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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...

A hierarchical pathway of protein folding can enable segmental unfolding by design. A monomeric insulin analogue containing pairwise substitution of internal A6-A11 cystine with serine [[Ser(A6),Ser(A11),Asp(B10),Lys(B28),Pro(B29)]insulin (DKP[A6-A11](Ser))] was previously investigated as a model of an oxidative protein-folding intermediate [Hua, Q. X., et al. (1996) J. Mol. Biol. 264, 390-403]. Its structure exhibits local unfolding of an adjoining amphipathic alpha-helix (residues A1-A8), leading to a 2000-fold reduction in activity. Such severe loss of function, unusual among mutant insulins, is proposed to reflect the cost of induced fit: receptor-directed restoration of the alpha-helix and its engagement in the hormone's hydrophobic core. To test this hypothesis, we have synthesized and characterized the corresponding alanine analogue [[Ala(A6),Ala(A11),Asp(B10),Lys(B28), Pro(B29)]insulin (DKP[A6-A11](Ala))]. Untethering the A6-A11 disulfide bridge by either amino acid causes similar perturbations in structure and dynamics as probed by circular dichroism and (1)H NMR spectroscopy. The analogues also exhibit similar decrements in thermodynamic stability relative to that of the parent monomer as probed by equilibrium denaturation studies (Delta Delta G(u) = 3.0 +/- 0.5 kcal/mol). Despite such similarities, the alanine analogue is 50 times more active than the serine analogue. Enhanced receptor binding (Delta Delta G = 2.2 kcal/mol) is in accord with alanine's greater helical propensity and more favorable hydrophobic-transfer free energy. The success of an induced-fit model highlights the applicability of general folding principles to a complex binding process. Comparison of DKP[A6-A11](Ser) and DKP[A6-A11](Ala) supports the hypothesis that the native A1-A8 alpha-helix functions as a preformed recognition element tethered by insulin's intrachain disulfide bridge. Segmental unfolding by design provides a novel approach to dissecting structure-activity relationships.

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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Proinsulin contains six cysteines whose specific pairing (A6-A11, A7-B7, and A20-B19) is a defining feature of the insulin fold. Pairing information is contained within A and B domains as demonstrated by studies of insulin chain recombination. Two insulin isomers containing non-native disulfide bridges ([A7-A11,A6-B7,A20-B19] and [A6-A7,A11-B7,A20-B19]), previously prepared by directed chemical synthesis, are metastable and biologically active. Remarkably, the same two isomers are preferentially formed from native insulin or proinsulin following disulfide reassortment in guanidine hydrochloride. The absence of other disulfide isomers suggests that the observed species exhibit greater relative stability and/or kinetic accessibility. The structure of the first isomer ([A7-A11,A6-B7,A20-B19], insulin-swap) has been described [Hua, Q. X., Gozani, S. N., Chance, R. E., Hoffmann, J. A., Frank, B. H., and Weiss, M. A. (1995) Nat. Struct. Biol. 2, 129-138]. Here, we demonstrate that the second isomer (insulin-swap2) is less ordered than the first. Nativelike elements of structure are retained in the B chain, whereas the A chain is largely disordered. Thermodynamic studies of guanidine denaturation demonstrate the instability of the isomers relative to native insulin (DeltaDeltaG(u) > 3 kcal/mol). In contrast, insulin-like growth factor I (IGF-I) and the corresponding isomer IGF-swap, formed as alternative products of a bifurcating folding pathway, exhibit similar cooperative unfolding transitions. The insulin isomers are similar in structure and stability to two-disulfide analogues whose partial folds provide models of oxidative folding intermediates. Each exhibits a nativelike B chain and less-ordered A chain. This general asymmetry is consistent with a hierarchical disulfide pathway in which nascent structure in the B chain provides a template for folding of the A chain. Structures of metastable disulfide isomers provide probes of the topography of an energy landscape.

The insulins of eutherian mammals contain histidines at positions B5 and B10. The role of His B10 is well defined: although not required in the mature hormone for receptor binding, in the islet ␤ cell this side chain functions in targeting proinsulin to glucose-regulated secretory granules and provides axial zincbinding sites in storage hexamers. In contrast, the role of His B5 is less well understood. Here, we demonstrate that its substitution with Ala markedly impairs insulin chain combination in vitro and blocks the folding and secretion of human proinsulin in a transfected mammalian cell line. The structure and stability of an Ala B5 -insulin analog were investigated in an engineered monomer (DKP-insulin). Despite its impaired foldability, the structure of the Ala B5 analog retains a native-like T-state conformation. At the site of substitution, interchain nuclear Overhauser effects are observed between the methyl resonance of Ala B5 and side chains in the A chain; these nuclear Overhauser effects resemble those characteristic of His B5 in native insulin. Substantial receptor binding activity is retained (80 ؎ 10% relative to the parent monomer). Although the thermodynamic stability of the Ala B5 analog is decreased (⌬⌬G u ‫؍‬ 1.7 ؎ 0.1 kcal/mol), consistent with loss of His B5 -related interchain packing and hydrogen bonds, control studies suggest that this decrement cannot account for its impaired foldability. We propose that nascent long-range interactions by His B5 facilitate alignment of Cys A7 and Cys B7 in protein-folding intermediates; its conservation thus reflects mechanisms of oxidative folding rather than structure-function relationships in the native state.Insulin is a small globular protein containing two chains, A (21 residues) and B (30 residues). The mature hormone is the post-translational product of a single-chain precursor, proinsulin (1), in which a connecting domain extends from the C-terminal residue of the B domain (Thr B30 ) to the N terminus of the A chain (Gly A1 ) (Fig. 1A). In the pancreatic ␤ cell, proinsulin folds in the endoplasmic reticulum (ER) 4 to form three specific disulfide bridges: A6 -A11, A7-B7, and A20 -B19 (Fig. 1A, orange bars). Although the three-dimensional structure of proinsulin has not been determined, a variety of evidence indicates that it consists of a folded insulin moiety (Fig. 1B, red and blue ribbons) and a disordered connecting region (dashed black line). Upon transit through the Golgi apparatus and entry into immature secretory granules (2), the C-peptide (Fig. 1A, black open circles) is excised by a specific set of prohormone convertases (3). The mature hormone is stored as Zn 2ϩ -stabilized hexamers within specialized secretory granules (4). Insulin hexamers dissociate upon secretion into the portal circulation, enabling the circulating hormone to function as a Zn 2ϩ -free monomer. The stability and receptor binding activity of insulin require maintenance of its three disulfide bridges (5-10).Classical structure-function relationships in ins...

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