Design of an active ultrastable single-chain insulin analog: synthesis, structure, and therapeutic implications.

Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (<3 residues) prevent induced fit upon receptor binding and so are essentially without biological activity. Substantial but incomplete activity can be regained with increasing linker length. Here, we describe the design, structure, and function of a single-chain insulin analog (SCI-57) containing a 6-residue linker (GGGPRR). Native receptor-binding affinity (130 +/- 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of SCI-57 is markedly increased (DeltaDeltaG(u) = 0.7 +/- 0.1 kcal/mol relative to the corresponding two-chain analog and 1.9 +/- 0.1 kcal/mol relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects demonstrates that a native-like fold is maintained in solution. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts Val(A3) at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 alpha-helix. Comparison between SCI-57 and its parent two-chain analog reveals striking enhancement of multiple native-like nuclear Overhauser effects within the tethered protein. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultrastable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world.

The A-chain of insulin contacts the insert domain of the insulin receptor. Photo-cross-linking and mutagenesis of a diabetes-related crevice.

The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of Val(A3), which projects within a crevice between the A- and B-chains. Engineered receptor alpha-subunits containing specific protease sites ("midi-receptors") are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a Pap(B25)-insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and alpha-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (Gly(A3) and Ser(A3)) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of Aba(A3) and allo-Thr(A3) analogs are reduced at least 10-fold; the activity of Thr(A3)-DKP-insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (Aba(A3)) or by altered polarity (allo-Thr(A3) and Thr(A3)). Our results provide evidence that Val(A3), a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the alpha-subunit in a hormone-receptor complex.

Chiral mutagenesis of insulin. Contribution of the B20-B23 beta-turn to activity and stability.

Insulin contains a beta-turn (residues B20-B23) interposed between two receptor-binding elements, the central alpha-helix of the B chain (B9-B19) and its C-terminal beta-strand (B24-B28). The turn contains conserved glycines at B20 and B23. Although insulin exhibits marked conformational variability among crystal forms, these glycines consistently maintain positive phi dihedral angles within a classic type-I beta-turn. Because the Ramachandran conformations of GlyB20 and GlyB23 are ordinarily forbidden to L-amino acids, turn architecture may contribute to structure or function. Here, we employ "chiral mutagenesis," comparison of corresponding D- and L-Ala substitutions, to investigate this turn. Control substitutions are introduced at GluB21, a neighboring residue exhibiting a conventional (negative) phi angle. The D- and L-Ala substitutions at B23 are associated with a marked stereospecific difference in activity. Whereas the D-AlaB23 analog retains native activity, the L analog exhibits a 20-fold decrease in receptor binding. By contrast, D- and L-AlaB20 analogs each exhibit high activity. Stereospecific differences between the thermodynamic stabilities of the analogs are nonetheless more pronounced at B20 (delta deltaG(u) 2.0 kcal/mole) than at B23 (delta deltaG(u) 0.7 kcal/mole). Control substitutions at B21 are well tolerated without significant stereospecificity. Chiral mutagenesis thus defines the complementary contributions of these conserved glycines to protein stability (GlyB20) or receptor recognition (GlyB23).

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

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 hormone-to 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 beta-turn and exhibits a positive phi angle (like a d amino acid); in the alternative R state, Gly(B8) is part of an alpha-helix and exhibits a negative phi 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 phi angle exerts kinetic control in a rugged landscape to distinguish between trajectories associated 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 assembly, and receptor binding appear to utilize the same residue as a structural switch; an "ambidextrous" glycine unhindered by the chiral restrictions of the Ramachandran plane. We speculate that this switch operates to protect insulin-and the beta-cell-from protein misfolding.

Enhancing the activity of insulin at the receptor interface: crystal structure and photo-cross-linking of A8 analogues.

The receptor-binding surface of insulin is broadly conserved, reflecting its evolutionary optimization. Neighboring positions nevertheless offer an opportunity to enhance activity, through either transmitted structural changes or introduction of novel contacts. Nonconserved residue A8 is of particular interest as Thr(A8) --> His substitution (a species variant in birds and fish) augments the potency of human insulin. Diverse A8 substitutions are well tolerated, suggesting that the hormone-receptor interface is not tightly packed at this site. To resolve whether enhanced activity is directly or indirectly mediated by the variant A8 side chain, we have determined the crystal structure of His(A8)-insulin and investigated the photo-cross-linking properties of an A8 analogue containing p-azidophenylalanine. The structure, characterized as a T(3)R(3)(f) zinc hexamer at 1.8 A resolution, is essentially identical to that of native insulin. The photoactivatable analogue exhibits efficient cross-linking to the insulin receptor. The site of cross-linking lies within a 14 kDa C-terminal domain of the alpha-subunit. This contact, to our knowledge the first to be demonstrated from the A chain, is inconsistent with a recent model of the hormone-receptor complex derived from electron microscopy. Optimizing the binding interaction of a nonconserved side chain on the surface of insulin may thus enhance its activity.

How insulin binds: the B-chain alpha-helix contacts the L1 beta-helix of the insulin receptor.

Binding of insulin to the insulin receptor plays a central role in the hormonal control of metabolism. Here, we investigate possible contact sites between the receptor and the conserved non-polar surface of the B-chain. Evidence is presented that two contiguous sites in an alpha-helix, Val(B12) and Tyr(B16), contact the receptor. Chemical synthesis is exploited to obtain non-standard substitutions in an engineered monomer (DKP-insulin). Substitution of Tyr(B16) by an isosteric photo-activatable derivative (para-azido-phenylalanine) enables efficient cross-linking to the receptor. Such cross-linking is specific and maps to the L1 beta-helix of the alpha-subunit. Because substitution of Val(B12) by larger side-chains markedly impairs receptor binding, cross-linking studies at B12 were not undertaken. Structure-function relationships are instead probed by side-chains of similar or smaller volume: respective substitution of Val(B12) by alanine, threonine, and alpha-aminobutyric acid leads to activities of 1(+/-0.1)%, 13(+/-6)%, and 14(+/-5)% (relative to DKP-insulin) without disproportionate changes in negative cooperativity. NMR structures are essentially identical with native insulin. The absence of transmitted structural changes suggests that the low activities of B12 analogues reflect local perturbation of a "high-affinity" hormone-receptor contact. By contrast, because position B16 tolerates alanine substitution (relative activity 34(+/-10)%), the contribution of this neighboring interaction is smaller. Together, our results support a model in which the B-chain alpha-helix, functioning as an essential recognition element, docks against the L1 beta-helix of the insulin receptor.

Diabetes-associated mutations in insulin: consecutive residues in the B chain contact distinct domains of the insulin receptor.

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.

Diabetes-associated mutations in insulin identify invariant receptor contacts.

Mutations in human insulin cause an autosomal-dominant syndrome of diabetes and fasting hyperinsulinemia. We demonstrate by residue-specific photo cross-linking that diabetes-associated mutations occur at receptor-binding sites. The studies use para-azido-phenylalanine, introduced at five sites by total protein synthesis. Because two such sites (Val(A3) and Phe(B24)) are largely buried in crystal structures of the free hormone, their participation in receptor binding is likely to require a conformational change to expose a hidden functional surface. Our results demonstrate that this surface spans both chains of the insulin molecule and includes sites of rare human mutations that cause diabetes.

A divergent INS protein in Caenorhabditis elegans structurally resembles human insulin and activates the human insulin receptor.

Caenorhabditis elegans contains a family of putative insulin-like genes proposed to regulate dauer arrest and senescence. These sequences often lack characteristic sequence features of human insulin essential for its folding, structure, and function. Here, we describe the structure and receptor-binding properties of INS-6, a single-chain polypeptide expressed in specific neurons. Despite multiple nonconservative changes in sequence, INS-6 recapitulates an insulin-like fold. Although lacking classical receptor-binding determinants, INS-6 binds to and activates the human insulin receptor. Its activity is greater than that of an analogous single-chain human insulin analog.

Chiral mutagenesis of insulin's hidden receptor-binding surface: structure of an allo-isoleucine(A2) analogue.

The hydrophobic core of vertebrate insulins contains an invariant isoleucine residue at position A2. Lack of variation may reflect this side-chain's dual contribution to structure and function: Ile(A2) is proposed both to stabilize the A1-A8 alpha-helix and to contribute to a "hidden" functional surface exposed on receptor binding. Substitution of Ile(A2) by alanine results in segmental unfolding of the A1-A8 alpha-helix, lower thermodynamic stability and impaired receptor binding. Such a spectrum of perturbations, although of biophysical interest, confounds interpretation of structure-activity relationships. To investigate the specific contribution of Ile(A2) to insulin's functional surface, we have employed non-standard mutagenesis: inversion of side-chain chirality in engineered monomer allo-Ile(A2)-DKP-insulin. Although the analogue retains native structure and stability, its affinity for the insulin receptor is impaired by 50-fold. Thus, whereas insulin's core readily accommodates allo-isoleucine at A2, its activity is exquisitely sensitive to chiral inversion. We propose that the Ile(A2) side-chain inserts within a chiral pocket of the receptor as part of insulin's hidden functional surface.

Non-standard insulin design: structure-activity relationships at the periphery of the insulin receptor.

The design of insulin analogues has emphasized stabilization or destabilization of structural elements according to established principles of protein folding. To this end, solvent-exposed side-chains extrinsic to the receptor-binding surface provide convenient sites of modification. An example is provided by an unfavorable helical C-cap (Thr(A8)) whose substitution by favorable amino acids (His(A8) or Arg(A8)) has yielded analogues of improved stability. Remarkably, these analogues also exhibit enhanced activity, suggesting that activity may correlate with stability. Here, we test this hypothesis by substitution of diaminobutyric acid (Dab(A8)), like threonine an amino acid of low helical propensity. The crystal structure of Dab(A8)-insulin is similar to those of native insulin and the related analogue Lys(A8)-insulin. Although no more stable than native insulin, the non-standard analogue is twice as active. Stability and affinity can therefore be uncoupled. To investigate alternative mechanisms by which A8 substitutions enhance activity, multiple substitutions were introduced. Surprisingly, diverse aliphatic, aromatic and polar side-chains enhance receptor binding and biological activity. Because no relationship is observed between activity and helical propensity, we propose that local interactions between the A8 side-chain and an edge of the hormone-receptor interface modulate affinity. Dab(A8)-insulin illustrates the utility of non-standard amino acids in hypothesis-driven protein design.

Protein structure and the spandrels of San Marco: insulin's receptor-binding surface is buttressed by an invariant leucine essential for its stability.

Insulin provides a model of induced fit in macromolecular recognition: the hormone's conserved core is proposed to contribute to a novel receptor-binding surface. The core's evolutionary invariance, unusual among globular proteins, presumably reflects intertwined constraints of structure and function. To probe the architectural basis of such invariance, we have investigated hydrophobic substitutions of a key internal side chain (Leu(A16)). Although the variants exhibit perturbed structure and stability, moderate receptor-binding activities are retained. These observations suggest that the A16 side chain provides an essential structural buttress but unlike neighboring core side chains, does not itself contact the receptor. Among invertebrate insulin-like proteins, Leu(A16) and other putative core residues are not conserved, suggesting that the vertebrate packing scheme is not a general requirement of an insulin-like fold. We propose that conservation of Leu(A16) among vertebrate insulins and insulin-like growth factors is a side consequence of induced fit: alternative packing schemes are disallowed by lack of surrounding covariation within the hormone's hidden receptor-binding surface. An analogy is suggested between Leu(A16) and the spandrels of San Marco, tapering triangular spaces at the intersection of the dome's arches. This celebrated metaphor of Gould and Lewontin emphasizes the role of interlocking constraints in the evolution of biological structures.

A cavity-forming mutation in insulin induces segmental unfolding of a surrounding alpha-helix.

To investigate the cooperativity of insulin's structure, a cavity-forming substitution was introduced within the hydrophobic core of an engineered monomer. The substitution, Ile(A2)-->Ala in the A1-A8 alpha-helix, does not impair disulfide pairing between chains. In accord with past studies of cavity-forming mutations in globular proteins, a decrement was observed in thermodynamic stability (DeltaDeltaG(u) 0.4-1.2 kcal/mole). Unexpectedly, CD studies indicate an attenuated alpha-helix content, which is assigned by NMR spectroscopy to selective destabilization of the A1-A8 segment. The analog's solution structure is otherwise similar to that of native insulin, including the B chain's supersecondary structure and a major portion of the hydrophobic core. Our results show that (1) a cavity-forming mutation in a globular protein can lead to segmental unfolding, (2) tertiary packing of Ile(A2), a residue of low helical propensity, stabilizes the A1-A8 alpha-helix, and (3) folding of this segment is not required for native disulfide pairing or overall structure. We discuss these results in relation to a hierarchical pathway of protein folding and misfolding. The Ala(A2) analog's low biological activity (0.5% relative to the parent monomer) highlights the importance of the A1-A8 alpha-helix in receptor recognition.