Surface Charge Can Modulate Phase Separation of Multidomain Proteins

Phase separation has emerged as an important mechanism explaining the formation of certain biomolecular condensates. Biological phase separation is often driven by the multivalent interactions of modular protein domains. Beyond valency, the physical features of folded domains that promote phase separation are poorly understood. We used a model system—the small ubiquitin modifier (SUMO) and its peptide ligand, the SUMO interaction motif (SIM)—to examine how domain surface charge influences multivalency-driven phase separation. Phase separation of polySUMO and polySIM was altered by pH via a change in the protonation state of SUMO surface histidines. These effects were recapitulated by histidine mutations, which modulated SUMO solubility and polySUMO–polySIM phase separation in parallel and were quantitatively explained by atomistic modeling of weak interactions among proteins in the system. Thus, surface charge can tune the phase separation of multivalent proteins, suggesting a means of controlling phase separation biologically, evolutionarily, and therapeutically.

). Basic residues including H16, H36, K34, and R35 of SUMO3 make greater contributions to self-interaction than the prominent basic residues in SUMO1 (Figure 4C).D) Same as panel C but calculated at high pH.At high pH, H16 and H36 lose importance; instead, other basic residues, R55 and R58, gain prominence.

Figure S1 :
Figure S1: Binding of SIM to WT SUMO as measured by ITC at pH 6.5, 7.0, and 8.0.

Figure S2 :
Figure S2: WT SUMO makes large soluble aggregates in solution at pH 6. A) Dynamic light scattering autocorrelation plot and B) the regularization graph of the scattering autocorrelation of WT SUMO at pH 6.Note that WT SUMO has a large scattering intensity around a radius of ~ 100 nm at pH 6. C) and D) Corresponding results at pH 6.5, demonstrating that this aggregate is not present at the higher pH.

Figure S3 :
Figure S3: pH only affects A2, not the affinity of SIM for the SUMO3 isoform; and salt dependence of SUMO1 A2 values.A) Kd measured between WT SUMO3 and SIM at pH 7 and 8 by using ITC.The affinity is identical within error, indicating that the ability to form networks is unchanged with pH.B) The A2 of WT SUMO3 at pH 7 and 8 determined by static light scattering, indicating a change in weak self-association with pH.C) The A2 of WT SUMO1 at pH 6.5 is dependent on salt concentration, suggesting that the interaction is electrostatic in nature.

Figure S4 :
Figure S4: Static light scattering data for SUMO mutants at pH 7 as a function of concentration.

Figure S5 :
Figure S5: pH titration of WT SUMO assessed by NMR spectroscopy.A) pH dependence of the 1 He chemical shifts and B) the pH dependence of the 13 Ce chemical shifts.The fitted pKa values are shown in the inset of the plot.Although sequence specific chemical shift assignments are not available for these moieties, all three histidine 1 He and 13 Ce signals in the NMR spectra had measured pKa values between 6.27 and 6.53, and are identical for each histidine within experimental error.

Figure S6 :
FigureS6: T-test of the histidine to serine mutations at pH 8 compared to WT SUMO.The t-test shows that the H35S and H43S mutations caused a statistically significant change of A2 (p < 0.05 for H35S and p < 0.01 H43S), while the H75S mutation did not cause a statistically significant difference in A2.

Figure S7 :
Figure S7: Second virial coefficients of the SUMO H35R/H43R double mutant at pH 7 (left) and pH 8 (right).Values for the SUMO H35R and H43R single mutants are included for comparison.

Figure S8 :
Figure S8: Results similar to those presented in Fig. 4, but calculated at high pH, i.e., with all His residues deprotonated.A) Surface electrostatic potential of SUMO1.B) Decomposition of the binary self-interaction energy of SUMO.Values for prominent basic residues (H35, K37, K39, H43, K46, and R54) and acidic residues (E67, E83, E84, E85, D86, and E89) are shown as blue and red bars, respectively.C) The difference between high pH and low pH results for inter-residue interactions.Residues showing the highest pH dependence are indicated by blue (K16, H35, H43) or red (E67, E83, and D86) bars; H75 is also shown by a blue bar.

Figure S9 :
Figure S9: Results similar to those presented in Fig. 4, but for SUMO3.A) Surface electrostatic potential of SUMO3 calculated at low pH, i.e., with all the His residues protonated.The negative potential on the back face is more intense and spreads more widely than the SUMO1 counterpart (Figure 4A, bottom right), due to substitutions to acidic residues (D70 and E76 in SUMO3).B) Sequence alignment of SUMO1 and SUMO3.Conserved basic and acidic residues are shown in blue and red, respectively; His residues are in green.C) Decomposition of the binary selfinteraction energy of SUMO3 at low pH.The highest contributions are shown as blue bars (H16, K34, R35, and H36) or red bars (D62, E76, E78,and D81).Basic residues including H16, H36, K34, and R35 of SUMO3 make greater contributions to self-interaction than the prominent basic residues in SUMO1 (Figure4C).D) Same as panel C but calculated at high pH.At high pH, H16 and H36 lose importance; instead, other basic residues, R55 and R58, gain prominence.

Figure S10 :
Figure S10: Change in binding affinity and weak self-association of the SUMO E67R mutant.A)The A2 of E67R is higher than that of WT SUMO and B) the binding affinities of SUMO E67R for SIM at pH 7 and 8 are similar to those of WT SUMO for SIM.

Figure S11 :
Figure S11: Linear correlation analysis of calculated and experimental results for A2 values of SUMOs at different pHs and with a variety of mutations.The calculated results here assumed the N-tail open conformation of SUMO.Symbols colors are: SUMO1, black; SUMO3, yellow; H35 mutants, purple; H43 mutants, dark blue; H75 mutants, cyan; H35/H43 double mutants, royal blue; and E67 mutants, red.

Figure S12 :
Figure S12: Turbidity measurements at 340 nm for mutant polySUMO and polySUMO3 titration with an equal module concentration of polySIM at pH 7 (green) and 8 (blue).The extrapolation to determine the phase separation threshold was performed by calculating a linear fit from the first three concentrations with an A340 > 0.1.

Figure S13 :
Figure S13: The affinities of SUMO mutants for SIM measured by ITC at A) pH7 and B) pH 8. Asterisks indicate that no heat was detected at the indicated temperature.ITC binding experiments did not show substantial deviation in measured affinity between 25°C and 30°C.

Figure S14 :
Figure S14: Principal component analysis of FMAP results.A) Eigenvalues of four sets of FMAP results.Each set of results comprised A2 or A23 values calculated using the open or closed conformations of SUMO, for 30 combinations of protein variant and pH (29 listed in Fig. 5 plus SUMO at pH 6).The four orthogonal eigenvectors have normalized amplitudes in the range of 0.3 to 0.7 along the four virial coefficients.PC1 has positive values for all its four components, while the higher PCs each have two positive and two negative components.In particular, PC4 has positive components along A2 closed and A23 open.B) Projection of the FMAP results for 14 combinations of protein variant and pH along two of the principal components, PC1 and PC4.These 14 combinations were those for which phase-separation threshold concentrations were measured.The two PCs were selected because they showed the highest correlations with the measured threshold concentrations.