How insulin engages its primary binding site on the insulin receptor

Abstract

Insulin receptor signalling has a central role in mammalian biology, regulating cellular metabolism, growth, division, differentiation and survival. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer's disease; aberrant signalling occurs in diverse cancers, exacerbated by cross-talk with the homologous type 1 insulin-like growth factor receptor (IGF1R). Despite more than three decades of investigation, the three-dimensional structure of the insulin-insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein. Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs. The direct interaction of insulin with the first leucine-rich-repeat domain (L1) of insulin receptor is seen to be sparse, the hormone instead engaging the insulin receptor carboxy-terminal α-chain (αCT) segment, which is itself remodelled on the face of L1 upon insulin binding. Contact between insulin and L1 is restricted to insulin B-chain residues. The αCT segment displaces the B-chain C-terminal β-strand away from the hormone core, revealing the mechanism of a long-proposed conformational switch in insulin upon receptor engagement. This mode of hormone-receptor recognition is novel within the broader family of receptor tyrosine kinases. We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titration calorimetry data that dissect the hormone-insulin receptor interface. Together, our findings provide an explanation for a wealth of biochemical data from the insulin receptor and IGF1R systems relevant to the design of therapeutic insulin analogues.

Figure 1. Structure of insulin, insulin receptor and the site 1 complexes

a, Insulin. InsA, A chain; InsB, B chain. b, Insulin receptor. CR, Cys-rich domain; FnIII-1, FnIII-2, FnIII-3, first, second and third fibronectin type III domains; ID, insert domain; L1, L2, first and second leucine-rich-repeat domains; TK, tyrosine kinase; TM, JM, transmembrane and juxtamembrane segments. c, d, IR310.T and IR593.αCT domain structure, respectively. e, f, Insulin-bound site 1 in complexes A and D, respectively. Blue, red spheres, observed chain termini; orange sphere, FnIII-1–αCT junction. g, Overlay of insulin-bound site 1 in complexes A (coloured as in e), B (red), C (green) and D (white). h, Sample 2F_obs – F_calc map volumes (B_sharp = −160 Ų; contours = 1.1–1.5σ) for complex A.

Figure 2. The insulin–site 1 interaction

a, Altered disposition of αCT with respect to that in apo-insulin receptor. b, Superposition (via the A- and B-chain helices) of receptor-free insulin onto the insulin–site 1 complex, indicating steric clash of the B-chain C-terminal segment (green) with αCT. See also Supplementary Fig. 3a. c, Interaction (at the bulk side-chain level of detail) between αCT(704–719) and insulin. View direction is parallel to L1–β2 surface (cyan); white shading shows insulin surface. d, e, Interaction (at the bulk side-chain level of detail) between L1 and αCT and between L1 and insulin B-chain helix, respectively. Panels based on complex A.

Figure 3. Insulin interactions in L1–CR–L2 mini-insulin receptor and holo insulin receptor

a, Helical wheel representation of ITC-derived insulin affinities for IR485 in the presence of Ala-substituted αCT(704–719) (red, > 100× reduction upon Ala substitution; green, > 10× reduction; grey, < 10× reduction; open circle, not determined). b, Reducing gel autoradiograms obtained from holo-insulin receptor after photo-crosslinking of αCT helix to bound ¹²⁵I-[Tyr^A14]-insulin. Arrowed band indicates crosslinked insulin receptor α-chain–insulin A chain. Colours indicate crosslinking efficiency (red, strong; green, medium; blue, weak; grey, none). c, d, Qualitative crosslinking efficiency from b mapped onto αCT segment within the site 1 complexes. e, Helical wheel representation of crosslinking data presented in b–d