, would be mainly positioned on the same side of the a-helix . The binding of cations such as Zn ++ to such an array of His residues could increase the formation of a-helix, although this point is difficult to test due to the Orb2A PLD aggregation that accompanies cation binding, even at low concentrations used for CD spectroscopy . The resulting helical conformations may drive association processes such as the formation of coiled-coils, as seen for other proteins which undergo liquid-liquid phase separation via intermolecular helical associations (Conicella et al., 2016). Q-rich coiled-coils and higher oligomeric species have been proposed as intermediates in amyloidogenesis of Aplysia and mammalian CPEBs (Fiumara et al., 2010;Pelassa et al., 2014;Hervás et al, 2020b;Ramírez de Mingo, et al., 2020a), which are probably heterogeneous as strongly suggested by kinetic modeling (Vitalis and Pappu, 2011). A hypothetical working model which incorporates these ideas is shown in Figure 5. Whereas Aplysia and mammalian CPEB homologs share a similar domain organization with Drosophila Orb2, they lack the multiple His residues interspersed in their Q-rich segments (Hervás et al, 2020b;Ramírez de Mingo, et al., 2020a) which are found in Orb2. This suggests that the pH-regulated nature of Orb2 amyloid formation and dissociation may be substituted by other mechanisms in higher organisms. Proline 54, which interrupts the helices formed by the Q/H-rich segments and residues 55-60, may play an important role in preventing premature amyloid formation.
Similar proline residues which delimit a-helices have been recently described in human CPEB3 (Ramírez de Mingo et al., 2020a).
The role of the initial hydrophobic segment ( Figure 1A) in Orb2A on amyloidogenesis has been debated. It was found to be important for Orb2 mediated memory, as even the conservative substitution of Phe 5 for Tyr affected activity (Majumdar et al., 2012). Moreover, this segment, and not the amyloidogenic Q/H-rich stretch, was found to adopt an a-helix which later evolved into amyloid in vitro as monitored by circular dichorism spectroscopy and solid state NMR spectroscopy (Cervantes et al., 2016). However, this segment was not observed in the physiological amyloid, which is composed chiefly of Orb2B, formed in Drosophila heads . Instead, the amyloid is composed of the longer Q/H-rich stretch. Here, this Orb2A N-terminal hydrophobic segment was found to be devoid of detectable populations of secondary structure, at both pH 4.0 and pH 7.0. By constrast, this segment was reported to form an amphiphilic a-helix in the presence of membrane mimetics . Therefore, it is possible that in in vivo conditions, this segment may interact with membranes to favor the formation of an a-helix which could promote association events that facilitate functional amyloid formation by the Q/H-rich stretch ( Figure 5). Once formed, the Orb2A amyloid would nucleate amyloid formation in Orb2B.
The 200-residue long G/S-rich region links the PLD to the folded RRM and ZnF domains of Orb2 (Sup. Fig. 1A). Although the physical linkage of the PLD to the RNA-binding domains is required for Orb2 function in memory , our characterization of the first 20 residues of this region suggests that it is highly disordered and flexible. Nevertheless, as Orb2 forms granules in cells (Hervás et al., 2016) and considering that the disordered region between the PLD and first RRM in human CPEB3 has been recently found to be necessary and sufficient for condensate formation by liquid-liquid phase separation ( M of NaCl was present in the buffer. The resulting protein is electrophoretically pure and was stored in concentrated urea at -80 ºC until use. Its purity and identity were subsequently corroborated by NMR spectroscopy. Just prior to NMR spectroscopy, the urea was removed and the protein was transferred to 1.0 mM deuterated acetic acid buffer containing 15 % D2O, pH 4.0 using a PD-10 (GE-Biosciences) desalting column. By refractive index, the sample was found to contain less than 10 mM residual urea. Following NMR spectral acquisition at pH 4.0, the sample was transferred to pH 7.0 by adding a 10X buffer stock of PBS prepared in D2O. Upon adding the PBS stock, the initial pH was 6.2; it was increased to 7.01 by adding small alquots of 0.3 M KOH. The final buffer content of the pH 7 sample was 0.9 mM deuterated acetate, 25 mM Na2HPO4, 25 mM NaH2PO4, 100 mM KCl and 1 mM TCEP as the reducing agent. The final sample volumes were 0.201 mL (pH 4) and 0.226 mL (pH 7). A final series of 2D and 3D spectra were recorded following the addition of ZnCl2 to a final concentration of 2 mM.
All spectra were recorded in a water-matched, 5 mm diameter Shigemi NMR tube.

NMR Spectral Assignments
All NMR spectra were recorded on a Bruker Avance NMR spectrometer operating at 800 MHz ( 1 H), except the last series examining the effects of Zn ++ binding, which were recorded with a Bruker 800 MHz Neo Avance NMR spectrometer. This instrument is equipped with a triple resonance ( 1 H, 13 C, 15 N) cryoprobe and Z-gradients. The program Topspin (Bruker Biospin) versions 2.1 and 4.0.8 were used to record, process and analyze the spectra. Because the Orb2A's N-terminal region is disordered and since its sequence is of low complexity and contains many repeated consecutive residues, it presents special challenges for NMR spectral assignment. Therefore, instead of the standard approach based by 1 HN excitation and 13 Ca and 13 Cb connectivities (Sattler et al., 1999), we have applied an unconventional proton-less approach based on a pair of 3D 13 C-detected spectra which provide consecutive 13 CO and 15 N backbone connectivities as these nuclei retain more dispersion in IDPs (Pantoja-Uceda & Santoro, 2014). Even with this approach, some cases of sequential residues with identical 13 CO and 15 N chemical shift values were observed.
Therefore, to overcome these ambiguities, an additional 3D 1 H-detected spectrum which yields consecutive 1 HN-1 HN connectivities was recorded (Sun et al., 2005;Pantoja-Uceda & Santoro, 2013). The analysis of these data led to the essentially complete spectral assignment at pH 4.0 as described in the Results section. After measuring relaxation measurements at pH 4.0, the sample pH was increased to pH 7.0. As described in the Results section, the sample tends to aggregate at neutral pH. Because of its lower solution concentration, the assignment was carried out by conventional 1 H-detected 2D 1 H-15 N HSQC and 3D CBCAcoNH, HNCO and HNCA spectra and comparison with the pH 4 spectra. A list of the NMR experiments and their parameters is shown in Sup. Table 1. 1 H peak widths in 1 H-15 N HSQC spectra were measured using a Gaussian function in the program NMRFAM Sparky 1.4. 3 JHNHA coupling constants were calculated based on the relative signal intensities of the 1 HN/ 1 Ha crosspeak : 1 HN/ 1 HN diagonal peak using either peak integration in Topspin 4.0 or peak heights in NMRFAM Sparky 1.4 as previously described (Vuister & Bax, 1993).
The expected chemical shift values for the 13 Ca, 13 Cb, 1 Ha, 1 HN, 15 N and 13 CO nuclei in statistical coil ensembles were calculated using the parameters tabulated by Kjaergaard and Poulsen (2011) and Kjaergaard et al., (2011), and implemented on the https://spin.niddk.nih.gov/bax/nmrserver/Poulsen_rc_CS/ server at the Bax laboratory.
These values were subtracted from the experimentally measured chemical shift values (d) to calculate conformational chemical shifts (Dd).

NMR Relaxation Measurements:
The dynamics on the ps-ns time scales were probed by measuring the heteronuclear 15 N{ 1 H} NOE (hNOE) of backbone amide groups as the ratio of spectra recorded with and without saturation in an interleaved mode. An eleven second recycling delay was employed. Uncertainties in peak integrals were estimated from the standard deviation of intensities from spectral regions lacking signal and containing only noise. R1r relaxation rates, which sense dynamic processes on slower µs-ms timescales, were measured by recording two sets of ten 1 H-15 N correlation spectra with relaxation delays at 8, 300, 36, 76, 900, 100, 500, 156, 200 and 700 ms. The R1r relaxation rates were calculated by least-squares fitting of an exponential decay function to peak integral data, which were obtained using Topspin 4.0.8.  S-1 corresponds to the Ser residue resulting from cloning and tag cleavage that precedes M1. The bold His and Gln labels refer to overlapped His and Gln peaks; these arise from residues in the amyloidogenic segment. The multiplication factor between contour levels is 1.4 here and in panel B as well as Figure 4A.
B. 2D 13 C-15 N CON spectrum of Orb2A PLD recorded at pH 4.0, 25ºC. The labels shown correspond to the 15 N signals; the 13 CO correlations arise from the preceding residue. Note that the y-axis scale is longer in panel B than A to include Pro residues. The bold His, Gln and Gln' labels are for overlapped His and Gln residues; the Gln' cluster comes from i His 13 CO /i+1 Gln 15 N correlations. For intense or overlapped peaks, a satellite peak is often seen slightly to the right and above the main peak. These satellite peaks arise from deuteration and some examples are indicated with blue circles.  -In panels B-E, the different zones of the Orb2 PLD are shaded as follows: N-terminal hydrophobic stretch = gray, Q/H-rich = light magenta, residues 55-60= blue, H·Q-rich= light magenta, G-rich=yellow. In the Q/H or H·Q-rich regions most of the values shown come from averaging of overlapped His or Gln peaks.
A. 2D 1 H-15 N HSQC spectrum of Orb2A PLD registered at pH 7.0, 25ºC. The bold His, Gln and Gln' labels refer to overlapped His and Gln peaks; these arise from residues in the amyloidogenic segment. The Gln' peak arises from Gln which follow a His along the sequence. The signal of G11 (boxed) is beneath the lowest selected contour level. To afford a fair appreciation of the wider peak widths observed here relative to the spectrum at pH 4, the same axes, aspect ratio and multiplication factor between contour levels (1.4) are used here as in Figure 2A. B. 1 H peak width of Orb2 PLD 1 H-15 N HSQC peaks at 25 ºC and pH 7 (green diamonds) and pH 4 (red circles). The dotted lines represent the average peak widths at pH 7 (green) and pH 4 (red). Overlapped peaks are excluded. The uncertainties are approximately ± 1.5 Hz. C. Conformational chemical shifts for 13 Ca (black bars) 13 Cb (red bars) and 13 CO (blue bars) at pH 7.0, 25ºC. For clarity, the y-axis scale is shifted for the 13 CO Dd values. The 13 CO of Gly 11 and the 13 Cb of Cys are outliers. The 13 CO Dd values of the Q-rich region have large uncertainties due to the broad nature of the overlapped peak of Q residues. D. Fast ps/ns dynamics assessed by the heteronuclear { 1 H}-15 N NOE. Uncertainties are about ± 0.015. E. Transverse relaxation rates in the rotating frame (R1r) for Orb2 PLD at pH 7.0, 25ºC (green squares). For comparison, the R1r value obtained at pH 4.0, 25 ºC and already shown in Fig. 3C are also shown here by open red diamonds. F. At pH 7, the Q/H segments (magenta cylinders) and the 55-60 segment (blue cylinder) adopts a small but detectable population of a-helical conformers separated by proline 54 (red).

Figure 4: Zn ++ binding to His residues promotes further oligomerization
A. 1 H-15 N HSQC before (green) and after (blue) the 2 month pause. Some distinct residues are labeled. B. 1 H-15 N before (blue) and after (black) the addition of ZnCl2.. His and Gln signals (labeled) weaken sharply after addition of Zn ++ . C. The largest drop in peak intensity upon Zn addition occurs for residues of the hydrophobic segment (gray band) and the Q/H-rich segments (rose band). D. Conformational chemical shifts for 13 Ca (black), 13 Cb (red) and 13 CO (blue) strongly suggest that the molecules remaining in solution lack helical zones and are disordered. A. The first N-terminal hydrophobic segment (black) interacts with membrane which promotes partial a-helix formation as reported by Soria et al., (2017). The proximity of phosphorylated Tob protein (top, red) RNA (bottom, red), and/or possibly other polyanions, creates a negatively charged milieu which favors the cationic form of the eleven His residues (His +) of the Q/H-rich segments (magenta). This keeps the Q/H-rich segments disordered. Pro 54 (red stop sign) would also act to prevent premature amyloidogenesis. The 55-60 residue segment (blue) adopts a partial a-helix while the G/S-rich segment (yellow) remains disordered and flexible.

B.
Following neural stimulation, the entry of Zn ++ and other cations could displace H + and bind to the neutral form of His residues (His) as proposed by Bajakian et al., (2017); releasing Tob and RNA (red). The neutral form of His allows partial a-helix formation of the Q/H-rich segments, unlocking pathways to amyloid formation through coiled-coil intermediates.
Sup. Analysis with the program PONDR predicts that Orb2A's PLD and G/S-rich regions will be unfolded. The folded RRM and ZnF domains, which begin near residue 300 (as shown in Sup. Fig. 1A) are correctly predicted to be ordered.