Fig. 2: IRPAs induce distinct conformational changes and HDX signatures on IR-extracellular domain.

Conformational changes of IR-ECD upon binding to RHI or IRPAs were evaluated using single particle cryo-EM and HDX-MS. Upper panel shows the ribbon representation of IR-ECD dimer: IR-ECD dimer complexed with two RHIs (a), two IRPA-3 partial agonists (b; PDB code 7MD4) and two IRPA-9 full agonists (c; PDB code 7MD5). Each IR-ECD monomer is colored in green or cyan. RHI or insulin from IRPAs are colored in magenta, red, purple or blue, respectively. The α-CT helix is colored in orange. Lower left panel shows zoom view of a and b to illustrate relative positions of L1, α-CT helix, L2 domain and the bound RHI or IRPA. Zoom view for RHI bound structure (d): RHI (magenta); and IRPA-3 bound structure (e): insulin1 (red), and insulin2 (purple). Lower middle panel are reconstructed maps for IR-ECD bound with RHIs, IRPA-3 and IPRA-9 (f–h), respectively. Colored circles represent RHI or one of the insulin monomers in the covalently linked insulin dimer molecules (IRPAs), which correspond to the ones shown in a–c. The same insulin in IRPAs is colored the same for illustration purpose. Lower right panel shows hydrogen/deuterium exchange difference plots for RHI and IRPAs (i). Region 1 consists of sequences in IR L2 domain: IRGGNN (344–349) and RSYAL (372–376), which differentiate IRPA partial agonist from full agonist binding (within the dashed line). Region 2 consists of sequences in IR α-CT helix: FRKTFEDYLHNVVF (701–714). Underlined amino acids were the interaction sites detected in cryo-EM studies. Deuterium uptake protection at region 1 was clearly detected for RHI and IRPA-9, both of which are IR full agonists. In contrast, no difference in deuterium uptake was seen for IRPA-3, an IR partial agonist.