A covalent homodimer probing early oligomers along amyloid aggregation

Early oligomers are crucial in amyloid aggregation; however, due to their transient nature they are among the least structurally characterized species. We focused on the amyloidogenic protein beta2-microglobulin (β2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing β2m molecules was repeatedly observed, suggesting that such interface may be relevant for β2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt β2m under native conditions, it shows enhanced amyloid aggregation propensity. Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity. Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation. The present data provide new insight into β2m early steps of amyloid aggregation.

. Crystal structure of DimC33 and DD strand interface. (A) Ribbon model of one of the two chains in DimC33 (blue) superposed on the structure of wt β 2m (green). β -strands are labeled according to the standard β 2m nomenclature, Ser33/Cys33 are shown in sticks. (B) A zoomed view into the DD interface of four superposed crystal structures of DimC33_ThT (green, pdb code: 4RA3), hexameric structure of H13F β 2m (magenta, pdb code: 3CIQ), DimC50 (cyan, pdb code: 3TM6) and DimC20 (yellow, pdb code: 3TLR), showing the main residues involved in the DD interface as sticks model. (C) Superposition of three β 2m non-covalently assembled dimers built through the DD strand interface with DimC33: DimC33_ThT in green, hexameric structure of H13F β 2m in magenta, DimC50 structure in cyan, and DimC20 structure in yellow. (D) Stereo view of the DD strand interface built by the facing β 2m molecules as observed in the structure of DimC33_high. The main residues involved in the interface are shown as sticks.
addition of seeds allows to study fibril growth, but it fully abolishes the key early process of oligomer formation.
Several independent reports suggest that the first oligomeric species formed during β 2m aggregation is an elongated dimer: extended oligomers have been observed by mass-spectrometry, and an elongated dimer was proposed to be the starting species in β 2m oligomerisation [13][14][15][16] . Among several studies aimed at uncovering the structure of β 2m oligomers 17,18 , Miranker and coworkers reported the crystal structure of a hexameric form of the H13F β 2m mutant 17 , formed under aggregating conditions; such hexameric assembly is however not amyloidogenic 17 . We recently tested a different approach to the study of β 2m oligomer formation. Single Cys mutations were inserted in different β 2m surface regions, and disulphide-linked covalent homodimers were prepared 19 . The rationale was that a covalent bond between two β 2m molecules acts as a constraint for the architecture of the dimer, which can be aggregation permissive or non-permissive. The crystal structures of two of such dimers showed a common antiparallel interface between covalent dimers (named the DD strand interface): these dimers also proved to be amyloidogenic and assembled as dimer of dimers in solution. Notably the S-S bond location in a third engineered homodimer hampers the formation of the DD strand interface resulting in a non-amyloidogenic, purely dimeric species in solution. Based on such observations, the DD strand interface was proposed as a key association interface involved in the early stages of β 2m association under native and non-native conditions 19 .
The DD strand interface involves the apical region of two β 2m molecules. The D-strands belonging to the facing molecules are antiparallel and the two β 2m molecules involved display the same conformation and are well superposable. Within each β 2m monomer the DD strand interface involves the D-strand, the DE and BC loops, namely regions that have been reported as major players in β 2m amyloid aggregation 19 (Fig. 1). Such intermolecular association interface had also been noted in the hexameric structure of the H13F β 2m mutant 17 .
Following our previous approach, in the present study we engineered a β 2m homodimer where the DD strand interface is specifically locked by a disulphide bond (linking the engineered C33 residues of two β 2m molecules -DimC33). We speculated that analysis of such a covalently stabilized dimer should allow us to assess more directly the role played by association through the DD strand interface in β 2m oligomerisation and amyloid formation. DimC33 has been characterized in solution and under aggregation conditions; moreover, we determined three different crystal structures of the covalent dimer, one of which hosts the DimC33 complex with Thioflavin (ThT), the hallmark fluorescent dye for amyloid aggregates. The data here reported show that a covalently stabilized DD strand interface facilitates β 2m aggregation under denaturating and non-denaturating conditions,, suggesting that DimC33 may be used as a model system to study β 2m early oligomerisation steps in vitro. Our results recapitulate previous data indicating the DD strand interface as the first and favourite intermolecular contact region between β 2m molecules under native or aggregating conditions. Furthermore, analysis of the crystal structure of DimC33 in complex with ThT strongly points to a second β 2m association interface that may be involved in amyloid aggregation.

Results
Recombinant S33C variant β2m homodimers. In order to engineer a β 2m covalent dimer displaying a locked DD strand interface, a detailed analysis of the crystal structures displaying such intermolecular association interface (pdb codes: 3TM6, 3TLR, 3CIQ) was performed, and the mutation of Ser33 to Cys was chosen based on the following rationale. Firstly, two Ser33 residues belonging to two facing β 2m molecules (in the examined crystal structures) fall at a suitable distance to be mutated to Cys and yield a disulphide bridge without disrupting or altering the DD interface. Secondly, among the residues, which could satisfy the above conditions, a Ser residue was chosen because it is essentially isosteric with Cys.
The S33C β 2m variant was expressed and purified under denaturating conditions and then refolded, according to our standard protocols 20 . In our previous study on covalent β 2m homodimers, in order to promote the formation of covalent disulphide linked homodimers, an oxidation reaction proved necessary. To this purpose, after refolding, the monomeric β 2m variants were incubated at high protein concentrations in the presence of H 2 O 2 19 , indicating that random encounters between molecules are not sufficient for the formation of disulphide linked dimers. Conversely, in the case of the S33C β 2m variant, a covalent dimeric species was abundantly present at the end of the purification, in the absence of further oxidation reactions. The spontaneous formation of disulphide bonds greatly depends on proper juxtaposition of the two Cys residues involved 21 . Therefore, the unprompted dimerization of S33C implies that in solution, under native conditions, the DD association interface spontaneously brings together two monomeric β 2m molecules, and only once the DD interface is properly assembled, the two facing Cys33 establish the disulphide bond, which results in the formation of the covalent dimeric species (DimC33).
The stability of DimC33 in solution was assessed by circular dichroism. Thermal unfolding monitored by Far-UV indicates that, as for wt β 2m, DimC33 unfolds according to a simple sigmoidal curve and displays a Tm value close to that of the wt protein (Tm DimC33 60.2 ± 0.3 °C; Tm wt 62.4 ± 0.3 °C) (Fig. 2a). Then, Far-UV spectra of DimC33 and of wt β 2m in phosphate buffer, in 10% and 20% TFE were collected, showing that native spectra are well superposable and that both proteins display a native secondary structure content in 10% TFE; conversely the β 2m fold is perturbed in 20% TFE (Fig. 2b). Thus, the Scientific RepoRts | 5:14651 | DOi: 10.1038/srep14651 spectroscopic data suggest that the engineered mutation and the achievement of a covalent dimeric state do not alter β 2m thermodynamic stability or its overall fold in DimC33.

Aggregation kinetics of DimC33 and wt ß2m in vitro.
We studied DimC33 aggregation propensity in vitro at physiological pH conditions (pH 7.4) in the absence of any pre-fibrillar seeds. In 20% (v/v) TFE, DimC33 at a final concentration of 1 mg mL −1 aggregates promptly without a lag phase, reaching equilibrium within the first 4 hours, and yielding a high ThT binding signal (Fig. 3a); under the same conditions wt β 2m remains soluble and does not aggregate. Interestingly, in 10% (v/v) TFE-where DimC33 and wt β 2m display CD spectra indicative of native-like conformations-a 5 mg mL −1 solution of DimC33 also aggregated. The AFM analysis of the samples after 1 week aggregation showed that DimC33 formed fibrils both in 20% TFE (Fig. 3b, left) and in 10% TFE (Fig. 3b, right). The fibril heights measured in the two conditions were similar and were in the range between 2.0 and 5.5 nm. Doxycycline is a known inhibitor of wt β 2 m aggregation 22 . In order to assess doxycycline inhibitory effect on DimC33 aggregation, a DimC33 solution was tested for aggregation in the presence of 100 μ M and 400 μ M Doxycycline, in 20% TFE. A 100 μ M final concentration of Doxycycline was sufficient to block DimC33 aggregation by approximately 85%, compared with a DimC33_20% TFE control solution, whereas 400 μ M Doxycycline completely inhibited DimC33 aggregation (Fig. 3c). No fibrillar aggregates were detected by AFM in DimC33 samples incubated for 1 week in the presence of doxycycline (not shown). Such results are in keeping with previous data on wt protein reporting an IC50 of 47 μ M for doxycycline as aggregation inhibitor 22 . X-ray crystal structures of DimC33. In order to assess fine details of the molecular structure of DimC33, two crystal structures of DimC33 were determined to 1.9 Å and 1.4 Å resolution, hereafter named DimC33_low and DimC33_high, respectively; additionally a crystal structure of DimC33 in complex with ThT was determined to 2.8 Å resolution, and named DimC33_ThT. Data collection and refinement statistics for the crystal structures are shown in Table 1.
The three crystal structures display three different space groups and crystal packings. The electron density is of excellent quality and all β 2m molecules are completely traceable in all three structures. Overall, no major structural effects on the β 2m fold are induced by the S33C mutation, either locally or globally, in all DimC33 structures, (Fig. 1b,c). The monomeric components of DimC33 most closely match the conformation of wt β 2m as observed in its physiologic class I major histocompatibility complex ( Table 2). The only noticeable difference occurs in the DE loop (residues 57-60) that displays a slightly modified conformation so that Trp60 participates in stacking interactions with Phe56, of the  same chain, and with His51 and Tyr66 of the second chain within DimC33; the rotation of Tyr63 side chain of about 90 degrees is also observed (see Fig. 1b). The covalent DD strand interface is structurally well conserved in all three DimC33 structures, where it maintains the same contact surface of 560 Å 2 . The overall dimer organization is conserved (r.m.s.d. 0.57 Å/195 Cα , 1.17 Å/195 Cα , DimC33_high versus DimC33_ThT and versus DimC33_low, respectively); the main regions involved in the association interface are the BC loop (residues 31-37), the D strand (residues 51-57), the DE loop (residues 57-6), Phe62, the E strand (residues 64 and 66), as observed in previous structures (Fig. 1b-d). The superposition of DimC33 with previously reported β 2m dimers, non-covalently associated through the DD strand interface, shows a very similar overall monomer-monomer orientation ( Fig. 1c and Table 2) and conserved conformations for the residues involved ( Fig. 1b-d and Table 2). All such data indicate that the S33C mutation minimally affects the β 2m fold, and that the C33 -33C disulphide covalently locks the DD strand interface with negligible structural effects Fig. 1b-d. The DimC33 ThT complex and the ABDE sheet. DimC33 was also crystallized in the presence of 5 mM ThT. Intriguingly, the crystallographic analysis showed that five ThT molecules are hosted in the crystal asymmetric unit (AU), which contains one DimC33 moiety and two halves of a second one (i.e. two additional and independent β 2m chains). As a result of crystal packing, β 2m molecules belonging to independent DimC33 units interact via their ABDE β -sheets (Fig. 4a); however, two distinct ABDE interfaces can be distinguished. In one of these, four ThT molecules are wedged between the ABDE sheets of two facing β 2m molecules (4 ThT site); in the other, only one ThT molecule is sandwiched between two facing ABDE sheets (1 ThT site) (Fig. 4a). In either case, binding of the ThT molecules does not induce any conformational adjustments in the β 2m fold, or in the DimC33 assembly. At the 1 ThT site, the ThT molecule is sandwiched between Tyr10 residues from two facing β 2m molecules; moreover, Tyr26 and Pro14 from both molecules establish van der Waals interactions with ThT (Fig. 4b). At the 4 ThT site, the four ThT molecules are stacked on each other, and in stacking contacts with Tyr10 and Tyr63 of the β 2m molecules defining the binding site. Tyr26 from both β 2m molecules also help accommodate the ThT hydrophobic rings (Fig. 4c).
In order to house one or four ThT molecules, the β 2m chains, which belong to two distinct DimC33 units, are differently juxtaposed. In particular, the two β 2m molecules defining the 4 ThT site move about 20 Å apart compared to the 1 ThT site, to make room for the four ThT molecules. Thus, very limited direct interactions connect the two β 2m molecules at the 4 ThT site, whereas more direct protein-protein contacts take place at the 1 ThT site. Despite such translational adjustments, the two ThT sites maintain very similar structural arrangements. The relative orientation of the two β 2m chains defining the ThT sites is comparable, and the protein regions involved in ThT binding are conserved. More specifically, Tyr10, Tyr26 and Tyr63, the three central residues in the ABDE interface, cross diagonally the sheet and confer substantial surface hydrophobicity (Fig. 4). In particular, at both ThT sites Tyr10 establishes direct stacking interactions with ThT molecules, even though the ThT molecules do not share the same orientation in the two sites.
It must be noted that wide intermolecular packing contacts through the ABDE sheet are established also in the absence of ThT. In the structure of DimC33_low (present work) and in the hexameric H13F mutant 17 , highly hydrophobic association interfaces are built via facing ABDE sheets (surface areas of 805 and 970 Å 2 , respectively). Although such two ABDE assemblies are not superposable, the two interfaces are structurally similar and residues Tyr10, Tyr26 and Tyr63 are clustered in their hydrophobic cores (see Fig. 4d,e).
In summary, in the four ABDE packing interfaces examined (the 1 ThT and 4 ThT sites, and the ABDE interfaces in DimC33_low and in hexameric β 2m) the orientations of each β 2m monomeric chain may vary, the distances between protein chains are somewhat different, and the packing interactions between the ABDE sheets can be direct or mediated through ThT molecules. However, the overall conservation of protein-protein interaction surface, the binding of ThT, the size of the surfaces, the structural data and the marked hydrophobicity of the residues involved, all point at this sheet as a favored site for β 2m association during oligomer formation.

Discussion
Elucidating the structure and the underlying interactions of on-pathway oligomers that lead to amyloid aggregation is a crucial and challenging task. A structural characterization of the first steps of oligomer formation would be the key for a deeper understanding of the aggregation process, and potentially for the design of inhibitors hampering the oligomerisation process, and the related protein cytotoxicity.
We selected β 2m as a model of amyloid aggregation, since the protein has been extensively characterized, and several structural determinants of β 2m amyloidogenicity have been uncovered 7 . In order to shed light on the intermolecular interactions that drive the first steps of aggregation, a disulphide-linked β 2m homodimer was designed according to a previously proposed strategy 19 . The engineered S-S bond was positioned in order to juxtapose two β 2m monomers along the DD strand interface, which was observed as an intermolecular association region 17,19 . The mutation Ser33 to Cys is structurally conservative, and designed to bring together two β 2m molecules through the DD strand interface, without altering its structure. From the in vitro biophysical data, and from the crystallographic analysis here reported, all evidences suggest that the mutation and the engineered disulphide bond do not affect β 2m properties to a significant extent.
Under native conditions, we and others have shown that a minor population of wt β 2m is oligomeric, mainly dimeric 14,23 . The observation that the disulphide bridge stabilizing DimC33 forms spontaneously in solution (in the absence of an added oxidizing agent) is indicative of the spontaneous juxtaposition of two facing C33 residues; it thus strongly suggests that the DD interface is the prevalent association interface underlying β 2m dimerisation under such conditions.
The aggregation data show that DimC33 is much more amyloidogenic than wt β 2m, even though its thermodynamic stability and its 3D structure are virtually identical to those of the wt protein. Using a standard β 2m aggregation protocol in 20% TFE, we have shown that DimC33 aggregates abundantly, but, opposite to wt β 2m, does not require fibril seeds for aggregation to start, an indication that DimC33 can spontaneously form the early oligomers required for the onset of fibrillogenesis. Analogously to wt β 2m early oligomer formation 22 , doxycyclin proved to be an inhibitor of DimC33 aggregation. However, DimC33 was shown to aggregate also in 10% TFE, a condition under which β 2m is properly folded (Fig. 2b). The data available to date indicate that the amyloidogenic intermediates display a native-like structure 13,14,24 ; on these bases, a β 2m variant, such as DimC33, which aggregates starting from non-denaturating conditions may prove to be an insightful system allowing the study in vitro of wt β 2m early oligomerisation.
Recent reports indicate that the first wt β 2m oligomer formed on the pathway to fibril association should be a head-to-head elongated dimer 5,[13][14][15] . In particular, Rennella et al. also provided evidence that the dimer association interface should involve the BC loop region 14 , a finding that is in keeping with our Scientific RepoRts | 5:14651 | DOi: 10.1038/srep14651 previous data and with a recently published report 13,19 . Thus, the data here reported for DimC33, together with previous results, point to the DD strand interface as the firstly established protein-protein association interface during β 2m aggregation. The increased aggregation propensity of DimC33 compared to wt β 2m, would then be the result of the immediate availability of the first on-pathway amyloidogenic oligomer, that is DimC33.
Interestingly, the crystal structure of DimC33 in complex with ThT provides further information on the β 2m aggregation path. ThT fluorescence is the most widely accepted spectroscopic method to discern cross-β fibrils from amorphous aggregate. The ThT molecules are held to intercalate the cross-β structure in the fibrils, resulting into a gain of fluorescence. Only two structures of ThT in complex with amyloidogenic proteins have been so far reported, and both concern β 2m 25 . In one of these structures (pdb code: 3 MYZ) the authors suggest that ThT molecules are simply trapped between β 2m molecules packed in the crystal lattice, while the second structure (pdb code: 3 MZT) presents technical issues that prevent a thorough discussion of ThT-β 2m interactions (See Materials and Methods). In this context, the DimC33_ThT structure here reported adds substantially new information on ThT binding mode to an amyloidogenic protein.
In the DimC33_ThT complex structure, the ThT molecules are nestled between the ABDE sheets of two facing β 2m molecules, in a highly hydrophobic environment. Notably, an extended ABDE interface between facing β 2m molecules can also be observed in the absence of ThT in DimC33_low (this work), and in the hexameric β 2m structure reported earlier 17 . The reciprocal orientation of the two facing ABDE β -sheets displays a high level of variability, but in all cases ABDE β -sheets face each other and the same three aromatic residues (Tyr10, Tyr26 and Tyr63) build the hydrophobic core of the association interface. Such variability can be accounted for by the presence of several Tyr residues and by the capability of hydrophobic interactions to form under different orientations of the contributing residues. The above observations point at the ABDE β -sheet as a second key interface involved in β 2m oligomer formation, in keeping with a previous report supporting the role of surface aromatic residues in determining β 2m amyloid propensity 26 . The structural adaptability of the ABDE surfaces may also explain the formation of several different dimeric/tetrameric building blocks, recently proposed by the EM reconstruction of mature β 2m fibrils 16 .
In summary, our study presents an engineered β 2m covalent dimer, DimC33, displaying the same biophysical properties of wt β 2m in solution, such dimer mimics the first oligomer formed during β 2m aggregation. Unlike wt β 2m, DimC33 does not require the addition of seeds to start fibrillogenesis, and aggregates under conditions where β 2m retains native secondary structure content. Given that in vivo wt β 2m likely aggregates from a native-like folded species, DimC33 could then be considered the first model system to study β 2m oligomerisation in vitro, resembling more closely the aggregation steps occurring in vivo, compared to the current available aggregation protocols based on denaturing conditions. Therefore DimC33 may also be an insightful system to test anti-oligomerisation inhibitors. Finally, the properties and the structures of DimC33 recapitulate previous evidences and indicate how oligomerisation may proceed during aggregation. The first dimeric oligomer would be built across the DD strand interface; at a later stage, the aromatic residues located on the ABDE sheet would contribute to the formation of a hydrophobic core for further association through a structurally adjustable ABDE interface.

Materials and Methods
Mutagenesis, expression and purification. The synthetic gene coding for the Ser33 to Cys β 2m variant was purchased from Eurofins genomics. The gene of interest was inserted into pET21B expression vector and transformed into BL21 (DE3) E. coli strain. The mutated β 2 m was expressed and purified as previously reported 27 . At the end of the procedure, an additional purification step was introduced to separate the spontaneously formed DimC33 by size-exclusion chromatography (Superdex75 16/60 GE healthcare) and eluted with 20 mM Sodium Phosphate buffer pH 7.4. Circular dichroism. Circular dichroism (CD) studies were carried out on a Jasco J810 spectropolarimeter equipped with a Peltier system for temperature control and analyzed by means of Jasco software. All measurements were recorded at 0.1 mg/mL protein concentration in 100 mM sodium chloride, 50 mM sodium phosphate buffer pH 7.4 by using a 0.1 cm path length cuvette. Spectra were recorded in plain buffer or in buffer supplied with 10% or 20% TFE at 37 °C. Temperature ramp measurements were recorded at 202 nm from 20 to 95 °C (temperature slope 50 °C/h). Tm values were calculated from the first derivative of the recorded traces.
Aggregation kinetics. DimC33 aggregation experiments were performed as unseeded reaction by incubating samples of 100 μ L at 37 °C for two weeks, without agitation. The following aggregation conditions were tested: DimC33_20% TFE (1 mg mL −1 DimC33 in 20% TFE, 100 mM Sodium chloride, 50 mM Sodium phosphate buffer pH 7.4) according to a standard protocol 28 ; DimC33_10% TFE (5 mg mL −1 DimC33 in 10% TFE, 100 mM Sodium chloride, 50 mM Sodium phosphate buffer pH 7.4). Unseeded aggregation experiments on wt β 2m were also performed as controls. Aggregation kinetics were monitored by means of VARIAN Cary Eclipse spectrofluorimeter by measuring ThT fluorescence signal at excitation and emission wavelength of 445 and 480 nm, respectively 29 .
Scientific RepoRts | 5:14651 | DOi: 10.1038/srep14651 The inhibitory effect of doxycycline on DimC33 aggregation was studied under the same aggregation condition as employed for DimC33_20% TFE, by monitoring ThT fluorescence binding signal and inspecting sample morphology by AFM after one week of incubation at 37 °C without agitation. Two doxycycline concentrations were screened: DimC33_Doxy100 containing 100 μ M Doxycycline; and DimC33_Doxy400 containing 400 μ M Doxycycline. DimC33_20% TFE aggregation was used as control model. The measurements are the average of three independent experiments. Doxycycline, TFE and ThT were purchased from SIGMA-Aldrich.
Atomic force microscopy. For AFM inspection, DimC33 samples were diluted 500-fold. A 10 μ l aliquot was deposited on a freshly cleaved mica substrate and dried under mild vacuum. Tapping mode AFM images were acquired in air using a Dimension 3100 Scanning Probe Microscope equipped with a 'G' scanning head (maximum scan size 100 μ m) and driven by a Nanoscope IIIa controller, and a Multimode Scanning Probe Microscope equipped with "E" scanning head (maximum scan size 10 μ m), driven by a Nanoscope IV controller (Digital Instruments -Bruker). Single beam uncoated silicon cantilevers (type OMCL-AC160TS, Olympus) were used. The drive frequency varied between 270 and 330 kHz, the scan rate was between 0.5 and 0.8 Hz. Aggregate size was measured from the height in cross section of topographic AFM images.
Crystallization and structure determination. DimC33 was crystallized under three different conditions by mixing equal amounts of 8 mg mL −1 protein and reservoir solutions containing: (i) DimC33_ low: 25% v/v PEG 4 K, 0.2 M Imidazole-Malate pH7.0; (ii) DimC33_high: 28% PEG 400, 0.2 M Calcium chloride dihydrate, and 0.1 M Hepes sodium pH7.5; (iii) DimC33_ThT: 25% PEG 4 K, 0.1 M Sodium chloride, 5 mM ThT, and 0.1 M Hepes sodium pH8.0-8.2. All DimC33 crystals were grown at 20 °C, using the sitting drop vapor diffusion crystallization method. DimC33_low and DimC33_ThT crystals were cryo-protected in 20-33% glycerol solution containing the respective crystallization mother liquor, and cryo-cooled in liquid nitrogen. DimC33_low and DimC33_high X-ray diffraction data were collected at ID23-1 beam-line (ESRF Grenoble), and DimC33_ThT X-ray diffraction data were collected at P13 beamline (PETRA Hamburg). DimC33_low X-ray data were processed using MOSFLM 30 and SCALA from the CCP4 software suite 31 and XDS 32 for DimC33_high and DimC33_ThT. The 3D structures of DimC33 were determined by molecular replacement using the Balbes software suite 33 . All structures were refined using Phenix.refine 34 and REFMAC5 35 . A twin fraction of 0.46 was estimated by Xtriage 34 . Therefore an amplitude based twin refinement protocol was applied during DimC33_ThT refinement process in REFMAC5. Model building and structural analysis for all DimC33 structures were carried out with COOT 36 and figures were prepared with Pymol and CCP4MG 37,38 . Omit-map has been generated by Phaser suite 34 using the annealing option.

Analysis of 3 MZT Structure.
The structure with the PDB code 3 MZT was reported as a hexameric H13F β 2m mutant in complex with Thioflavin (ThT) 25 . The structure was determined at 2.70 Å resolution. The three ThT molecules modeled in the complex are sandwiched at three intermolecular interfaces present in the hexameric assembly; each ThT molecule is presented in two alternative, but almost superimposable, conformations each at 0.5 occupancy. Temperature factors for ThT atoms range between 80 and 120 Å 2 for each of the two conformations; neighboring protein residues, all modeled with full occupancy, display B-factors mainly in the 45-70 Å 2 range.
When the 3 MZT structure was refined (in our lab, using data from the PDB database) in the absence of the ThT molecules, the residual ThT electron density for each binding site was reduced to a roughly spheroidal blob, much smaller than the size of a ThT molecule. Based on such observations, the 3 MZT molecular model of ThT interaction with β 2m was not considered for the structural comparisons here reported.
Structure deposition. Structure factors and coordinates have been deposited in the Protein Data Bank under accession codes: 4R9H for DimC33_low, 4RAH for DimC33_high and 4RA3 for DimC33_ThT.