Phase Separation and Fibrillization of Human Annexin A7 Are Mediated by Its Proline-Rich Domain

Human annexin A7, a calcium- and phospholipid-binding protein, governs calcium homeostasis, plasma membrane repair, apoptosis, and tumor progression. A7 contains an N-terminal proline-rich domain (PRD; 180 residues, ∼24% prolines) that determines its functional specificity. Using microscopy and dye-binding assays, we show that recombinant A7 and its isolated PRD spontaneously phase separate into spherical condensates, which subsequently transform into β-sheet-rich fibrils. We demonstrate that fibrillization of A7-PRD proceeds via primary nucleation and fibril-catalyzed secondary nucleation processes, as determined by chemical kinetics, providing a mechanistic basis for its amyloid assembly. This study confirms and highlights a subclass of eukaryotic PRDs prone to forming aggregates with important physiological and pathological implications.


Recombinant protein expression and purification.
Codon-optimized A7 constructs, namely A7, A7PRD, and A7 PRD Strep , were custom synthesized from Azenta Life Sciences; see Fig. S4A for the design and mass-spectrometry (MS) analysis and Table S1 for subcloning of each construct.A7 was expressed with a tobacco etch virus (TEV) cleavable C-terminal twin-strep tag. 1 The primary sequence of the twin-strep tag is as follows: GSGSGSGSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK; underlined residues represent the binding motif for strep-tactin, a derivative of streptavidin.Both truncated constructs, namely A7PRD, and A7 PRD Strep , were expressed with the N-terminal B1 domain of protein G (GB1) tag, 2 used to enhance the expression levels, followed by a spacer sequence, a polyhistidine (6xHis) affinity tag, and a TEV cleavage site.Additionally, A7 PRD Strep carried a non-cleavable C-terminal strep tag.
The primary sequence of the strep tag is as follows: WSHPQFEK.All A7 plasmids reported in this study were deposited in the Addgene repository, https://www.addgene.org(accession no.
TEV protease was expressed at 37 °C as described previously. 3All A7 constructs were expressed at 16 °C.Cells were grown at 37 °C in 1 L Luria-Bertani (LB; MP Biomedicals, catalog no.3002-036) medium at natural isotopic abundance.About 30 min before induction, the temperature of the cell culture was reduced to 16 °C.Cells were induced with 1 mM isopropyl β-d-1thiogalactopyranoside (IPTG) at an absorbance of 0.8 at 600 nm and harvested after ca.24 h.
The purification scheme of TEV protease has been described previously. 3A7 was purified using affinity chromatography (ÄKTA Pure protein purification system, Cytiva) whereas A7PRD, and A7 PRD Strep , were purified using a combination of affinity and size-exclusion chromatography (ÄKTA Pure and Start protein purification systems, Cytiva) and reverse-phase high-performance liquid chromatography (HPLC; 1260 Infinity II liquid chromatography system, Agilent Technologies).
In the case of A7, cells were resuspended in a lysis buffer comprising 50 mM Tris, pH 8, 10 mM  2.5 mM d-desthiobiotin.The eluted protein was mixed with recombinant TEV protease (molar ratio 50:1) to hydrolyze the N-terminal GB1 fusion tag (completion of proteolysis was assessed using SDS-PAGE electrophoresis).The proteolysis reaction was carried out at room temperature (~20 h) and produced a poorly soluble hydrolyzed product.The precipitated product was solubilized by the addition of 6 M GdmCl and further purified using reverse-phase HPLC (Jupiter 10 μm C18 300 Å column) with a 25-42% acetonitrile gradient comprising 0.1% trifluoroacetic acid (TFA).The eluted protein fractions were pooled, lyophilized, and stored at -80 °C.
For A7PRD, a similar lysis and HisTrap column purification procedures as described above for A7 PRD Strep were carried out.The eluted protein was further purified using size-exclusion chromatography on a HiLoad 26/600 Superdex 75 prep-grade column (Cytiva) pre-equilibrated with 50 mM Tris, pH 8.0, and 250 mM NaCl.Relevant A7PRD fractions were pooled and incubated with recombinant TEV protease to cleave off the N-terminal GB1 fusion tag.The hydrolyzed product was further purified by reverse-phase HPLC using the above-described conditions.Eluted A7PRD fractions were aliquoted, lyophilized, and stored at -80 °C. ][5]

Sedimentation velocity analytical ultracentrifugation (AUC).
Sedimentation velocity experiments on A7 were carried out at 50,000 rpm and 20 °C on a Beckman Coulter ProteomeLab XL-I analytical ultracentrifuge and an An-50-Ti rotor following standard protocols. 6A7 stock solution was prepared as described above (see Recombinant protein expression and purification section).Samples were diluted to ~2 and ~7 μM using a buffer comprising 25 mM HEPES, pH 7, and 1 mM TCEP.][9] Sedimentation profiles showed the presence of a monomeric A7.
In the case of A7, protein was mixed with a 3-molar equivalent of ATTO-647N maleimide in 25 mM HEPES, pH 7, and 1 mM TCEP.The reaction was allowed to proceed at 4 °C overnight.The excess dye was removed by PD midiTrap G-25 columns (Cytiva).This sample was mixed with the corresponding unconjugated protein (concentration of fluorophore-labeled protein = 5 molar percent).2 mM CaCl2 was then added to induce phase separation of A7 and the resultant sample (final protein concentration = 40 μM) was visualized using fluorescence microscopy (see below).
For A7 PRD Strep , the lyophilized protein was dissolved in a small volume of dimethylsulfoxide (DMSO) and diluted immediately in a buffer containing 25 mM HEPES and 5 mM CaCl2 to induce phase separation (final protein concentration = 50 μM).0.02 mg/mL Streptavidin Alexa-Fluor488 conjugate was then added to this solution, which was visualized using fluorescence microscopy (see below).To determine the effects of calcium and protein concentration on the phase separation of A7 PRD Strep , the turbidity of A7 PRD Strep solutions with varying protein and calcium concentrations was recorded at an optical density (OD) of 330 nm using a DU 730 UV-Vis spectrophotometer (Beckman Coulter) in 1 cm quartz cuvettes (Starna Cells Inc.).Additionally, these samples were visualized using fluorescence microscopy.The condensates of A7PRD formed in the presence of calcium were monitored using differential interference contrast (DIC) imaging.A7PRD condensates were prepared using the same procedure as that of A7 PRD Strep , sans Streptavidin Alexa-Fluor488.

Microscopy imaging and FRAP assays.
1][12] Briefly, DIC imaging was performed on a Nikon Ti2 widefield microscope equipped with a DS-Qi2 CMOS camera and 100x/1.49NAoil DIC N2 Objective; Nikon Imaging Center, UC San Diego.The condenser prism and the polarizer cube were controlled by the Nikon Elements software.Samples of A7 condensates were excited by a 640 nm laser controlled by a Lumencor SpectraX for imaging of ATTO-647N.In the case of A7 PRD Strep + Streptavidin Alexa-Fluor488 mixtures, a 488 nm laser was used.Microscopy image of aged droplets of A7PRD shown in Fig. 3E, main text, was acquired using a similar procedure.Briefly, droplets of 50 μM A7PRD with 20 μM ThT were incubated at 37 ºC for one day.Images were taken using a 488 nm laser.
3] Briefly, slides were cleaned by sonication in 3% v/v Hellmanex III (Sigma-Aldrich), rinsed in 0.5 M NaOH, dried using a nitrogen stream, and placed in a vacuum oven (VWR) at 90 °C for 10 min.PEG-Silane was dissolved in DMSO at 5% w/v and was sandwiched between the slide and coverslip, and incubated at 90 °C for 20 min.Passivated slides were subsequently rinsed with water and dried with a nitrogen stream.Imaging chambers composed of two passivated coverslips and a 9-mm silicone mold (Grace BioLabs) were used for all microscopy analyses.
FRAP measurements of ATTO-647N-labeled A7 were performed on a Nikon point scanning confocal C2 with 2 GaAsP PMTs using a Plan Apo λ 100x/1.45NA Oil objective.Photobleaching of each sample was achieved using 2 iterations of 50% 640 nm laser power directed at the bleaching area for 10 s, and subsequent recovery was imaged at 2 s intervals over 150 frames using 0.1% 640 nm laser power.Images were corrected for background fluorescence, and intensity from the bleached region was normalized against an unbleached region on a nearby condensate of similar size and intensity.A similar procedure was used for A7 PRD Strep + Streptavidin Alexa-Fluor488 mixtures, except for the use of 488 nm laser.

GUV preparation.
GUVs were prepared using the inverted emulsion method. 14Briefly, for the oil phase, the chloroform solutions of DOPC, DOPS, and 16:0 Liss Rhod PE were mixed in a glass vial under dark conditions (69.5, 30, and 0.5%, respectively).The resultant mixture was dried under nitrogen, followed by vacuum desiccation (vacuum oven; VWR) for ~2 h at room temperature.1 mL of mineral oil (Thermo Fisher Scientific, catalog no.O121-1) was added to the vial and sonicated for 1 h at room temperature.For the aqueous phase, phosphate buffered saline (PBS; osmolarity: 280 mOsm, pH 7.4) was mixed with Ficoll 400 (Sigma-Aldrich; 35% w/v in water) to give a final concentration of 3.5% Ficoll-400. 10 μL of this solution was added to 100 μL oil phase and emulsified.The emulsion was then mixed with the aqueous phase, followed by centrifugation (10,000g, 10 min, 4 °C).The GUVs at the bottom of the tube were collected and stored at 4 °C for further analysis.

CR assay.
Briefly, CR was dissolved in MilliQ water (MilliQ IQ 7000 purification system, Millipore-Sigma).CR stock solution (0.2% w/v) was filtered through a 0.22 μm filter and used immediately.A7PRD samples were prepared by dissolving ~1 mg lyophilized protein in 20 μL DMSO and diluting this solution to 50 μM using a buffer comprising 25 mM HEPES, pH 7.0, and 5 mM CaCl2.These freshly prepared samples were mixed with CR stock solution (50:1 dilution; protein vs. CR).Additionally, samples of A7PRD were incubated for ~3 h at room temperature to induce fibrillization, followed by mixing with CR stock solution (same dilution as above).In both cases, the absorption spectra of three replicates were measured using an DeNovix DS-11+ (M/C) Spectrophotometer.

Transmission electron microscopy (TEM).
TEM samples of fibrils of A7 and A7PRD constructs were prepared using our published protocols. 4- 5 EM images were acquired using a JEM-1400 Plus transmission electron microscope (JEOL) and recorded on a OneView digital camera (Gatan), Electron Microscopy Core Facility, UC San Diego.

X-ray diffraction.
X-ray diffraction was carried out as described previously. 12A 50 μM stock solution of A7PRD was incubated at 37 °C for a week.A7PRD fibrils were pelleted for 30 min at 259,000g and 20°C using Optima XE Ultracentrifuge and SW 55 Ti swinging bucket rotor (Beckman Coulter).A small amount of the sample was dried and loaded onto a Cryoloop.The sample was mounted on a Bruker Microstar 592 diffractometer equipped with an APEX II CCD detector and Cu Kα radiation (λ = 1.54178Å); UC San Diego Crystallography Facility.X-ray data was collected using a 360° ϕ scan with an exposure time of 300 s.

Fibril formation and dissolution kinetics.
In the case of A7PRD, samples were prepared by dissolving the lyophilized A7PRD in DMSO and rapidly diluting the DMSO stock in a buffer comprising 25 mM HEPES, pH 7, and 5 mM CaCl2 to achieve final concentrations ranging from 5-50 μM.Measurements were carried out at 37 °C under non-agitated conditions using a microplate reader (Infinite M Plex; Tecan) and sealed 96well flat bottom plates (Corning; catalog no.3370) containing 100 μl sample per well.ThT (20 μM) fluorescence was recorded as a function of time.Excitation and emission wavelengths were 415 and 480 nm, respectively.For A7, similar experimental conditions were used, except that measurements were carried out with continuous linear shaking (1.5 mm, 335.8 rpm).To determine the effect of seeding on the aggregation kinetics, 50 μM of A7PRD was allowed to aggregate overnight at room temperature.The resultant fibrils were resuspended using a pipette tip and were added to the freshly prepared 5 μM A7PRD solutions, such that the seeds represented 1 and 5% of the total protein mass.These samples were allowed to aggregate under non-agitating conditions; 5 μM A7PRD solution without the seeds served as a control.Experimental and buffer conditions were the same as described above.Global fitting of the experimental ThT curves for A7PRD was carried out using the protocol described by Meisl et al.   (ranging between 12-33%).In addition, the following domains/regions harbor high proline content (>15%): A7 (residues 2-180; ~24% prolines), A11 (residues 2-196; ~30% prolines), ALIX (residues 800-868; ~29% prolines), UBQLN2 (residues 491-537, ~33% prolines), and tau (residues 151-246; ~17% prolines).Note that although the precise molecular code that governs biomolecular phase separation is unclear, it is primarily mediated by disordered proteins or domains. 22PRDs are therefore often involved in phase separation due to their lack of structure and their ability to form dynamic multivalent complexes. 10Additionally, the presence of interspersed glycine and aromatic residues (e.g., tyrosine and phenyl alanine) may modulate phase separation of the PRDs as well as their fibrillization via increased degrees of freedom and hydrophobic CH/π interactions, respectively.[m0] γ .The obtained value of γ (i.e., -0.66) suggests that in the case of A7PRD fibrillization, the primary nucleation process is partially dependent on initial monomer concentration, whereas the secondary process of fibril propagation is independent of monomer concentration.c) The yield represents the total amount of TEV-cleaved protein obtained from a liter of bacterial culture.

15 S9Figure S1 .
Figure S1.Primary sequence comparison of representative A7-head domains among vertebrate species.A blue-to-red gradient is used to denote conservation, with blue and red colors depicting the least and most conserved residues, respectively.The following sequences were used for analysis: H. sapiens (Uniprot accession no.P20073), M. musculus (Uniprot accession no.Q07076), and B. taurus (Uniprot accession no.P20072).

Figure S2 .
Figure S2.Primary sequence comparison of head domains of A7 and A11.Same color gradient as Fig. S1.The uniprot accession no. for A11 is P50995.

Figure S3 .
Figure S3.Comparison of A7-head domain with other known human proteindomains/regions that undergo phase separation and/or fibrillization.(A) Analysis of the primary sequence of A7-head domain using the webserver CIDER (classification of intrinsically disordered ensemble relationships)16 and the corresponding diagram of states.The CIDER webserver calculates the fractions of positively and negatively charged residues in a given polypeptide sequence (f+ and f-, respectively), and uses these fractions to partition the sequence into one of the five distinct conformational classes, namely globules and tadpoles (R1), collapsed or expanded (R2), coils and hairpins (R3), and swollen coils (R4 and R5).The A7-head domain is

Figure S4 .
Figure S4.Recombinant A7 constructs used in current study.(A) List of recombinant A7 constructs, namely A7, A7 PRD Strep and A7PRD.Each construct is designated by a circled number.The locations of the TEV cleavage sites are marked by blue vertical dashed lines and scissors.In the case of A7 (construct no.1), the TEV cleavage site is located at the C-terminus, sandwiched between a spacer sequence (SGSENLYFQ) and the twin-strep tag 1 .Note that native residue Q488 of A7 was not included in this construct to accommodate the spacer and TEV cleavage site.For the truncated constructs, GB1-6xHis denotes the N-terminal GB1 tag, used to enhance the expression levels, followed by a spacer sequence and a polyhistidine (6xHis) affinity tag.Additionally, A7 PRD Strep (construct no. 2) carried a non-cleavable C-terminal strep tag 23 .(B) Analysis of TEV-cleaved constructs (same numbering as panel A) using liquid chromatographyelectrospray ionization-time-of-flight mass spectrometry (LC-ESI-TOFMS); the numbers in parenthesis represent the corresponding theoretical masses.

Figure S5 .
Figure S5.Sedimentation analysis of recombinant A7.Absorbance sedimentation c(s) profiles of recombinant A7 (15 µM).All measurements were carried out at 20 ºC in 25 mM HEPES, pH 7, and 1 mM TCEP.Note that unlike the profiles shown in Fig.1C(main text) that were acquired using 12 mm cell, the above data were acquired using 3 mm cell.

Figure S6 .
Figure S6.Confocal microscopy analysis of membrane binding properties of A7.GUVs were made using DOPC (99.95%) and spiked with 16:1 Liss Rhod PE dye (0.05%).The concentration of ATTO-647N-labeled A7 was 200 nM.The buffer conditions were as follows: 25 mM HEPES, pH 7, and 100 nM calcium.Representative microscopy images of the respective fluorescent channels and their overlay are shown.The lack of fluorescence signal from A7 at the membrane surface indicated that A7 did not colocalize with zwitterionic GUVs containing DOPC.

Figure S7 .
Figure S7.The interplay between calcium, protein concentration, and phase separation of A7 PRD Strep .Phase separation of A7 PRD Strep was monitored by turbidity assay at varying protein and calcium concentrations; n = 2. Representative images of 50 µM A7 PRD Strep with and without calcium are shown in the dashed square on the right.Note that in the absence and presence of 0.1 mM calcium, the condensates formed by 50 µM A7 PRD Strep were on a submicrometer scale and, thus, were difficult to visualize using fluorescence microscopy.A stepwise increase in calcium concentrations (0.5, 1, and 5 mM) resulted in the formation of correspondingly larger droplets, further confirming the regulatory roles of calcium and consequently, of hydrophobic interactions in the modulation of A7 phase separation.Microscopy images were acquired at 25 °C.The buffer composition was as follows: 25 mM HEPES, pH 7, and 0.02 mg/mL Streptavidin Alexa-Fluor488.

Figure S8 .
Figure S8.DIC images of droplets made by A7PRD.Images were acquired at 25 °C.The buffer composition as follows: 25 mM HEPES, pH 7, and 5 mM calcium.

Figure S9 .
Figure S9.Aggregation kinetics of A7PRD.ThT fluorescence was monitored to determine the effects of concentration on the aggregation kinetics of A7PRD, n = 2, mean (solid line), SD (shaded region).All measurements were carried out at 37 °C under non-agitated conditions.The buffer composition as follows: 25 mM HEPES, pH 7, 5 mM calcium, and 20 μM ThT.

Figure S10 .
Figure S10.Dependence of half-times, t1/2, on the initial monomer concentration of A7PRD.Double logarithmic plot, log t1/2 = γlog[m0] + constant, where γ is the scaling exponent and m0 is A7PRD monomer at time 0. The raw data used to generate this plot is shown in Fig. S9.The values of t1/2 and γ were extracted using the program Amylofit 15 by fitting the power-law function, t1/2 ~

Figure S11 .
Figure S11.Microscopic processes involved in protein fibrillization, and the associated rate constants.kn is the rate constant for the formation of primary nuclei, k+ is the fibril elongation rate constant, and k2 is the rate of formation of secondary nuclei, adapted from Meisl et al.15

Table S1 . Recombinant constructs used in current study (a,b) . ______________________________________________________________________________
Cultures were grown in LB media overnight upon induction with 1 mM IPTG.LB capsules were obtained from MP Biomedicals (catalog no.3002-036), and were used according to the manufacturers' protocols.