Chiral mutagenesis of insulin. Foldability and function are inversely regulated by a stereospecific switch in the B chain.

Insulin, a small globular protein containing three disulfide bridges, plays a central role in the regulation of vertebrate metabolism. The hormone is stored in the pancreatic β cell as a zinc hexamer and functions in the bloodstream as a zinc-free monomer. The functional surface of insulin has long been the object of speculation. Despite decades of investigation by mutagenesis and X-ray crystallography (1, 2), structures of insulin and insulin analogues do not consistently predict relative potencies (3–5). Such anomalies suggest that a change in structure occurs on receptor binding. Here, chiral mutagenesis–comparison of corresponding d and l amino acid substitutions of an invariant glycine–is employed to investigate the interrelation of structure and function in the B chain.1 Experimental design is motivated by the classical T → R transition, an allosteric feature of zinc-insulin hexamers (6–8).2 Remarkably, chiral stabilization of the native T state (the predominant conformation in solution; refs 9 and 10) markedly impairs its binding to the insulin receptor (IR).3 Because the extent of impairment exceeds that ordinarily encountered in studies of mutant insulins (1, 2, 11), we suggest that the d substitution impairs an R-like conformational switch required for high-affinity receptor binding. We focus on an invariant glycine in the B chain (GlyB8; arrow in Figure 1A). This glycine follows a motif-specific cysteine (CysB7) and is broadly conserved among vertebrate insulin-like polypeptides (Figure 2). The environment of GlyB8 differs between the two classes of crystallographic protomers, T and R (Figure 1B). Whereas residues B7–B10 form a type II′ β turn in the T state (Figure 1C; ref 1), the same residues lie within an extended α helix in the alternative R state (Figure 1D; refs 6 and 7). GlyB8 lies at the junction of this chameleon segment (12) and the central α helix (B9–B19). Although the B8 junction is exposed on the surface of the T and R state protomers, local packing schemes thus differ among crystal forms (Figure 3A,B). These contrasting environments are associated with different B8 ϕ dihedral angles (Figure 3C,D): positive in the T state (like a d amino acid; 56.4° ± (4.1 among multiple crystal structures) and negative in the R state (like an l amino acid; −63.0° ± 3.2) (see Supporting Information). Such Ramachandran relationships motivate investigation of whether d and l amino acid substitutions would induce stereospecific perturbations of folding or function. In particular, because the structure of the insulin monomer in solution closely resembles the crystallographic T state (4, 10, 13), would d substitutions at B8 stabilize the β turn, and if so, would such a chiral “lock” enhance or impair biological activity? Conversely, what would be the effects of l substitutions? Molecular modeling suggests that in a T-like protomer d or l side chains would project into solvent (Figure 1E); in a putative R-like protomer, l substituents would be exposed, whereas d side chains would be buried (Figure 1F). Figure 1 Overview of insulin structure. (A) Sequence of human insulin indicating invariant glycine in B chain (GlyB8; arrow). Substitutions in monomeric DKP-insulin template are shown in magenta. Disulfide bridges are indicated by lines. (B) Cylinder model of ... Figure 2 Glycine is invariant at position B8 among insulin sequences and also conserved among insulin-like growth factors (IGF–I and IGF–II). Comparison of representative B chain or B domain sequences is shown; B8 residues are boxed (asterisk). ... Figure 3 Structural relationships in crystal structures of insulin surrounding GlyB8. (A and B) In T state protomers GlyB8 participates in a β turn (red asterisk in A) whereas in R state protomers, B8 is part of an α-helix (red asterisk in B). ... To address these questions, we first employ combinatorial peptide chemistry to compare effects of l and d amino acid substitutions on insulin foldability. A novel in vitro selection is designed based on chain combination (14); mass spectrometry (MS) is used to distinguish between allowed and disallowed B chain sequences 15). Respective peptide 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. We then extend these findings to characterize representative diastereomeric pairs of B8 analogues. d Substitutions enhance the thermodynamic stability of insulin but markedly impair its biological activity, whereas unstable l analogues can be highly active.4 Remarkably, a single d methyl group attached to the surface of insulin is shown to stabilize the native structure of insulin in solution (ΔΔGu > 1 kcal/mol) but impair its receptor binding by 1000-fold. Because this decrement far exceeds effects of mutations on the protein surface at neighboring sites (1, 11), these observations suggest that the canonical T state of insulin represents an inactive conformation: the introduced d methyl group acts as a spanner in the works to block an R-like change in B8 conformation on receptor binding. We propose that this invariant glycine in the B chain functions as a Ramachandran switch between folding-competent and active conformations, mirroring aspects of the classical T → R allosteric transition (6, 7). We thus envisage that the flexibility of GlyB8 both protects the β-cell from toxic protein misfolding and enables high-affinity hormone-receptor recognition.