Wenhua Jia

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 (ΔΔGu = 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 ValA3 at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 α-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 ValA3, which projects within a crevice between the A- and B-chains. Engineered receptor α-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 PapB25-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 α-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (GlyA3 and SerA3) 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 AbaA3 and allo-ThrA3 analogs are reduced at least 10-fold; the activity of ThrA3-DKP-insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (AbaA3) or by altered polarity (allo-ThrA3 and ThrA3). Our results provide evidence that ValA3, a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the α-subunit in a hormone-receptor complex.

The Folding Nucleus of the Insulin Superfamily A FLEXIBLE PEPTIDE MODEL FORESHADOWS THE NATIVE STATE

Oxidative folding of insulin-like growth factor I (IGF-I) and single-chain insulin analogs proceeds via one- and two-disulfide intermediates. A predominant one-disulfide intermediate in each case contains the canonical A20–B19 disulfide bridge (cystines 18–61 in IGF-I and 19–85 in human proinsulin). Here, we describe a disulfide-linked peptide model of this on-pathway intermediate. One peptide fragment (19 amino acids) spans IGF-I residues 7–25 (canonical positions B8-B26 in the insulin superfamily); the other (18 amino acids) spans IGF-I residues 53–70 (positions A12–A21 and D1–D8). Containing only half of the IGF-I sequence, the disulfide-linked polypeptide (designated IGF-p) is not well ordered. Nascent helical elements corresponding to native α-helices are nonetheless observed at 4 °C. Furthermore, 13C-edited nuclear Overhauser effects establish transient formation of a native-like partial core; no non-native nuclear Overhauser effects are observed. Together, these observations suggest that early events in the folding of insulin-related polypeptides are nucleated by a native-like molten subdomain containing CysA20 and CysB19. We propose that nascent interactions within this subdomain orient the A20 and B19 thiolates for disulfide bond formation and stabilize the one-disulfide intermediate once formed. Substitutions in the corresponding region of insulin are associated with inefficient chain combination and impaired biosynthetic expression. The intrinsic conformational propensities of a flexible disulfide-linked peptide thus define a folding nucleus, foreshadowing the structure of the native state.

Toward the Active Conformation of Insulin: Stereospecific modulation of a structural switch in the B chain.

How insulin binds to the insulin receptor has long been a subject of speculation. Although the structure of the free hormone has been extensively characterized, a variety of evidence suggests that a conformational change occurs upon receptor binding. Here, we employ chiral mutagenesis, comparison of corresponding d and l amino acid substitutions, to investigate a possible switch in the B-chain. To investigate the interrelation of structure, function, and stability, isomeric analogs have been synthesized in which an invariant glycine in a β-turn (GlyB8) is replaced by d- or l-Ser. The d substitution enhances stability (ΔΔGu 0.9 kcal/mol) but impairs receptor binding by 100-fold; by contrast, the l substitution markedly impairs stability (ΔΔGu -3.0 kcal/mol) with only 2-fold reduction in receptor binding. Although the isomeric structures each retain a native-like overall fold, the l-SerB8 analog exhibits fewer helix-related and long range nuclear Overhauser effects than does the d-SerB8 analog or native monomer. Evidence for enhanced conformational fluctuations in the unstable analog is provided by its attenuated CD spectrum. The inverse relationship between stereospecific stabilization and receptor binding strongly suggests that the B7-B10 β-turn changes conformation on receptor binding.

Enhancing the activity of a protein by stereospecific unfolding. Conformational life cycle of insulin and its evolutionary origins

A central tenet of molecular biology holds that the function of a protein is mediated by its structure. An inactive ground-state conformation may nonetheless be enjoined by the interplay of competing biological constraints. A model is provided by insulin, well characterized at atomic resolution by x-ray crystallography. Here, we demonstrate that the activity of the hormone is enhanced by stereospecific unfolding of a conserved structural element. A bifunctional β-strand mediates both self-assembly (within β-cell storage vesicles) and receptor binding (in the bloodstream). This strand is anchored by an invariant side chain (PheB24); its substitution by Ala leads to an unstable but native-like analog of low activity. Substitution by d-Ala is equally destabilizing, and yet the protein diastereomer exhibits enhanced activity with segmental unfolding of the β-strand. Corresponding photoactivable derivatives (containing l- or d-para-azido-Phe) cross-link to the insulin receptor with higher d-specific efficiency. Aberrant exposure of hydrophobic surfaces in the analogs is associated with accelerated fibrillation, a form of aggregation-coupled misfolding associated with cellular toxicity. Conservation of PheB24, enforced by its dual role in native self-assembly and induced fit, thus highlights the implicit role of misfolding as an evolutionary constraint. Whereas classical crystal structures of insulin depict its storage form, signaling requires engagement of a detachable arm at an extended receptor interface. Because this active conformation resembles an amyloidogenic intermediate, we envisage that induced fit and self-assembly represent complementary molecular adaptations to potential proteotoxicity. The cryptic threat of misfolding poses a universal constraint in the evolution of polypeptide sequences.

Chiral mutagenesis of insulin.contribution of the B20-B23 β -turn to activity and stability

Insulin contains a β-turn (residues B20-B23) interposed between two receptor-binding elements, the central α-helix of the B chain (B9-B19) and its C-terminal β-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 φ dihedral angles within a classic type-I β-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) φ 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 (ΔΔGu 2.0 kcal/mole) than at B23 (ΔΔGu 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).

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

To investigate the cooperativity of insulins structure, a cavity‐forming substitution was introduced within the hydrophobic core of an engineered monomer. The substitution, IleA2→Ala in the A1–A8 α‐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 (ΔΔGu 0.4–1.2 kcal/mole). Unexpectedly, CD studies indicate an attenuated α‐helix content, which is assigned by NMR spectroscopy to selective destabilization of the A1–A8 segment. The analogs solution structure is otherwise similar to that of native insulin, including the B chains 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 IleA2, a residue of low helical propensity, stabilizes the A1–A8 α‐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 AlaA2 analogs low biological activity (0.5% relative to the parent monomer) highlights the importance of the A1–A8 α‐helix in receptor recognition.

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 hormones conserved core is proposed to contribute to a novel receptor-binding surface. The cores 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 hormones 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 domes arches. This celebrated metaphor of Gould and Lewontin emphasizes the role of interlocking constraints in the evolution of biological structures.

Conformational Dynamics of Insulin

We have exploited a prandial insulin analog to elucidate the underlying structure and dynamics of insulin as a monomer in solution. A model was provided by insulin lispro (the active component of Humalog®; Eli Lilly and Co.). Whereas NMR-based modeling recapitulated structural relationships of insulin crystals (T-state protomers), dynamic anomalies were revealed by amide-proton exchange kinetics in D2O. Surprisingly, the majority of hydrogen bonds observed in crystal structures are only transiently maintained in solution, including key T-state-specific inter-chain contacts. Long-lived hydrogen bonds (as defined by global exchange kinetics) exist only at a subset of four α-helical sites (two per chain) flanking an internal disulfide bridge (cystine A20–B19); these sites map within the proposed folding nucleus of proinsulin. The anomalous flexibility of insulin otherwise spans its active surface and may facilitate receptor binding. Because conformational fluctuations promote the degradation of pharmaceutical formulations, we envisage that “dynamic re-engineering” of insulin may enable design of ultra-stable formulations for humanitarian use in the developing world.

Deciphering the Hidden Informational Content of Protein Sequences FOLDABILITY OF PROINSULIN HINGES ON A FLEXIBLE ARM THAT IS DISPENSABLE IN THE MATURE HORMONE

Protein sequences encode both structure and foldability. Whereas the interrelationship of sequence and structure has been extensively investigated, the origins of folding efficiency are enigmatic. We demonstrate that the folding of proinsulin requires a flexible N-terminal hydrophobic residue that is dispensable for the structure, activity, and stability of the mature hormone. This residue (PheB1 in placental mammals) is variably positioned within crystal structures and exhibits 1H NMR motional narrowing in solution. Despite such flexibility, its deletion impaired insulin chain combination and led in cell culture to formation of non-native disulfide isomers with impaired secretion of the variant proinsulin. Cellular folding and secretion were maintained by hydrophobic substitutions at B1 but markedly perturbed by polar or charged side chains. We propose that, during folding, a hydrophobic side chain at B1 anchors transient long-range interactions by a flexible N-terminal arm (residues B1–B8) to mediate kinetic or thermodynamic partitioning among disulfide intermediates. Evidence for the overall contribution of the arm to folding was obtained by alanine scanning mutagenesis. Together, our findings demonstrate that efficient folding of proinsulin requires N-terminal sequences that are dispensable in the native state. Such arm-dependent folding can be abrogated by mutations associated with β-cell dysfunction and neonatal diabetes mellitus.