Functional selectivity of insulin receptor revealed by aptamer-trapped receptor structures

Abstract

Activation of insulin receptor (IR) initiates a cascade of conformational changes and autophosphorylation events. Herein, we determined three structures of IR trapped by aptamers using cryo-electron microscopy. The A62 agonist aptamer selectively activates metabolic signaling. In the absence of insulin, the two A62 aptamer agonists of IR adopt an insulin-accessible arrowhead conformation by mimicking site-1/site-2’ insulin coordination. Insulin binding at one site triggers conformational changes in one protomer, but this movement is blocked in the other protomer by A62 at the opposite site. A62 binding captures two unique conformations of IR with a similar stalk arrangement, which underlie Tyr1150 mono-phosphorylation (m-pY1150) and selective activation for metabolic signaling. The A43 aptamer, a positive allosteric modulator, binds at the opposite side of the insulin-binding module, and stabilizes the single insulin-bound IR structure that brings two FnIII-3 regions into closer proximity for full activation. Our results suggest that spatial proximity of the two FnIII-3 ends is important for m-pY1150, but multi-phosphorylation of IR requires additional conformational rearrangement of intracellular domains mediated by coordination between extracellular and transmembrane domains.

Structural Conservation of Insulin/IGF Signalling Axis at the Insulin Receptors Level inDrosophilaand humans

“…Two binding sites equivalent to insulin’s sites 1 and 2 have also been identified on hIGF-1 and 2 31, 32 , with site 1 extending into their C-domain. The role of these sites was ultimately confirmed in the last decade in over 40 crystal and cryoEM structures of complexes of these hormones (as well as insulin mimetic peptides and aptamers) with their cognate receptors, and their extensive functional studies 22, 33–54; for reviews, see refs. 55, 56 ).…”

“…Two binding sites equivalent to insulin's sites 1 and 2 have also been identified on IGF-I and II by site-directed mutagenesis (Gauguin et al, 2008;Alvino et al, 2009), with site 1 extending into their C-domain. The engagement of insulin/IGFs site 1 and 2 in hIR/mIR/IGF-1R binding was ultimately confirmed in the last decade in over 40 crystal and cryoEM structures of complexes of these hormones (as well as insulin mimetic peptides and aptamers) with their cognate receptors and their extensive functional studies ( Kavran et al, 2014, Croll et al, 2016Weis et al, 2018;Xu et al, 2018Xu et al, , 2020Xu et al, , 2022Scapin et al, 2018;Gutmann et al, 2018Gutmann et al, , 2020Uchikawa et al, 2019;Li et al, 2019Li et al, , 2022Blyth et al, 2020;Zhang et al, 2020Xiong et al, 2022;Nielsen et al, 2022;Wu et al, 2022;Kirk et al, 2022;Park et al, 2022;Kim et al, 2022; for reviews, see Lawrence, 2021;Forbes, 2023).…”

“…The copyright holder for this this version posted February 17, 2023. ; https://doi.org/10.1101/2023.02.17.528932 doi: bioRxiv preprint Kirk et al, 2022;Park et al, 2022;Kim et al, 2022; for reviews, see Lawrence, 2021;Forbes, 2023).…”

Aptamer–Protein Interactions: From Regulation to Biomolecular Detection

“…Kim's group developed two chemically modified aptamers, A43 and A62, as agonists of the insulin receptor (IR) to modulate IR autophosphorylation and downstream signaling pathways. 356 The structure of A43/A62-IR complex was determined by cryo-EM in the range of 3.62 to 4.27 Å resolution. The obtained structural information elucidated the conformational dynamics of IR activation related to signaling pathways, providing a structural basis for the design of functionally selective agonists for IR.…”

“…The types of interactions that occur at the tJBA8.1–TfR1 binding interface between individual protein residues and nucleotides include hydrophobic interactions between Leu619 and the base of T21 as well as Phe650 and the base and sugar of T23 (Figure E). Kim’s group developed two chemically modified aptamers, A43 and A62, as agonists of the insulin receptor (IR) to modulate IR autophosphorylation and downstream signaling pathways . The structure of A43/A62-IR complex was determined by cryo-EM in the range of 3.62 to 4.27 Å resolution.…”