Extended Data Fig. 5. Symmetry of the EREG-induced sEGFR^R84K dimer and implications for negative cooperativity in EGFR.

a. Overlay of the EREG-induced sEGFR^R84K dimer (dark green ribbons) with the symmetric dimers of sEGFR^WT induced by TGFα (1MOX¹⁵, gold ribbons) or EGF (3NJP^14,17, grey ribbons). EREG, TGFα and EGF are coloured magenta, orange, and black respectively.

b. Schematic of half-of-the-sites negative cooperativity in ligand binding to WT EGFR^20,54,55 for any ligand (blue). As we previously described in detail for the Drosophila EGFR²⁰, and as also seems to apply to human EGFR⁵⁴, binding of a single ligand can promote formation of asymmetric sEGFR dimers (left-hand side of cartoon) with autoinhibitory domain I/II interactions broken (open red star) only in one protomer. This asymmetric dimerisation is driven by contacts involving N-terminal regions of domain II as well as altered dimer arm docking^5,20 – together restraining domain II in the unliganded protomer. When a second ligand binds to this dimer, it must ‘wedge’ apart the two ligand-binding domains (I and III) in the right-hand protomer to drive formation of the symmetric dimer (top right in cartoon). This requires disruption of autoinhibitory domain I/II interactions in both molecules (both red stars are open). It also requires disruption of domain II dimer interface contacts – with a resulting bend in domain II (Extended Data Fig. 6d) – giving rise to the symmetric 2:2 dimer. This is readily achieved by high-affinity ligands such as EGF and TGFα, but low-affinity EGFR ligands like EREG⁵⁶ cannot disrupt the autoinhibitory domain I/II interactions or bend the restrained domain II to optimise dimer arm contacts. As a consequence, low-affinity ligands fail to wedge apart domains I and III in the right-hand protomer – instead binding to an unaltered asymmetric dimer (lower right in cartoon) through a compromised set of ligand/receptor interactions (i.e. a remodeled binding site⁵: see Extended Data Fig. 8d). The R84K mutation lowers this barrier to dimer ‘symmetrisation’ by weakening autoinhibitory domain I/II interactions so that the second ligand-binding event more readily bends domain II and symmetrises dimers. This appears to be the origin of the R84K mutation’s ability to selectively stabilise dimers induced by low-affinity EGFR ligands. Weakening of autoinhibitory domain I/II interactions may also explain the enhanced ligand-binding affinity seen for R84K EGFR (Extended Data Fig. 7b). The ability of the R84K mutation to equalise the two EREG-binding sites in a dimer, and to increase EREG affinity also argues that this mutation removes a barrier to ligand binding, and may diminish the half-of-the-sites negative cooperativity seen in wild-type EGFR^20,54,57,58.