The Structure of a Mutant Insulin Uncouples Receptor Binding from Protein Allostery

Abstract

The zinc insulin hexamer undergoes allosteric reorganization among three conformational states, designated T 6 , T 3 R 3 f , and R 6 . Although the free monomer in solution (the active species) resembles the classical T-state, an R-like conformational change is proposed to occur upon receptor binding. Here, we distinguish between the conformational requirements of receptor binding and the crystallographic TR transition by design of an active variant refractory to such reorganization. Our strategy exploits the contrasting environments of His B5 in wild-type structures: on the T 6 surface but within an intersubunit crevice in R-containing hexamers. The TR transition is associated with a marked reduction in His B5 pK a , in turn predicting that a positive charge at this site would destabilize the R-specific crevice. Remarkably, substitution of His B5 (conserved among eutherian mammals) by Arg (occasionally observed among other vertebrates) blocks the TR transition, as probed in solution by optical spectroscopy. Similarly, crystallization of Arg B5 -insulin in the presence of phenol (ordinarily a potent inducer of the TR transition) yields T 6 hexamers rather than R 6 as obtained in control studies of wild-type insulin. The variant structure, determined at a resolution of 1.3 Å , closely resembles the wild-type T 6 hexamer. Whereas Arg B5 is exposed on the protein surface, its side chain participates in a solvent-stabilized network of contacts similar to those involving His B5 in wild-type T-states. The substantial receptor-binding activity of Arg B5 -insulin (40% relative to wild type) demonstrates that the function of an insulin monomer can be uncoupled from its allosteric reorganization within zinc-stabilized hexamers.Insulin is a small globular protein containing two chains, A (21 residues) and B (30 residues). In pancreatic ␤-cells, the hormone is stored as Zn 2ϩ -stabilized hexamers, which form microcrystalline arrays within specialized secretory granules (1). The hexamers dissociate upon secretion into the portal circulation, enabling the hormone to function as a zinc-free monomer. Structure-function relationships have been inferred from patterns of sequence conservation (2) and extensively probed by mutagenesis (2-10). A variety of evidence suggests that insulin undergoes a change in conformation on binding to the insulin receptor (IR) 2 (11). A model for induced fit is provided by the TR transition, a long range allosteric reorganization of zinc insulin hexamers (12). The structural basis of the TR transition has been extensively investigated by x-ray crystallography (13-15). In this paper, we investigate the relationship between such allosteric reorganization and biological activity. Experimental design exploits the contrasting structures of classical hexamers to introduce an electrostatic block to the TR transition.The TR transition encompasses three families of zinc insulin hexamers, designated T 6 , T 3 R 3 f , and R 6 (Fig. 1). Interconversion among these families is regulated by ionic strength (13, 16...

Biophysical Optimization of a Therapeutic Protein by Nonstandard Mutagenesis

“…1E), so named by analogy to hemoglobin. Although the functional significance of the T 3 R transition is not well understood (46,57), studies of insulin analogs have suggested that such allostery exploits sites of conformational change relevant to induced fit of the hormone on receptor binding (54,58).…”

Supramolecular Protein Engineering

“…Wild-type S-sulfonate B-chain derivatives were obtained by oxidative sulfitolysis of insulin (23). Insulin analogs were prepared by chain combination and purified as described (22,23). The paired His A-chain substitutions were also incorporated into engineered monomer DKP-insulin (His B10 3 Asp, Pro B28 3 Lys, and Lys B29 3 Pro) (24).…”

“…Synthesis of Insulin Analog-Variant insulin chains were prepared by solid-phase synthesis (22). Wild-type S-sulfonate B-chain derivatives were obtained by oxidative sulfitolysis of insulin (23).…”

“…Insulin glargine was obtained from Lantus (Sanofi-Aventis, Paris, France). Reagents for peptide synthesis were as described (22).…”

Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I

“…Such impairment stands in contrast to the general robustness of chain combination to substitutions, especially in the C-terminal B-chain segment and N-terminal A-chain segment (65 (41,91), which like B5 are sites of neonatal diabetesassociated mutations (12,14). It is possible that substitutions at , which is shifted from 6.0 to 7.0 in native insulin (93,94). The neonatal diabetes-associated mutation at B5 is Asp (12), whose negative charge would be expected to destabilize a thiolate reaction intermediate.…”