Fig. 4: C-loop and C-helix stabilize SOS1 in an occluded conformation.

a Occluded conformation of SOS1, stabilized by the C-loop and the C-helix. The intracellular cavity between the dimerization ___domain and core ___domain is displayed with the electrostatic surface, and the depth of the cavity is labeled. The putative cation binding sites, D201^TM6, are shown as sticks. The C-loop and C-helix are displayed as tubes in purple. b, c Alignment of the core ___domain of inward-facing human NHE1-CHP1^IF (deep blue) and SOS1 (orange). The core ___domain is shown in cartoon, and the dimerization ___domain of SOS1 is shown in a solid surface. The directions and distances of TM5b displacement are indicated. d Interactions among TM5b, TM6, and the N-terminus of HC8′ from the other protomer. Charged residues involved in the interactions are shown in sticks. The dashed line indicates the charge-dipole interaction between K704′ and the C-terminal of TM5b. e Salt tolerance test of AXT3K cells expressing solely the wild-type SOS1 (SOS1-WT), SOS1 together with SOS2-SOS3 (SOS1 + SOS2 + SOS3) and SOS1 mutants as indicated. Decimal dilutions of saturated cultures were plated in AP medium supplemented with 1 mM KCl and 0, 50, 100, 200, or 400 mM NaCl. The concentration of NaCl is labeled above the image. The growth of all transformants was indistinguishable in plates without NaCl. f Hydrophobic interactions between the C-loop and α-CTD. The hydrophobic cavity formed by HC1, HC3, and HC5 is shown as the surface. g Polar interactions between the C-loop and α-CTD. Hydrogen and salt-bridge bonds are shown as dashed lines. h C-helix interacts with HC1, HC2, and CNBLD′ from another subunit. i Intracellular Na⁺ content in the untransformed and transgenic yeast cells. Units are milligram of ion per gram dry weight of cell samples. DW: dry weight. Data are expressed as the means ± SEM (n = 3) and asterisks above the columns indicate significant differences. Each spot represents a single data value. Statistical significance was determined by two-side and unpaired t-test, without making any adjustments for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; ***p < 0.0001). P value, SOS1-WT vs. ΔC-loop, 0.0001; SOS1-WT vs. W1013A, 0.0001; SOS1-WT vs. ΔC-loop/C-helix, <0.0001; W1013A vs. ΔC-helix, 0.0007; W1013A vs. ΔC-loop/C-helix, 0.0140; ΔC-helix vs. ΔC-loop/C-helix, 0.0001. Source data are provided as a Source Data file.