Insulin receptor signalling has a central role in mammalian biology, regulating cellular metabolism, growth, division, differentiation and survival1,2. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer’s disease3; aberrant signalling occurs in diverse cancers, exacerbated by crosstalk with the homologous type 1 insulin-like growth factor receptor (IGF1R)4. 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 kinases5. 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.

Background: Insulin-like growth factor (IGF) II interacts with IR-A and is a more powerful mitogen than insulin. Results: IGF-II and insulin differ in regulating insulin receptor (IR)-A trafficking and stability. Conclusion: Compared with insulin, IGF-II induces lower IR-A and downstream effectors activation but protects IR-A and IRS-1 from down-regulation, thereby evoking a sustained mitogenic stimulus. Significance: These results further elucidate the mechanisms controlling IR-A biological responses.

Background:The structure of the C-terminal B21-B30 chain of insulin bound to the insulin receptor remains undetermined. Results: The structures of B24-modified insulins were determined. Conclusion: The structural integrity of Phe B24 but flexibility of B25-B30 insulin residues are important for receptor binding. Significance: The knowledge of the receptor-bound structure of insulin is important for the design of new insulin receptor agonists.

The structural characterization of the insulin-insulin receptor (IR) interaction still lacks the conformation of the crucial B21-B30 insulin region, which must be different from that in its storage forms to ensure effective receptor binding. Here, it is shown that insulin analogues modified by natural amino acids at the TyrB26 site can represent an active form of this hormone. In particular, [AsnB26]-insulin and [GlyB26]-insulin attain a B26-turn-like conformation that differs from that in all known structures of the native hormone. It also matches the receptor interface, avoiding substantial steric clashes. This indicates that insulin may attain a B26-turn-like conformation upon IR binding. Moreover, there is an unexpected, but significant, binding specificity of the AsnB26 mutant for predominantly the metabolic B isoform of the receptor. As it is correlated with the B26 bend of the B-chain of the hormone, the structures of AsnB26 analogues may provide the first structural insight into the structural origins of differential insulin signalling through insulin receptor A and B isoforms.

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22–B30 segment, using the CuI-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26–B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone’s B-chain C-terminus for its IR-B specificity.

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