Chiral Mutagenesis of Insulin. Foldability and Function Are Inversely Regulated by a Stereospecific Switch in the B Chain,
- 12 March 2005
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 44 (13), 4984-4999
- https://doi.org/10.1021/bi048025o
Abstract
How insulin binds to its receptor is unknown despite decades of investigation. Here, we employ chiral mutagenesiscomparison of corresponding d and l amino acid substitutions in the hormoneto define a structural switch between folding-competent and active conformations. Our strategy is motivated by the T → R transition, an allosteric feature of zinc-hexamer assembly in which an invariant glycine in the B chain changes conformations. In the classical T state, GlyB8 lies within a β-turn and exhibits a positive φ angle (like a d amino acid); in the alternative R state, GlyB8 is part of an α-helix and exhibits a negative φ angle (like an l amino acid). Respective B chain libraries containing mixtures of d or l substitutions at B8 exhibit a stereospecific perturbation of insulin chain combination: l amino acids impede native disulfide pairing, whereas diverse d substitutions are well-tolerated. Strikingly, d substitutions at B8 enhance both synthetic yield and thermodynamic stability but markedly impair biological activity. The NMR structure of such an inactive analogue (as an engineered T-like monomer) is essentially identical to that of native insulin. By contrast, l analogues exhibit impaired folding and stability. Although synthetic yields are very low, such analogues can be highly active. Despite the profound differences between the foldabilities of d and l analogues, crystallization trials suggest that on protein assembly substitutions of either class can be accommodated within classical T or R states. Comparison between such diastereomeric analogues thus implies that the T state represents an inactive but folding-competent conformation. We propose that within folding intermediates the sign of the B8 φ angle exerts kinetic control in a rugged landscape to distinguish between trajectories associated with productive disulfide pairing (positive T-like values) or off-pathway events (negative R-like values). We further propose that the crystallographic T → R transition in part recapitulates how the conformation of an insulin monomer changes on receptor binding. At the very least the ostensibly unrelated processes of disulfide pairing, allosteric assembly, and receptor binding appear to utilize the same residue as a structural switch; an “ambidextrous” glycine unhindered by the chiral restrictions of the Ramachandran plane. We speculate that this switch operates to protect insulinand the β-cellfrom protein misfolding.Keywords
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