Solution structure of an ultra-stable single-chain insulin analog connects protein dynamics to a novel mechanism of receptor binding

Abstract

Domain-minimized insulin receptors (IRs) have enabled crystallographic analysis of insulin-bound "micro-receptors." In such structures, the C-terminal segment of the insulin B chain inserts between conserved IR domains, unmasking an invariant receptor-binding surface that spans both insulin A and B chains. This "open" conformation not only rationalizes the inactivity of single-chain insulin (SCI) analogs (in which the A and B chains are directly linked), but also suggests that connecting (C) domains of sufficient length will bind the IR. Here, we report the high-resolution solution structure and dynamics of such an active SCI. The hormone's closed-to-open transition is foreshadowed by segmental flexibility in the native state as probed by heteronuclear NMR spectroscopy and multiple conformer simulations of crystallographic protomers as described in the companion article. We propose a model of the SCI's IR-bound state based on molecular-dynamics simulations of a micro-receptor complex. In this model, a loop defined by the SCI's B and C domains encircles the C-terminal segment of the IR α-subunit. This binding mode predicts a conformational transition between an ultra-stable closed state (in the free hormone) and an active open state (on receptor binding). Optimization of this switch within an ultra-stable SCI promises to circumvent insulin's complex global cold chain. The analog's biphasic activity, which serendipitously resembles current premixed formulations of soluble insulin and microcrystalline suspension, may be of particular utility in the developing world.

Evolution of insulin at the edge of foldability and its medical implications

“…For Western blot assays, MCF-7 cells (ATCC) were cultured to 70 to 75% confluence, serum-starved for 24 h, and then exposed to insulin analogs at doses 5, 10, 20, and 50 nM. Blotting protocols were as described previously (41). The plate-based in-cell fluorescence-based immunoblotting pIR assay employed HepG2 cells (ATCC; ∼8,000 cells per well).…”

“…Main-chain dihedral angle restraints were generated using TALOS + . Structures were calculated using X-PLOR-NIH (81) as described previously (41). Ensembles were visualized using insightII and molmol software (82).…”

A thing of beauty: Structure and function of insulin's “aromatic triplet”

“…The receptor‐bound open conformation of the hormone may be particularly susceptible to such degradation, including formation of amyloid (insulin fibrillation). A promising structure‐based route to the engineering of ultra‐stable analogs is provided by foreshortened C domains . In such single‐chain insulins (SCIs) the aromatic triplet resides in a closed conformation—tethered by a foreshortened connecting peptide—and yet sufficient “play” is provided to enable its detachment on receptor binding.…”

“…A promising structure-based route to the engineering of ultra-stable analogs is provided by foreshortened C domains. 104,105 In such singlechain insulins (SCIs) the aromatic triplet resides in a closed conformation-tethered by a foreshortened connecting peptide-and yet sufficient "play" is provided to enable its detachment on receptor binding. It would be of future interest to investigate crystal-or cryo-EM structures of SCI-receptor complexes as probes of the closedopen transition.…”

Modulating Insulin Fibrillation Using Engineered B-Chains with Mutated C-Termini

“…Although the C-terminus of B-chain is known to act as an interface during insulin dimerization, this region has no important role in binding of insulin to its membrane receptor (21)(22)(23). Taking into account this aspect, we decided to incorporate positively charged residue in this segment that could produce a repulsive force, interfering with insulin association.…”