Fig. 1: Cooperative assembly of the SHOC2–MRAS–PP1Cα ternary complex.
a, The affinity (KD) and associated cooperativity (α) values derived from SPR sensorgrams with surface-immobilized MRAS–GppNHp (WT or Q71R) and the indicated analytes. Cooperativity is defined as the ratio of binary and ternary KD values, α = KDSHOC2/KDSHOC2–PP1C. The increase in response units (RU) signal is consistent with the saturation of MRAS with both proteins Rmax, SHOC2, ~800–1,000 RU; Rmax, SHOC2/PP1Cα, ~1,250–1,500 RU). b, Sedimentation coefficient distributions c(s) derived from sedimentation velocity profiles of MRAS(Q71R) (black); PP1Cα (bronze); SHOC2(80–582, M173I) (blue); mixture of MRAS(Q71R) and SHOC2(80–582, M173) (gold); mixture of SHOC2(80–582, M173I), MRAS(Q71R) and PP1Cα (red). The raw sedimentation signal was acquired over time by absorbance at 280 nm at a rotor speed of 42,000 rpm at 20 °C. All proteins were sedimented at equimolar concentrations of 10 μM. Data are representative of two independent experiments. c, Ultraviolet light absorbance at 280 nm traces from semi-preparative SEC (top). SDS–PAGE analysis of individual fractions corresponding to the ternary complex trace (bottom). The SEC experiment was independently repeated three times with similar results. d, Surface representation of the ternary complex of SHOC2 (pale blue), MRAS (grey) and PP1Cα (maroon). The phosphate group bound at the active site is shown as red spheres, and Mn2+ is shown as purple.
