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

Michael D Glidden,Yanwu Yang,Nicholas A Smith,Nelson B Phillips,Kelley Carr,Nalinda P Wickramasinghe,Faramarz Ismail-Beigi,Michael C Lawrence,Brian J Smith,Michael A Weiss

doi:10.1074/jbc.m117.808667

Michael D Glidden, Yanwu Yang + Show 8 more

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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.

Highlights

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
The tractability of the one-dimensional 1H NMR spectrum of single-chain insulin (SCI)-b at high protein concentration [9] motivated its present uniform 15N/13C-labeling in yeast Pichia pastoris; heteronuclear resonances assignments were obtained by standard methods [10, 27]
This study has described the high-resolution solution structure and dynamics of an active monomeric SCI

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Introduction

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. C domains encircles the C-terminal segment of the IR ␣-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. Ultra-stable insulin analogs promise to circumvent a costly “cold chain” underlying its global distribution [2] The salience of this issue has been sharpened by an emerging pandemic of DM in the developing world [5]

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