Fig. 4. Open conformation promotes SHP2 LLPS.

(A) Representative disease-associated SHP2 mutants. NS/JMML mutations (blue) and NS-ML (green). (B) A scheme representing the intrinsic propensity for open conformation in SHP2^WT and indicated SHP2^mut. (C) The correlation between in vitro LLPS capability and openness of SHP2 mutants. (D) Representative images of recombinant indicated SHP2^mut-mEGFP protein droplet formation in the presence of 10%(w/v) PEG3350. Scale bar, 5 μm. (E) A scheme illustrating allosteric inhibitor SHP099 locks SHP2 in the closed conformation. (F) Single molecule FRET results show the conformation change of SHP2^E76A-87/266 induced by SHP099. See Figure S4A for representative FRET time-trajectories. (G, H) Indicated SHP2^mut droplets were treated with SHP099 and ET070. Time course of droplet turbidity OD600 (means ± SEM, N = 3 experiments) **p<0.01; ***p<0.001. (G) and representative images (H). Scale bar, 5 μm. (I, J) KYSE520 cells stably expressing indicated SHP2^mut–mEGFP treated with SHP099 or ET070. Quantification of puncta/nuclei (means ± SEM) **p<0.01; ***p<0.001. N=46 cells. (I) and representative images of indicated SHP2^mut–mEGFP (J). Scale bar, 10 μm. (K) The cartoon for binding of the bis-P peptide to SHP2. (L) SHP2^Y279C proteins were stimulated w/o 1 μM bis-P peptide and the droplet turbidity (means ± SEM, N=2 experiments) was assessed. ***p<0.001. (M) Images of SHP2^Y279C droplets treated with DMSO and 1 μM bis-P peptide. Scale bar, 5 μm.