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[show abstract]
[hide abstract]
ABSTRACT: The primary hormone-binding surface of the insulin receptor spans one face of the N-terminal β-helix of the α-subunit (the L1 domain) and an α-helix in its C-terminal segment (αCT). Crystallographic analysis of the free ectodomain has defined a contiguous dimer-related motif in which the αCT α-helix packs against L1 β-strands 2 and 3. To relate structure to function, we exploited expanded genetic-code technology to insert photo-activatable probes at key sites in L1 and αCT. The pattern of αCT-mediated photo-cross-linking within the free and bound receptor is in accord with the crystal structure and prior mutagenesis. Surprisingly, L1 photo-probes in β-strands 2 and 3, predicted to be shielded by αCT, efficiently cross-link to insulin. Furthermore, anomalous mutations were identified on neighboring surfaces of αCT and insulin that impair hormone-dependent activation of the intracellular receptor tyrosine kinase (contained within the transmembrane β-subunit) disproportionately to their effects on insulin binding. Taken together, these results suggest that αCT, in addition to its hormone-recognition role, provides a signaling element in the mechanism of receptor activation.
Proceedings of the National Academy of Sciences 06/2012; 109(28):11166-71. · 9.68 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Insulin is susceptible to thermal fibrillation, a misfolding process that leads to nonnative cross-β assembly analogous to pathological amyloid deposition. Pharmaceutical formulations are ordinarily protected from such degradation by sequestration of the susceptible monomer within native protein assemblies. With respect to the safety and efficacy of insulin pumps, however, this strategy imposes an intrinsic trade-off between pharmacokinetic goals (rapid absorption and clearance) and the requisite physical properties of a formulation (prolonged shelf life and stability within the reservoir). Available rapid-acting formulations are suboptimal in both respects; susceptibility to fibrillation is exacerbated even as absorption is delayed relative to the ideal specifications of a closed-loop system. To circumvent this molecular trade-off, we exploited structural models of insulin fibrils and amyloidogenic intermediates to define an alternative protective mechanism. Single-chain insulin (SCI) analogs were shown to be refractory to thermal fibrillation with maintenance of biological activity for more than 3 months under conditions that promote the rapid fibrillation and inactivation of insulin. The essential idea exploits an intrinsic incompatibility between SCI topology and the geometry of cross-β assembly. A peptide tether was thus interposed between the A- and B-chains whose length was (a) sufficiently long to provide the "play" needed for induced fit of the hormone on receptor binding and yet (b) sufficiently short to impose a topological barrier to fibrillation. Our findings suggest that ultrastable monomeric SCI analogs may be formulated without protective self-assembly and so permit simultaneous optimization of pharmacokinetics and reservoir life.
Journal of diabetes science and technology 01/2012; 6(2):277-88.
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[show abstract]
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ABSTRACT: Mammalian testis-determining factor SRY contains a high mobility group box, a conserved eukaryotic motif of DNA bending. Mutations
in SRY cause XY gonadal dysgenesis and somatic sex reversal. Although such mutations usually arise de novo in spermatogenesis, some are inherited and so specify male development in one genetic background (the father) but not another
(the daughter). Here, we describe the biophysical properties of a representative inherited mutation, V60L, within the minor
wing of the L-shaped domain (box position 5). Although the stability and DNA binding properties of the mutant domain are similar
to those of wild type, studies of SRY-induced DNA bending by subnanosecond time-resolved fluorescence resonance energy transfer
(FRET) revealed enhanced conformational fluctuations leading to long range variation in bend angle. 1H NMR studies of the variant protein-DNA complex demonstrated only local perturbations near the mutation site. Because the
minor wing of SRY folds on DNA binding, the inherited mutation presumably hinders induced fit. Stopped-flow FRET studies indicated
that such frustrated packing leads to accelerated dissociation of the bent complex. Studies of SRY-directed transcriptional
regulation in an embryonic gonadal cell line demonstrated partial activation of downstream target Sox9. Our results have demonstrated a nonlocal coupling between DNA-directed protein folding and protein-directed DNA bending.
Perturbation of this coupling is associated with a genetic switch poised at the threshold of activity.
Journal of Biological Chemistry 10/2011; 286(42):36787-36807. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Mammalian testis-determining factor SRY contains a high mobility group box, a conserved eukaryotic motif of DNA bending. Mutations in SRY cause XY gonadal dysgenesis and somatic sex reversal. Although such mutations usually arise de novo in spermatogenesis, some are inherited and so specify male development in one genetic background (the father) but not another (the daughter). Here, we describe the biophysical properties of a representative inherited mutation, V60L, within the minor wing of the L-shaped domain (box position 5). Although the stability and DNA binding properties of the mutant domain are similar to those of wild type, studies of SRY-induced DNA bending by subnanosecond time-resolved fluorescence resonance energy transfer (FRET) revealed enhanced conformational fluctuations leading to long range variation in bend angle. (1)H NMR studies of the variant protein-DNA complex demonstrated only local perturbations near the mutation site. Because the minor wing of SRY folds on DNA binding, the inherited mutation presumably hinders induced fit. Stopped-flow FRET studies indicated that such frustrated packing leads to accelerated dissociation of the bent complex. Studies of SRY-directed transcriptional regulation in an embryonic gonadal cell line demonstrated partial activation of downstream target Sox9. Our results have demonstrated a nonlocal coupling between DNA-directed protein folding and protein-directed DNA bending. Perturbation of this coupling is associated with a genetic switch poised at the threshold of activity.
Journal of Biological Chemistry 08/2011; 286(42):36787-807. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Mammalian testis-determining factor SRY contains a high-mobility-group (HMG) box, a conserved eukaryotic motif of DNA bending.
Mutations in SRY cause 46, XY gonadal dysgenesis, leading to somatic sex reversal. Although such mutations usually arise
de novo in spermatogenesis, some are inherited: the latter specify male development in one genetic background (the father)
but not in another (the sterile daughter). Because such mutant DNA-bending domains may define a threshold for activation
of a genetic switch, we investigated the biophysical properties of a representative mutation: V60L within the minor wing of
the L-shaped domain (box position 5). Although the stability and specific DNA-binding properties of the mutant domain are
similar to those of the wild-type box, studies of SRY-induced DNA bending by subnanosecond time-resolved fluorescence resonance
energy transfer (FRET) revealed enhanced conformational fluctuations leading to long-range variation in bend angle. 1H-NMR
spectra of the variant protein-DNA complex demonstrated only local perturbations near the mutation site. Because the minor
wing of SRY folds on DNA binding, the inherited mutation presumably hinders induced fit. Stopped-flow FRET studies indicated
that such frustrated packing leads to accelerated dissociation of the bent complex. Studies of SRY-directed transcriptional
regulation in an embryogenic gonadal cell line demonstrated partial activation of downstream target Sox9. Our results have
demonstrated a non-local coupling between DNA-directed protein folding and protein-directed DNA bending. Perturbation of
this coupling leads to a genetic switch poised at the threshold of activity.
Journal of Biological Chemistry 08/2011; · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Bottom-up control of supramolecular protein assembly can provide a therapeutic nanobiotechnology. We demonstrate that the
pharmacological properties of insulin can be enhanced by design of “zinc staples” between hexamers. Paired (i, i+4) His substitutions
were introduced at an α-helical surface. The crystal structure contains both classical axial zinc ions and novel zinc ions
at hexamer-hexamer interfaces. Although soluble at pH 4, the combined electrostatic effects of the substitutions and bridging
zinc ions cause isoelectric precipitation at neutral pH. Following subcutaneous injection in a diabetic rat, the analog effected
glycemic control with a time course similar to that of long acting formulation Lantus®. Relative to Lantus, however, the analog
discriminates at least 30-fold more stringently between the insulin receptor and mitogenic insulin-like growth factor receptor.
Because aberrant mitogenic signaling may be associated with elevated cancer risk, such enhanced specificity may improve safety.
Zinc stapling provides a general strategy to modify the pharmacokinetic and biological properties of a subcutaneous protein
depot.
Journal of Biological Chemistry 04/2010; 285(16):11755-11759. · 4.77 Impact Factor
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Yanwu Yang,
Aneta Petkova,
Kun Huang,
Bin Xu,
Qing-Xin Hua,
I-Ju Ye,
Ying-Chi Chu,
Shi-Quan Hu, Nelson B Phillips,
Jonathan Whittaker,
Faramarz Ismail-Beigi,
Robert B Mackin,
Panayotis G Katsoyannis,
Robert Tycko,
Michael A Weiss
[show abstract]
[hide abstract]
ABSTRACT: Insulin fibrillation provides a model for a broad class of amyloidogenic diseases. Conformational distortion of the native monomer leads to aggregation-coupled misfolding. Whereas beta-cells are protected from proteotoxicity by hexamer assembly, fibrillation limits the storage and use of insulin at elevated temperatures. Here, we have investigated conformational distortions of an engineered insulin monomer in relation to the structure of an insulin fibril. Anomalous (13)C NMR chemical shifts and rapid (15)N-detected (1)H-(2)H amide-proton exchange were observed in one of the three classical alpha-helices (residues A1-A8) of the hormone, suggesting a conformational equilibrium between locally folded and unfolded A-chain segments. Whereas hexamer assembly resolves these anomalies in accordance with its protective role, solid-state (13)C NMR studies suggest that the A-chain segment participates in a fibril-specific beta-sheet. Accordingly, we investigated whether helicogenic substitutions in the A1-A8 segment might delay fibrillation. Simultaneous substitution of three beta-branched residues (Ile(A2) --> Leu, Val(A3) --> Leu, and Thr(A8) --> His) yielded an analog with reduced thermodynamic stability but marked resistance to fibrillation. Whereas amide-proton exchange in the A1-A8 segment remained rapid, (13)Calpha chemical shifts exhibited a more helical pattern. This analog is essentially without activity, however, as Ile(A2) and Val(A3) define conserved receptor contacts. To obtain active analogs, substitutions were restricted to A8. These analogs exhibit high receptor-binding affinity; representative potency in a rodent model of diabetes mellitus was similar to wild-type insulin. Although (13)Calpha chemical shifts remain anomalous, significant protection from fibrillation is retained. Together, our studies define an "Achilles' heel" in a globular protein whose repair may enhance the stability of pharmaceutical formulations and broaden their therapeutic deployment in the developing world.
Journal of Biological Chemistry 04/2010; 285(14):10806-21. · 4.77 Impact Factor
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Yanwu Yang,
Aneta Petkova,
Kun Huang,
Bin Xu,
Qing-xin Hua,
I-Ju Ye,
Ying-Chi Chu,
Shi-Quan Hu, Nelson B. Phillips,
Jonathan Whittaker,
Faramarz Ismail-Beigi,
Robert B. Mackin,
Panayotis G. Katsoyannis,
Robert Tycko,
Michael A. Weiss
[show abstract]
[hide abstract]
ABSTRACT: Insulin fibrillation provides a model for a broad class of amyloidogenic diseases. Conformational distortion of the native
monomer leads to aggregation-coupled misfolding. Whereas β-cells are protected from proteotoxicity by hexamer assembly, fibrillation
limits the storage and use of insulin at elevated temperatures. Here, we have investigated conformational distortions of an
engineered insulin monomer in relation to the structure of an insulin fibril. Anomalous 13C NMR chemical shifts and rapid 15N-detected 1H-2H amide-proton exchange were observed in one of the three classical α-helices (residues A1–A8) of the hormone, suggesting
a conformational equilibrium between locally folded and unfolded A-chain segments. Whereas hexamer assembly resolves these
anomalies in accordance with its protective role, solid-state 13C NMR studies suggest that the A-chain segment participates in a fibril-specific β-sheet. Accordingly, we investigated whether
helicogenic substitutions in the A1–A8 segment might delay fibrillation. Simultaneous substitution of three β-branched residues
(IleA2 → Leu, ValA3 → Leu, and ThrA8 → His) yielded an analog with reduced thermodynamic stability but marked resistance to fibrillation. Whereas amide-proton
exchange in the A1–A8 segment remained rapid, 13Cα chemical shifts exhibited a more helical pattern. This analog is essentially without activity, however, as IleA2 and ValA3 define conserved receptor contacts. To obtain active analogs, substitutions were restricted to A8. These analogs exhibit
high receptor-binding affinity; representative potency in a rodent model of diabetes mellitus was similar to wild-type insulin.
Although 13Cα chemical shifts remain anomalous, significant protection from fibrillation is retained. Together, our studies define an
“Achilles' heel” in a globular protein whose repair may enhance the stability of pharmaceutical formulations and broaden their
therapeutic deployment in the developing world.
Journal of Biological Chemistry 04/2010; 285(14):10806-10821. · 4.77 Impact Factor
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Youhei Sohma,
Qing-xin Hua,
Ming Liu, Nelson B. Phillips,
Shi-Quan Hu,
Jonathan Whittaker,
Linda J. Whittaker,
Aubree Ng,
Jr Charles T. Roberts,
Peter Arvan,
Stephen B. H. Kent,
Michael A. Weiss
[show abstract]
[hide abstract]
ABSTRACT: Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium.
Native insulin-related growth factor (IGF)-I has canonical cystines (A6—A11, A7–B7, and A20—B19) maintained by IGF-binding proteins;
IGF-swap has alternative pairing (A7–A11, A6—B7, and A20—B19) and impaired activity. Studies of mini-domain models suggest that residue
B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation
in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions
in the two proteins. In insulin, HisB5 → Thr markedly destabilizes the hormone (ΔΔGu 2.0 ± 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution
ThrB5 → His (residue 4) specifies a unique structure with native 1H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I
undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, HisB5-IGF-I retains canonical pairing. Chemical denaturation studies indicate that HisB5 does not significantly enhance thermodynamic stability (ΔΔGu 0.2 ± 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the
activity of ThrB5-insulin is decreased 5-fold, HisB5-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation
of ThrB5 in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast,
the conservation of HisB5 in insulin highlights its critical role in insulin biosynthesis.
Journal of Biological Chemistry 02/2010; 285(7):5040-5055. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Bottom-up control of supramolecular protein assembly can provide a therapeutic nanobiotechnology. We demonstrate that the pharmacological properties of insulin can be enhanced by design of "zinc staples" between hexamers. Paired (i, i+4) His substitutions were introduced at an alpha-helical surface. The crystal structure contains both classical axial zinc ions and novel zinc ions at hexamer-hexamer interfaces. Although soluble at pH 4, the combined electrostatic effects of the substitutions and bridging zinc ions cause isoelectric precipitation at neutral pH. Following subcutaneous injection in a diabetic rat, the analog effected glycemic control with a time course similar to that of long acting formulation Lantus. Relative to Lantus, however, the analog discriminates at least 30-fold more stringently between the insulin receptor and mitogenic insulin-like growth factor receptor. Because aberrant mitogenic signaling may be associated with elevated cancer risk, such enhanced specificity may improve safety. Zinc stapling provides a general strategy to modify the pharmacokinetic and biological properties of a subcutaneous protein depot.
Journal of Biological Chemistry 02/2010; 285(16):11755-9. · 4.77 Impact Factor
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Youhei Sohma,
Qing-Xin Hua,
Ming Liu, Nelson B Phillips,
Shi-Quan Hu,
Jonathan Whittaker,
Linda J Whittaker,
Aubree Ng,
Charles T. Roberts,
Peter Arvan,
Stephen B H Kent,
Michael A Weiss
[show abstract]
[hide abstract]
ABSTRACT: Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6—A11, A7–B7, and A20—B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7–A11, A6—B7, and A20—B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, HisB5 → Thr markedly destabilizes the hormone (ΔΔGu 2.0 ± 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution ThrB5 → His (residue 4) specifies a unique structure with native 1H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, HisB5-IGF-I retains canonical pairing. Chemical denaturation studies indicate that HisB5 does not significantly enhance thermodynamic stability (ΔΔGu 0.2 ± 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of ThrB5-insulin is decreased 5-fold, HisB5-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of ThrB5 in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of HisB5 in insulin highlights its critical role in insulin biosynthesis.
Journal of Biological Chemistry 12/2009; · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Insulin binds with high affinity to the insulin receptor (IR) and with low affinity to the type 1 insulin-like growth factor
(IGF) receptor (IGFR). Such cross-binding, which reflects homologies within the insulin-IGF signaling system, is of clinical
interest in relation to the association between hyperinsulinemia and colorectal cancer. Here, we employ nonstandard mutagenesis
to design an insulin analog with enhanced affinity for the IR but reduced affinity for the IGFR. Unnatural amino acids were
introduced by chemical synthesis at the N- and C-capping positions of a recognition α-helix (residues A1 and A8). These sites
adjoin the hormone-receptor interface as indicated by photocross-linking studies. Specificity is enhanced more than 3-fold
on the following: (i) substitution of GlyA1 by d-Ala or d-Leu, and (ii) substitution of ThrA8 by diaminobutyric acid (Dab). The crystal structure of [d-AlaA1,DabA8]insulin, as determined within a T6 zinc hexamer to a resolution of 1.35 Å, is essentially identical to that of human insulin. The nonstandard side chains project
into solvent at the edge of a conserved receptor-binding surface shared by insulin and IGF-I. Our results demonstrate that
modifications at this edge discriminate between IR and IGFR. Because hyperinsulinemia is typically characterized by a 3-fold
increase in integrated postprandial insulin concentrations, we envisage that such insulin analogs may facilitate studies of
the initiation and progression of cancer in animal models. Future development of clinical analogs lacking significant IGFR
cross-binding may enhance the safety of insulin replacement therapy in patients with type 2 diabetes mellitus at increased
risk of colorectal cancer.
Journal of Biological Chemistry 11/2009; 284(46):32178-32187. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Insulin binds with high affinity to the insulin receptor (IR) and with low affinity to the type 1 insulin-like growth factor (IGF) receptor (IGFR). Such cross-binding, which reflects homologies within the insulin-IGF signaling system, is of clinical interest in relation to the association between hyperinsulinemia and colorectal cancer. Here, we employ nonstandard mutagenesis to design an insulin analog with enhanced affinity for the IR but reduced affinity for the IGFR. Unnatural amino acids were introduced by chemical synthesis at the N- and C-capping positions of a recognition alpha-helix (residues A1 and A8). These sites adjoin the hormone-receptor interface as indicated by photocross-linking studies. Specificity is enhanced more than 3-fold on the following: (i) substitution of Gly(A1) by D-Ala or D-Leu, and (ii) substitution of Thr(A8) by diaminobutyric acid (Dab). The crystal structure of [D-Ala(A1),Dab(A8)]insulin, as determined within a T(6) zinc hexamer to a resolution of 1.35 A, is essentially identical to that of human insulin. The nonstandard side chains project into solvent at the edge of a conserved receptor-binding surface shared by insulin and IGF-I. Our results demonstrate that modifications at this edge discriminate between IR and IGFR. Because hyperinsulinemia is typically characterized by a 3-fold increase in integrated postprandial insulin concentrations, we envisage that such insulin analogs may facilitate studies of the initiation and progression of cancer in animal models. Future development of clinical analogs lacking significant IGFR cross-binding may enhance the safety of insulin replacement therapy in patients with type 2 diabetes mellitus at increased risk of colorectal cancer.
Journal of Biological Chemistry 09/2009; 284(46):32178-87. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Biological Chemistry 06/2008; 283(21):14703-16. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Biological Chemistry 05/2008; 283(21):14703-14716. · 4.77 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Maturity-onset diabetes of the young (MODY3), a monogenic form of type II diabetes mellitus, results most commonly from mutations in hepatocyte nuclear factor 1alpha (HNF-1alpha). Diabetes-associated mutation G20R perturbs the dimerization domain of HNF-1alpha, an intertwined four-helix bundle. In the wild-type structure G20 participates in a Schellman motif to cap an alpha-helix; its dihedral angles lie in the right side of the Ramachandran plot (alpha(L) region; phi 97 degrees). Substitutions G20R and G20A lead to dimeric molten globules of low stability, suggesting that the impaired function of the diabetes-associated transcription factor is due in large part to a main-chain perturbation rather than to specific features of the Arg side-chain. This hypothesis is supported by the enhanced stability of non-standard analogues containing D-Ala or D-Ser at position 20. The crystal structure of the D-Ala20 analogue, determined to a resolution of 1.4 A, is essentially identical to the wild-type structure in the same crystal form. The mean root-mean-square deviation between equivalent C(alpha) atoms (residues 5-28) is 0.3 A; (phi, psi) angles of D-Ala20 are the same as those of G20 in the wild-type structure. Whereas the side-chain of A20 or R20 would be expected to clash with the preceding carbonyl oxygen (thus accounting for its frustrated energy landscape), the side-chain of D-Ala20 projects into solvent without perturbation of the Schellman motif. Calorimetric studies indicate that the increased stability of the D-Ala20 analogue (DeltaDeltaG(u) 1.5 kcal/mol) is entropic in origin, consistent with a conformational bias toward native-like conformations in the unfolded state. Studies of multiple substitutions at G20 and neighboring positions highlight the essential contributions of a glycine-specific tight turn and adjoining inter-subunit side-chain hydrogen bonds to the stability and architectural specificity of the intertwined dimer. Comparison of L- and D amino acid substitutions thus provides an example of the stereospecific control of an energy landscape by a helix-capping residue.
Journal of Molecular Biology 10/2006; 362(3):414-29. · 4.00 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Systemic amyloidoses, an important class of protein misfolding diseases, are often due to fibrillation of disulfide-cross-linked globular proteins otherwise unrelated in sequence or structure. Although cross-beta assembly is regarded as a universal property of polypeptides, it is not understood how such amyloids accommodate diverse disulfide connectivities. Does amyloidogenicity depend on protein topology? A model is provided by insulin, a two-chain protein containing three disulfide bridges. The importance of chain topology is demonstrated by mini-proinsulin (MP), a single-chain analogue in which the C-terminus of the B chain (residue B30) is tethered to the N-terminus of the A chain (A1). The B30-A1 tether impedes the fiber-specific alpha --> beta transition, leading to slow formation of a structurally nonuniform amorphous precipitate. Conversely, fibrillation is robust to interchange of disulfide bridges. Whereas native insulin exhibits pairings [A6-A11, A7-B7, and A20-B19], metastable isomers with alternative pairings [A6-B7, A7-A11, A20-B19] or [A6-A7, A11-B7, A20-B1] readily undergo fibrillation with essentially identical alpha --> beta transitions. Respective pairing schemes are in each case retained. Isomeric fibrils and the amorphous MP precipitate are each able to seed the fibrillation of wild-type insulin, suggesting a structural correspondence between respective nuclei or modes of assembly. Together, our results demonstrate that effects of polypeptide topology on amyloidogenicity depend on structural context. Although the native structures and stabilities of single-chain insulin analogues are similar to those of wild-type insulin, the interchain tether constrains the extent of conformational distortion at elevated temperature, retards initial non-native aggregation, and is apparently incompatible with the mature structure of an insulin protofilament. We speculate that the general danger of fibrillation has imposed a constraint in protein evolution, selecting for topologies unfavorable to amyloid formation.
Biochemistry 08/2006; 45(34):10278-93. · 3.42 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Sex-reversal mutations in human SRY cluster within its high-mobility group box, a conserved motif of DNA bending. A classical substitution at the crux of this angular domain (M64I) has been reported to impair DNA bending but not DNA binding, implying that sharp bending is required for transcriptional activation and testis determination. Surprisingly, we report that this defect was an inadvertent consequence of protein truncation: in the intact protein, sharp DNA bending is restored by the basic tail of the high-mobility group box. Structural coupling between box and tail is tuned to the native DNA bend angle, damping conformational fluctuations and enabling bidirectional induced fit within the bent complex. M64I-associated sex reversal is instead caused by the impaired function of a flanking non-classical nuclear localization signal (NLS). Similar impairment is caused by M64A, suggesting that mislocalization is due to loss of an M64-specific function and not gain of a non-native I64-specific function. Transcriptional activity, attenuated by mislocalization, is rescued by fusion of a heterologous NLS. In a male embryonic gonadal cell line, M64I and M64A SRY-NLS fusion proteins exhibit native transcriptional activation of Sox9, a key step in testicular differentiation. Our results suggest that male development is robust to subtle alterations in SRY-DNA architecture but depends critically on nuclear localization. The previously unsuspected role of M64 within a non-classical NLS may contribute to its invariance among SOX-related and LEF-1-related transcription factors.
Journal of Molecular Biology 08/2006; 360(2):310-28. · 4.00 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Human testis-determining factor SRY contains a high-mobility-group (HMG) box, an alpha-helical DNA-binding domain that binds within an expanded minor groove to induce DNA bending. This motif is flanked on the C-terminal end by a basic tail, which functions both as a nuclear localization signal and accessory DNA-binding element. Whereas the HMG box is broadly conserved among otherwise unrelated transcription factors, tails differ in sequence and mode of DNA binding. Contrasting examples are provided by SRY and lymphoid enhancer factor 1 (LEF-1): whereas the SRY tail remains in the minor groove distal to the HMG box, the LEF-1 tail binds back across the center of the bent DNA site. The LEF-1 tail relieves electrostatic repulsion that would otherwise be incurred within the compressed major groove to enable sharp DNA bending with high affinity. Here, we demonstrate that the analogous SRY tail functions as a "kinetic clamp" to regulate the lifetime of the bent DNA complex. As in LEF-1, partial truncation of the distal SRY tail reduces specific DNA affinity and DNA bending, but these perturbations are modest: binding is reduced by only 1.8-fold, and bending by only 7-10 degrees . "Tailed" and truncated SRY complexes exhibit similar structures (as probed by NMR) and distributions of long-range conformational substates (as probed by time-resolved fluorescence resonance energy transfer). Surprisingly, however, the SRY tail retards dissociation of the protein-DNA complex by 20-fold. The marked and compensating changes in rates of association and dissociation observed on tail truncation, disproportionate to perturbations in affinity or structure, suggest that this accessory element functions as a kinetic clamp to regulate the lifetime of the SRY-DNA complex. We speculate that the kinetic stability of a bent DNA complex is critical to the assembly and maintenance of a sex-specific transcriptional pre-initiation complex.
Journal of Molecular Biology 05/2006; 358(1):172-92. · 4.00 Impact Factor
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ABSTRACT: Insulin is susceptible to fibrillation, a misfolding process leading to well ordered cross-beta assembly. Protection from fibrillation in beta cells is provided by sequestration of the susceptible monomer within zinc hexamers. We demonstrate that proinsulin is refractory to fibrillation under conditions that promote the rapid fibrillation of zinc-free insulin. Proinsulin fibrils, as probed by Raman microscopy, are nonetheless similar in structure to insulin fibrils. The connecting peptide, although not well ordered in native proinsulin, participates in a fibril-specific beta-sheet. Native insulin and proinsulin exhibit similar free energies of unfolding as inferred from guanidine denaturation studies: relative amyloidogenicities are thus not correlated with global stability. Strikingly, the susceptibility of proinsulin to fibrillation is increased by scission of the connecting peptide at single sites. We thus propose that the connecting peptide constrains a large scale conformational change in the misfolded protein. A tethering mechanism is proposed based on a model of an insulin protofilament derived from electron-microscopic image reconstruction. The proposed relationship between cross-beta assembly and protein topology is supported by studies of single-chain analogs (mini-proinsulin and insulin-like growth factor I) in which foreshortened connecting peptides further retard fibrillation. In addition to its classic function to facilitate disulfide pairing, the connecting peptide may protect beta cells from toxic protein misfolding in the endoplasmic reticulum.
Journal of Biological Chemistry 01/2006; 280(51):42345-55. · 4.77 Impact Factor
