In order to evaluate the cause of the greatly decreased receptor-binding potency of the naturally occurring mutant human insulin Insulin Wakayama ([LeuA3]insulin, 0.2% relative potency), we examined (by the semisynthesis of insulin analogues based on N alpha-PheB1,N epsilon-LysB29-bisacetyl-insulin) the importance of aliphatic side chain structure at positions A2 and A3 (Ile and Val, respectively) in directing the interaction of insulin with its receptor. Analogues bearing glycine, alanine, alpha-amino-n-butyric acid, norvaline, norleucine, valine, isoleucine, allo-isoleucine, threonine, tert-leucine, or leucine at positions A2 or A3 were assayed for their potencies in competing for the binding of 125I-labeled insulin to isolated canine hepatocytes, as were analogues bearing deletions from the A-chain amino terminus or the B-chain carboxyl terminus. Selected analogues were also analyzed by far-UV CD and absorption spectroscopy of Co2+ complexes. Our results identify that (a) Ile and Val serve well at position A2, whereas residues with other side chains (including those with straight chains, alternatively configured beta-branches, or a gamma-branch) exhibit relative receptor-binding potencies in the range 1-5%; (b) greater flexibility is allowed side-chain structure at position A3, with Ile, allo-Ile, alpha-amino-n-butyric acid, and tert-Leu exhibiting relative receptor-binding potencies in the range 11-36%; and (c) simultaneous replacements at positions A2 and A3, and deletions of the COOH-terminal domain of the insulin B chain in related analogues, yield cumulative effects. These findings are discussed with respect to a model for insulin-receptor interactions that involves a structure-orienting role for residue A2, the direct interaction of residue A3 with receptor, and multiple separately defined elements of structure and of conformational adjustment.

How insulin binds to its receptor is unknown despite decades of investigation. Here, we employ chiral mutagenesis-comparison 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, Gly B8 lies within a -turn and exhibits a positive ϕ angle (like a D amino acid); in the alternative R state, Gly B8 is part of an -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 † This work was supported in part by Diabetes Research and Training Center at the University of Chicago (S. H SUPPORTING INFORMATION AVAILABLENine figures illustrating disulfide pairing and structural relationships in insulin crystals, visible absorption spectra of cobalt-substituted hexamers, additional CD and NMR spectra, diagonal plot of NOEs, and summary of NMR sequential assignment. Nine tables providing B8 dihedral angles, summary of mutations at sites neighboring B8, crystallographic unit-cell dimensions, NMR resonance assignments, statistical information pertaining to DG/RMD ensemble, and restraints. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2013 December 02. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript 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 assemb...

The C-terminal segment of the human insulin receptor α-chain (designated αCT) is critical to insulin binding as has been previously demonstrated by alanine scanning mutagenesis and photo-crosslinking. To date no information regarding the structure of this segment within the receptor has been available. We employ here the technique of thermal-factor sharpening to enhance the interpretability of the electron-density maps associated with the earlier crystal structure of the human insulin receptor ectodomain. The αCT segment is now resolved as being engaged with the central β-sheet of the first leucine-rich repeat (L1) domain of the receptor. The segment is α-helical in conformation and extends 11 residues N-terminal of the classical αCTsegment boundary originally defined by peptide mapping. This tandem structural element (αCT-L1) thus defines the intact primary insulin-binding surface of the apo-receptor. The structure, together with isothermal titration calorimetry data of mutant αCT peptides binding to an insulin minireceptor, leads to the conclusion that putative "insulin-mimetic" peptides in the literature act at least in part as mimics of the αCT segment as well as of insulin. Photo-cross-linking by novel bifunctional insulin derivatives demonstrates that the interaction of insulin with the αCT segment and the L1 domain occurs in trans, i.e., these components of the primary binding site are contributed by alternate α-chains within the insulin receptor homodimer. The tandem structural element defines a new target for the design of insulin agonists for the treatment of diabetes mellitus.inding of insulin to the insulin receptor initiates a signaling cascade in target tissues as the first step in the regulation of metabolic homeostasis. However, a molecular description of how insulin binds and activates its receptor remains elusive. Whereas determination of the structure of insulin (Fig. 1A) represented an early triumph of protein crystallography (2), the structure of the much larger receptor ectodomain homodimer (in apo form) has only recently been crystallographically analyzed (3). The latter structure and its implications for the nature of the hormone-binding sites have been extensively reviewed (4-7). Briefly, the insulin receptor is a disulfide-linked dimer wherein each proreceptor monomer is proteolytically cleaved into an N-terminal α-chain and a C-terminal β-chain (Fig. 1B). A single disulfide bond links the α-and β-chains within each monomer. The extracellular portion of the insulin receptor includes both α-chains as well as 194 residues (Ser724-Lys917) of each β-chain. Each receptor monomer consists of several structural domains (Fig. 1B), including a leucine-rich repeat domain L1 (residues 1-157), a cysteine-rich region (CR, residues 158-310), a second leucine-rich repeat domain L2 (residues 311-470), and three fibronectin type-III domains: FnIII-1 (residues 471-595), FnIII-2 (residues 596-808), and FnIII-3 (residues 809-906). FnIII-2 contains a ∼120-residue insert domain (ID, residues 638-756) that co...

The landscape paradigm of protein folding can enable preferred pathways on a funnel-like energy surface. Hierarchical preferences may be manifest as a nonrandom pathway of disulfide pairing. Stepwise stabilization of structural subdomains among on-pathway intermediates is proposed to underlie the disulfide pathway of proinsulin and related molecules. Here, effects of pairwise serine substitution of insulin's exposed interchain disulfide bridge (Cys(A7)-Cys(B7)) are characterized as a model of a late intermediate. Untethering cystine A7-B7 in an engineered monomer causes significantly more marked decreases in the thermodynamic stability and extent of folding than occur on pairwise substitution of internal cystine A6-A11 [Weiss, M. A., Hua, Q. X., Jia, W., Chu, Y. C., Wang, R. Y., and Katsoyannis, P. G. (2000) Biochemistry 39, 15429-15440]. Although substantially disordered and without significant biological activity, the untethered analogue contains a molten subdomain comprising cystine A20-B19 and a native-like cluster of hydrophobic side chains. Remarkably, A and B chains make unequal contributions to this folded moiety; the B chain retains native-like supersecondary structure, whereas the A chain is largely disordered. These observations suggest that the B subdomain provides a template to guide folding of the A chain. Stepwise organization of insulin-like molecules supports a hierarchic view of protein folding.

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How insulin binds to and activates the insulin receptor has long been the subject of speculation. Of particular interest are invariant phenylalanine residues at consecutive positions in the B chain (residues B24 and B25). Sites of mutation causing diabetes mellitus, these residues occupy opposite structural environments: Phe(B25) projects from the surface of insulin, whereas Phe(B24) packs against the core. Despite these differences, site-specific cross-linking suggests that each contacts the insulin receptor. Photoactivatable derivatives of insulin containing respective p-azidophenylalanine substitutions at positions B24 and B25 were synthesized in an engineered monomer (DKP-insulin). On ultraviolet irradiation each derivative cross-links efficiently to the receptor. Packing of Phe(B24) at the receptor interface (rather than against the core of the hormone) may require a conformational change in the B chain. Sites of cross-linking in the receptor were mapped to domains by Western blot. Remarkably, whereas B25 cross-links to the C-terminal domain of the alpha subunit in accord with previous studies (Kurose, T., et al. (1994) J. Biol. Chem. 269, 29190-29197), the probe at B24 cross-links to its N-terminal domain (the L1 beta-helix). Our results demonstrate that consecutive residues in insulin contact widely separated sequences in the receptor and in turn suggest a revised interpretation of electron-microscopic images of the complex. By tethering the N- and C-terminal domains of the extracellular alpha subunit, insulin is proposed to stabilize an active conformation of the disulfide-linked transmembrane tyrosine kinase.

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