Extended Data Fig. 7: Competition between NRG1A and NRG1C for the interaction with the EDS1-SAG101 complex.

a, EDS1-SAG101 and NRG1C form a monomeric ternary complex in gel filtration. Shown in the left are gel filtration profiles (Superdex 200 Increase 10/300) of EDS1-SAG101 (purple), NRG1C (red), and EDS1-SAG101-NRG1C (green) proteins. A₂₈₀, absorbance at 280 nm; mAU, milli-absorbance units. Peak fractions in the left were visualized by SDS-PAGE followed by Coomassie blue staining and are shown on the right. b, Purification of NRG1A^∆CC protein. N terminal GST tagged NRG1A^∆CC (residues 160-809) was expressed in insect cells. GST beads were used to purify proteins, which were further purified by Superose 6 Increase 10/300 GL. Left: a gel filtration profile of NRG1A^∆CC. Right: SDS-PAGE analysis of peak fractions. c, NRG1C fails to efficiently outcompete NRG1A^WHD-LRR in EDS1-SAG101- NRG1A^WHD-LRR. 0.08 μmol the EDS1-SAG101-NRG1A^WHD-LRR (with a GST tag) complex protein was incubated with a varying amount of NRG1C (0, 0.04, 0.08, 0.16, 0.32, 0.64 μmol). The mixture was flowed through the Strep beads. After extensive washing, the beads were analysed by SDS-PAGE. In a-c, the experiments were repeated three times with similar results. For gel source data, see Supplementary Fig. 1. d, Structural comparison of EDS1-SAG101-NRG1C with AlphaFold2-predicted EDS1-SAG101-NRG1A^NOD-LRR. The cryo-EM structure of EDS1-SAG101-NRG1C (PDB code: 8YN0) was aligned with AlphaFold2-predicted EDS1-SAG101-NRG1A^NOD-LRR in PyMOL. The NBD of NRG1A is shown in surface and others in cartoon representation. The NBD of NRG1A clashes with EP domains of EDS1-SAG101.