Ethyl Tosyl-L-isoleucylg1ycinate.-A solution of tosyl-Lisoleucyl chloride (prepared from 2.9 g. of tosyl-L-isoleucine and-phosphorus pentachloride) in r5 ml. of anhydrous ether was added slowly to a suspension of 2.5 g. of ethyl glycinate hydrochloride and 3.5 ml. of triethylamine in 50 ml. of anhydrous ether and the mixture was allowed to stand at room temperature for 12 hours. The white precipitate was filtered off and washed with ether. After trituration with water to remove the triethylamine hydrochloride, 3.34 g. of product, m.p. 159-160°, was obtained; yield 89%. Another 0.25 g., m.p. 152-154', was afforded by extraction of the ether filtrate successively with water, dilute HCl, dilute aqueous KHCO, and water, followed by removal of the ether zn vacuo. Recrystallization of the second crop from ethanol raised the m.p. to 158-160". For analysis, the tosyl dipeptide ester was recrystallized twice from ethanol and then melted a t 160-161".

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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...

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.

The alpha-helix formed by the amino acid residues 9-19 of the B-chain of insulin is involved in the stabilization of its three-dimensional structure. We have shown that modification at positions B9, B10, B12, and B16 results in analogues possessing biological activities ranging from ca. 0.2% to ca. 500% relative to that of natural insulin. The lowest potency was displayed by [B12 Asn]insulin, in which the hydrophobic B12 Val residue was replaced by the hydrophilic Asn residue. We now report the synthesis of four insulin analogues in which hydrophobicity is retained, and only the spatial arrangement of atoms in the B12 region is altered. Substitution of B12 Val with alpha-aminoisobutyric acid (Aib), D-Ala, and Phe led to analogues possessing biological activities, in lipogenesis assays, of 8.5%, 2%, and 0.2%, respectively, relative to that of natural insulin. Inversion of the B11-B12 sequence, -Leu-Val-, led to an analogue displaying 3.3% activity. A synthetic B-chain in which the B11 Leu-B12 Val sequence was replaced by B11 Ala-B12 Ile was incapable of combining with the natural A-chain. We conclude that the Val residue in the B12 position in insulin fulfills special side-chain packing requirements involved in the stability of the structure of insulin. Even slight steric alteration at position B12 results in a distortion of the overall conformation of the B-chain which affects its ability to combine with the natural A-chain. This distortion is retained in the corresponding analogue, which is reflected in diminished biological potency.

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