LiteFold/DisProt huggingface dataset:
Protein: DP04141 (UniProt Q96E09)
Region: residues 73	to 95
Annotation: protein phosphatase 2A binding (Molecular function)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
An overlay of the 2D 1H,15N HSQC spectrum of FAM122AID with and without B55LL (Fig. 1h and Extended Data Fig. 5c) showed that N/HN cross-peaks with reduced intensities correspond to FAM122A residues 73–95, which bind solely to B55.
Protein: DP04141 (UniProt Q96E09)
Region: residues 81	to 111
Annotation: protein phosphatase 2A binding (Molecular function)
Evidence: cryogenic electron microscopy evidence used in manual assertion
Supporting statements:
FAM122A also binds exclusively to B55 and PP2Ac and does so, again, using helices (pre-populated in free FAM122A; Extended Data Fig. 3d,f). FAM122A binds a short surface on B55 (the B55 binding platform) and across the PP2Ac active site; the interaction buries 2,700 Å2 solvent-accessible surface area.
Cross-references: PDB:8SO0
EMDB:40644
Protein: DP04141 (UniProt Q96E09)
Region: residues 81	to 111
Annotation: order (Structural state)
Evidence: cryogenic electron microscopy evidence used in manual assertion
Supporting statements:
FAM122A also binds exclusively to B55 and PP2Ac and does so, again, using helices (pre-populated in free FAM122A; Extended Data Fig. 3d,f).
The interaction of FAM122A with PP2A:B55 is different to that of ARPP19, with FAM122A residues 81–111 binding PP2A:B55 with two helices (Fig. 4a).
Cross-references: PDB:8SO0
EMDB:40644
Protein: DP04141 (UniProt Q96E09)
Region: residues 81	to 111
Annotation: disorder to order (Structural transition)
Evidence: cryogenic electron microscopy evidence used in manual assertion
Supporting statements:
FAM122A also binds exclusively to B55 and PP2Ac and does so, again, using helices (pre-populated in free FAM122A; Extended Data Fig. 3d,f).
The interaction of FAM122A with PP2A:B55 is different to that of ARPP19, with FAM122A residues 81–111 binding PP2A:B55 with two helices (Fig. 4a).
Cross-references: PDB:8SO0
EMDB:40644
Protein: DP04141 (UniProt Q96E09)
Region: residues 29	to 80
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy evidence used in manual assertion
Supporting statements:
The lack of electron density shows this region is disordered.
Although FAM122A residues 29–66 were not sufficiently ordered to be modelled, our NMR and binding data suggest that they contribute to binding via a dynamic (fuzzy) charge–charge interaction24,31,32 (Extended Data Table 2 and Extended Data Fig. 2b).
Cross-references: PDB:8SO0
EMDB:40644
Protein: DP04141 (UniProt Q96E09)
Region: residues 84	to 89
Annotation: protein phosphatase 2A binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Mutating the basic–hydrophobic residue pairs—that is, 84RLHQIKQEE92 to 84AAHQIKQEE92 (84AA85) and 84RLHQAAQEE92 (88AA89)—reduced FAM122A binding by 1.6- and 2.0-fold, respectively (Fig. 4g and Extended Data Table 2). Pull-down assays using PP2A:B55 lysates incubated with 84AA85 and 88AA89 FAM122A showed similar reductions in binding compared with the wild-type protein (Fig. 4h). Consistent with their weaker affinities, the IC50 values of the 84AA85 and 88AA89 variants increased by 38- and 48-fold, respectively (Fig. 4g and Extended Data Table 1).
Protein: DP04141 (UniProt Q96E09)
Region: residues 90	to 95
Annotation: protein phosphatase 2A binding (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Because the FAM122A E92K mutation was identified in cancer tissues (cBioPortal), we also generated E91K and E92K variants and showed they also bound PP2A:B55 less strongly and were less potent inhibitors of PP2A:B55 (Extended Data Tables 1 and 2 and Extended Data Fig. 2a,b).
Protein: DP04141 (UniProt Q96E09)
Region: residues 97	to 111
Annotation: protein phosphatase inhibitor activity (Molecular function)
Evidence: cryogenic electron microscopy evidence used in manual assertion
Supporting statements:
FAM122A residues C-terminal to the B55 helix form a sharp turn with helix α2 (97INRETVHEREVQTAM111, the inhibition helix; Extended Data Fig. 9i) binding and blocking the PP2Ac active site. 
Cross-references: PDB:8SO0
EMDB:40644
Protein: DP04141 (UniProt Q96E09)
Region: residues 104	to 108
Annotation: protein phosphatase inhibitor activity (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
cBioPortal34 highlighted that FAM122A R105L, V107G variants are present in different cancers (FAM122A is a tumour suppressor, as patients with cancer who express low levels of FAM122A have significantly worse overall survival than those with high levels of expression8). FAM122A R105L and V107G variants showed 11- and 6-fold less inhibition than the wild-type protein, respectively (Fig. 4l and Extended Data Table 1), demonstrating that the probable mode of action of these cancer variants is due to a weaker inhibition of PP2A:B55, thereby disrupting PP2A:B55 cellular functions.
Protein: DP04141 (UniProt Q96E09)
Region: residues 1	to 124
Annotation: protein phosphatase 2A binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
We also performed a pull-down competition assay by affinity purification of PP2A:B55 (using GFP–B55) in the presence of FAM122A alone or FAM122A with a fivefold excess of tpARPP19 (Extended Data Fig. 10d)
Protein: DP04141 (UniProt Q96E09)
Region: residues 97	to 111
Annotation: protein phosphatase 2A binding (Molecular function)
Evidence: cryogenic electron microscopy evidence used in manual assertion
Supporting statements:
FAM122A residues C-terminal to the B55 helix form a sharp turn with helix α2 (97INRETVHEREVQTAM111, the inhibition helix; Extended Data Fig. 9i) binding and blocking the PP2Ac active site.
Cross-references: PDB:8SO0
EMDB:40644
Protein: DP04141 (UniProt Q96E09)
Region: residues 1	to 124
Annotation: protein phosphatase inhibitor activity (Molecular function)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
We quantified PP2A:B55 inhibition by ARPP19, thiophosphorylated ARPP19 (full-length (amino acids 1–112) and phosphorylated with ATPγS using MASTL kinase) and FAM122A (N-terminal domain (amino acids 1–124) (FAM122ANterm)) (Fig. 1b). Whereas PP2A:B55 was only moderately inhibited by ARPP19, it was strongly inhibited by both thiophosphorylated ARPP19 and FAM122ANterm (Extended Data Table 1 and Extended Data Fig. 2a), with thiophosphorylated ARPP19 inhibiting PP2A:B55 around 250-fold more potently than FAM122A.
Protein: DP04142 (UniProt Q09312)
Region: residues 570	to 632
Annotation: disorder (Structural state)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
Here, we report that an intrinsically disordered region (IDR) at the C-terminus of FBF-2 autoinhibits its RNA-binding affinity by increasing the off rate for RNA binding.
We report a 2.1-Å crystal structure of FBF-2 in complex with RNA that identifies a PIM within the intrinsically disordered FBF-2 C-terminal tail (CT) bound to the same site on the RBD as LST-1.
Protein: DP04142 (UniProt Q09312)
Region: residues 605	to 615
Annotation: order (Structural state)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
To explore possible roles of the FBF-2 CT, we expressed and purified an FBF-2 fragment containing both RBD and CT (FBF-2164–632). This FBF-2 RBD + CT had increased thermal stability compared to the RBD alone (Supplementary Table 1). We determined a crystal structure of FBF-2 RBD + CT in complex with a cFBE RNA (5′-CUGUGAAUG-3′) at 2.1 Å resolution (Supplementary Table 2, Fig. 1c). The electron density map revealed new density, not present in the previous RBD structures. This additional density appeared near a loop in the RBD that connects repeats 7 and 8 (Fig. 1d). This R7-R8 loop (residues 476-489) also binds to both LST-1 A and LST-1 B peptides in crystal structures of FBF-2 RBD in complex with LST-1 (Fig. 1c)17,18. The sequence of FBF-2 CT residues 607-613 matched this new electron density, but other C-terminal residues (aa 570-606 and 614-632) were disordered.
This evidence include the seven visible FBF-2 CT residues (SLMLEPR 607–613).
Cross-references: PDB:8SJ7
Protein: DP04142 (UniProt Q09312)
Region: residues 605	to 615
Annotation: disorder to order (Structural transition)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
To explore possible roles of the FBF-2 CT, we expressed and purified an FBF-2 fragment containing both RBD and CT (FBF-2164–632). This FBF-2 RBD + CT had increased thermal stability compared to the RBD alone (Supplementary Table 1). We determined a crystal structure of FBF-2 RBD + CT in complex with a cFBE RNA (5′-CUGUGAAUG-3′) at 2.1 Å resolution (Supplementary Table 2, Fig. 1c). The electron density map revealed new density, not present in the previous RBD structures. This additional density appeared near a loop in the RBD that connects repeats 7 and 8 (Fig. 1d). This R7-R8 loop (residues 476-489) also binds to both LST-1 A and LST-1 B peptides in crystal structures of FBF-2 RBD in complex with LST-1 (Fig. 1c)17,18. The sequence of FBF-2 CT residues 607-613 matched this new electron density, but other C-terminal residues (aa 570-606 and 614-632) were disordered.
This evidence include the seven visible FBF-2 CT residues (SLMLEPR 607–613).
Cross-references: PDB:8SJ7
Protein: DP04142 (UniProt Q09312)
Region: residues 605	to 615
Annotation: RNA binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
To explore possible roles of the FBF-2 CT, we expressed and purified an FBF-2 fragment containing both RBD and CT (FBF-2164–632). This FBF-2 RBD + CT had increased thermal stability compared to the RBD alone (Supplementary Table 1). We determined a crystal structure of FBF-2 RBD + CT in complex with a cFBE RNA (5′-CUGUGAAUG-3′) at 2.1 Å resolution (Supplementary Table 2, Fig. 1c). The electron density map revealed new density, not present in the previous RBD structures. This additional density appeared near a loop in the RBD that connects repeats 7 and 8 (Fig. 1d). This R7-R8 loop (residues 476-489) also binds to both LST-1 A and LST-1 B peptides in crystal structures of FBF-2 RBD in complex with LST-1 (Fig. 1c)17,18. The sequence of FBF-2 CT residues 607-613 matched this new electron density, but other C-terminal residues (aa 570-606 and 614-632) were disordered.
This evidence include the seven visible FBF-2 CT residues (SLMLEPR 607–613).
Cross-references: PDB:8SJ7
Protein: DP04142 (UniProt Q09312)
Region: residues 607	to 613
Annotation: protein binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
FBF-2 CT binds FBF-2 RBD on the same surface as LST-1
Protein: DP04142 (UniProt Q09312)
Region: residues 570	to 632
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
FBF-2 CT and partner proteins compete for binding to the RBD
We found that the FBF-2 CT greatly reduced FBF-2 affinity for LST-1 A and LST-1 B. Binding of LST-1 A to FBF-2 RBD + CT was too weak to be detected by ITC (Fig. 2c) and thus considerably weaker than the affinity of LST-1 A to the RBD alone (Kd = 2.1 µM)18. The binding of LST-1 B to RBD + CT was ~50-fold weaker than to the RBD alone (Kd = 2.7 µM for RBD + CT vs 0.05 µM for RBD, Fig. 2b, d)18. The lower protein interaction affinities for LST-1 A and LST-1 B caused by the FBF-2 CT suggested that the CT and LST-1 compete for binding to the RBD.
Protein: DP04142 (UniProt Q09312)
Region: residues 570	to 632
Annotation: protein binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
FBF-2 CT and partner proteins compete for binding to the RBD
To examine this idea, we next asked if the reduced binding affinity for LST-1 relies on the CT interaction with RBD. L610 in the FBF-2 CT is equivalent to the key leucines in LST-1 PIMs. Mutation of the key leucines in LST-1 disrupt interaction with FBF-217, and the L610A mutation was designed to test the importance of the interaction between the RBD and CT. We confirmed that the L610A mutation disrupts interaction with the FBF-2 RBD by ITC (Fig. 2e, Supplementary Table 3). The L610A substitution decreased the thermal stability of FBF-2 RBD + CT with a melting temperature of the mutated protein near that of the RBD alone (Supplementary Table 1), suggesting that interaction of the CT with the RBD is stabilizing. We found that FBF-2 RBD + CT L610A bound to LST-1 A and B with affinities similar to the FBF-2 RBD alone (Kd = 2.4 µM for LST-1 A and Kd = 0.06 µM for LST-1 B, Fig. 2b, f, g). Therefore, the interaction between the FBF-2 CT and RBD via L610 is critical for the reduced affinity of RBD + CT for LST-1 A and B, apparently by competing with LST-1 interaction.
Protein: DP04142 (UniProt Q09312)
Region: residues 570	to 632
Annotation: self-inhibition (Disorder function)
Evidence: electrophoretic mobility shift assay evidence used in manual assertion
Supporting statements:
FBF-2 CT autoinhibits RNA-binding affinity
The FBF-2 CT autoinhibits RNA-binding affinity.
Indeed, FBF-2 RBD + CT had a nearly 5-fold weaker affinity for gld-1 FBEa RNA than FBF-2 RBD alone (Kd = 334 nM for FBF-2 RBD + CT and 70 nM for RBD alone). This substantial decrease is greater than the effect of LST-1 B on RBD (Kd = 176 nM, Supplementary Fig. 1a), and the CT effect is intramolecular.
To confirm that the reduced RNA-binding affinity was due to the CT interacting with the RBD, we also measured RNA-binding affinity of FBF-2 RBD + CT proteins mutated in the R7-R8 loop (Y479A) or in the C-terminal tail (L610A). Y479 is a critical part of the hydrophobic pocket that binds to key leucine residues in partner proteins (Fig. 1f), and its mutation to an alanine (Y479A) disrupts interaction between FBF-2 and LST-117. Like L610A, the Y479A substitution decreased the thermal stability of FBF-2 RBD + CT (Supplementary Table 1).
We conclude that tethering of the FBF-2 CT to the RBD is autoinhibitory for RNA binding.
Electrostatic repulsion by acidic residues in the FBF-2 CT reduces RNA-binding affinity
The reduced negative charge in the FBF-2 CT loop increased binding affinity to RNA (Kd = 334 nM for RBD + CT vs 101 nM for RBD + CT Loop6A, Fig. 4d) and thus diminished CT autoinhibition. Therefore, both tethering of the CT and the cluster of charged residues contribute to CT autoinhibition.
Protein: DP04142 (UniProt Q09312)
Region: residues 570	to 632
Annotation: self-inhibition (Disorder function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
FBF-2 CT accelerates dissociation from the RNA
To further examine the effect of the CT on RNA-binding activity of FBF-2, we performed surface plasmon resonance (SPR) assays to measure the kinetics of FBF-2 binding to gld-1 FBEa RNA. We first compared the binding kinetics of FBF-2 RBD and FBF-2 RBD + CT. We found that the on rate for FBF-2 RBD binding was slightly faster than for RBD + CT: 13.6 × 104 M−1 s−1 vs 7.1 × 104 M−1 s−1. In contrast, the off rate for RBD + CT was ~5-fold faster than for RBD: 4.42 × 10−3 s−1 vs 0.86 × 10−3 s−1 (Fig. 5), indicating that the CT accelerates dissociation from the gld-1 FBEa RNA. We therefore measured the binding kinetics of FBF-2 RBD + CT mutants, Y479A, L610A, and Loop6A. Consistent with the differences in off rate for FBF-2 RBD vs RBD + CT and our EMSA results above, the off rates for the RBD + CT mutants were all slower than for wild type RBD + CT and similar to the off rate of FBF-2 RBD alone (Fig. 5c, Supplementary Fig. 3)
The SPR data further support the conclusion that the FBF-2 CT lowers RNA-binding affinity and does so by destabilizing protein-RNA interaction and accelerating dissociation.
Protein: DP04144 (UniProt P21707)
Region: residues 80	to 142
Annotation: disorder (Structural state)
Evidence: combinatorial experimental and author inference evidence used in manual assertion
Supporting statements:
Analysis of the linker, using the VL-XT algorithm and PONDR software47, revealed that residues 82-133 form an intrinsically disordered region (IDR; Fig. 2a). These structural predictions were further confirmed using AlphaFold48,49 (Supplementary Fig. 2).
Intrinsic disorder for the Syt 1 linker was experimentally confirmed in human (PMID: 27191789).
Protein: DP04144 (UniProt P21707)
Region: residues 80	to 142
Annotation: negative regulation of calcium ion binding (Biological process)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
Juxtamembrane linker of syt1 inhibits Ca2+ binding to the C2-domains
Intriguingly, inclusion of the complete linker resulted in a significant reduction in the intrinsic affinity for Ca2+ of all five sites compared to the construct lacking residues 80–95. In the case of the highest affinity site, the KD increased from 23.5 ± 2.1 to 80.0 ± 3.9 µM for Ca2+ upon removal of residues 80–95 (Fig. 1c, d, Table 1). Removal of the entire linker, resulting in the 143–421 construct, yielded relatively flat ITC traces due to the cancellation of the endo- and exothermic signals from each C2-domain, respectively, as documented previously46, precluding a determination of Ca2+ affinity (Fig. 1e).
These findings reveal that the juxtamembrane linker can negatively affect the Ca2+ binding activity of syt1 via a process that is disrupted by the lysine mutations.
Protein: DP04144 (UniProt P21707)
Region: residues 80	to 142
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
The juxtamembrane linker of syt1 mediates LLPS in vitro
These findings reveal that the lysine residues in the juxtamembrane linker are crucial for the cytoplasmic domain of syt1 to undergo LLPS. To confirm these results, we labeled both the complete and truncated cytoplasmic domain of syt1 (residues 80–421 and 143–421, respectively) with an organic dye (fluorescein) at native Cys residues 82 and 277. As expected, we observed droplet formation with the complete cytoplasmic domain of syt1 but not for the truncated protein lacking the linker (Supplementary Fig. 5). To assess whether the juxtamembrane linker alone is capable of undergoing LLPS, we subjected the constructs in Fig. 3b to the same conditions that were used in Fig. 3c. Interestingly, we found that the GFP-tagged juxtamembrane linker (80–142) formed droplets, and this activity was, again, abolished in the JuxtaK mutant (Fig. 3d). Hence, the juxtamembrane linker alone is sufficient to undergo LLPS, in a lysine residue-dependent manner. GFP alone failed to form droplets under any conditions tested (Fig. 3d).
Protein: DP04144 (UniProt P21707)
Region: residues 80	to 142
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: dynamic light scattering assay evidence used in manual assertion
Supporting statements:
As an independent measure of droplet formation, we conducted dynamic light scattering (DLS) experiments, again using the constructs shown in Fig. 3a, b, and found that WT syt1 C2AB, bearing the juxtamembrane linker, as well as the isolated juxtamembrane linker (residues 80–142), yielded two distinct peaks with diameters corresponding to monomers as well as higher ordered structures. In sharp contrast, a single peak, corresponding to monomers, was observed for the other constructs, as shown and quantified in Supplementary Fig. 6a, b, and Supplementary Table 3, respectively. These findings are consistent with the microscopy experiments in Fig. 3c, d. Since our DLS approach cannot discriminate between sizes above 1 µm, we returned to microscopy to establish the relationship between [syt1 C2AB (80–421)-GFP] and droplet size. As expected, based on LLPS of various proteins58, we observed that droplet size increased with protein concentration (Supplementary Fig. 7a–e). Because the droplet number is confounded by droplet-droplet fusion, this parameter was not further analyzed.
These additional findings, combined with results in Fig. 2, provide compelling evidence that the lysine-rich motif in the juxtamembrane linker of syt1 predominantly mediates LLPS.
Protein: DP04144 (UniProt P21707)
Region: residues 80	to 142
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence recovery after photobleaching evidence used in manual assertion
Supporting statements:
Furthermore, we conducted fluorescence recovery after photobleaching (FRAP) experiment on the syt1 droplets; a time series of representative FRAP images are shown in Fig. 4b (see Supplementary Movie 5 for the video), clearly demonstrating recovery. The fluorescence recovery curve was best fitted with a hyperbolic function, yielding a t1/2 of 64 ± 2 s (Fig. 4c; see Methods for analysis details), which is characteristic of the protein mobility in bona fide LLPS droplets. Together, these droplet fusions and FRAP results confirm that syt1 C2AB (80–421)-GFP undergoes LLPS.
Syt1 droplets fuse with each other, and recover after photobleaching.
Protein: DP04144 (UniProt P21707)
Region: residues 80	to 142
Annotation: non-membrane-bounded organelle assembly (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Syt1 undergoes LLPS in cells
Upon overexpression, droplet formation by syt1 C2AB (80–421)-GFP was observed in the HEK293T cells; as shown in Fig. 6a, ~5–10 droplets formed in the cytoplasm of each cell. Consistent with our in vitro biochemical assays (see Fig. 3c), truncation of the linker, or substitution of the lysine residues within the linker, abolished droplet formation (Fig. 6b).
Together, these experiments demonstrate that the cytoplasmic domain of syt1 forms droplets in cells, including neurons where syt1 is normally expressed.
Protein: DP04144 (UniProt P21707)
Region: residues 80	to 142
Annotation: identical protein binding (Molecular function)
Evidence: dynamic light scattering assay evidence used in manual assertion
Supporting statements:
As an independent measure of droplet formation, we conducted dynamic light scattering (DLS) experiments, again using the constructs shown in Fig. 3a, b, and found that WT syt1 C2AB, bearing the juxtamembrane linker, as well as the isolated juxtamembrane linker (residues 80–142), yielded two distinct peaks with diameters corresponding to monomers as well as higher ordered structures.
Protein: DP04144 (UniProt P21707)
Region: residues 86	to 139
Annotation: regulation of vesicle fusion (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
However, for the double or triple mutants we clearly observed the impairment of fusion pore opening (Figure 3B, middle and right-hand panels) in the presence of 500 μM Ca2+. The quantitative analysis revealed that after 1 min, content mixing for the double mutants was reduced by 20~30% when compared with that of wild-type Syt1, whereas the triple mutants had as much as a 50% reduction (Figure 3C). Thus the results of the present study suggest that the bipolar charge distribution contributes to fusion pore opening in a positive way.