ABSTRACT
The mutant proinsulin syndrome is a monogenic cause of diabetes mellitus due to toxic misfolding of insulin's biosynthetic precursor. Also designated mutant INS-gene induced diabetes of the young (MIDY), this syndrome defines molecular determinants of foldability in the endoplasmic reticulum (ER) of ß-cells. Here, we describe a peptide model of a key proinsulin folding intermediate and variants containing representative clinical mutations; the latter perturb invariant core sites in native proinsulin (LeuB15âPro, LeuA16âPro, and PheB24âSer). The studies exploited a 49-residue single-chain synthetic precursor (designated DesDi), previously shown to optimize in vitro efficiency of disulfide pairing. Parent and variant peptides contain a single disulfide bridge (cystine B19-A20) to provide a model of proinsulin's first oxidative folding intermediate. The peptides were characterized by circular dichroism and redox stability in relation to effects of the mutations on (a) in vitro foldability of the corresponding insulin analogs and (b) ER stress induced in cell culture on expression of the corresponding variant proinsulins. Striking correlations were observed between peptide biophysical properties, degree of ER stress and age of diabetes onset (neonatal or adolescent). Our findings suggest that age of onset reflects the extent to which nascent structure is destabilized in proinsulin's putative folding nucleus. We envisage that such peptide models will enable high-resolution structural studies of key folding determinants and in turn permit molecular dissection of phenotype-genotype relationships in this monogenic diabetes syndrome. Our companion study (next article in this issue) employs two-dimensional heteronuclear NMR spectroscopy to define site-specific perturbations in the variant peptides.
Subject(s)
Diabetes Mellitus , Proinsulin , Adolescent , Diabetes Mellitus/metabolism , Disulfides/chemistry , Disulfides/metabolism , Humans , Insulin/metabolism , Peptides , Proinsulin/chemistry , Proinsulin/genetics , Proinsulin/metabolism , Protein FoldingABSTRACT
Insulin-signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to structural reorganization. To test the functional coupling between insulin's "hinge opening" and receptor activation, we inserted an artificial ligand-dependent switch into the insulin molecule. Ligand-binding disrupts an internal tether designed to stabilize the hormone's native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (meta-fluoro-phenylboronic acid at GlyA1) and internal diol (3,4-dihydroxybenzoate at LysB28). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin-signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the hormone. Similarly, metabolite-regulated signaling was not observed in control studies of 1) an unmodified insulin analog or 2) an analog containing a diol sensor without internal tethering. Although secondary structure (as probed by circular dichroism) was unaffected by ligand-binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and allosteric holoreceptor signaling. In addition to this foundational finding, our results provide proof of principle for design of a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a "smart" insulin analog to mitigate hypoglycemic risk in diabetes therapy.
Subject(s)
Insulin/chemistry , Blotting, Western , Fructose/chemistry , Fructose/metabolism , Hep G2 Cells , Humans , Insulin/metabolism , Ligands , Models, Molecular , Protein Conformation , Signal TransductionABSTRACT
Proteins have evolved to be foldable, and yet determinants of foldability may be inapparent once the native state is reached. Insight has emerged from studies of diseases of protein misfolding, exemplified by monogenic diabetes mellitus due to mutations in proinsulin leading to endoplasmic reticulum stress and ß-cell death. Cellular foldability of human proinsulin requires an invariant Phe within a conserved crevice at the receptor-binding surface (position B24). Any substitution, even related aromatic residue TyrB24, impairs insulin biosynthesis and secretion. As a seeming paradox, a monomeric TyrB24 insulin analog exhibits a native-like structure in solution with only a modest decrement in stability. Packing of TyrB24 is similar to that of PheB24, adjoining core cystine B19-A20 to seal the core; the analog also exhibits native self-assembly. Although affinity for the insulin receptor is decreased â¼20-fold, biological activities in cells and rats were within the range of natural variation. Together, our findings suggest that the invariance of PheB24 among vertebrate insulins and insulin-like growth factors reflects an essential role in enabling efficient protein folding, trafficking, and secretion, a function that is inapparent in native structures. In particular, we envision that the para-hydroxyl group of TyrB24 hinders pairing of cystine B19-A20 in an obligatory on-pathway folding intermediate. The absence of genetic variation at B24 and other conserved sites near this disulfide bridge-excluded due to ß-cell dysfunction-suggests that insulin has evolved to the edge of foldability. Nonrobustness of a protein's fitness landscape underlies both a rare monogenic syndrome and "diabesity" as a pandemic disease of civilization.
Subject(s)
Insulin/metabolism , Amino Acid Substitution/physiology , Animals , Cell Line , Cell Line, Tumor , Diabetes Mellitus/metabolism , Disulfides/metabolism , Gene Regulatory Networks/physiology , HEK293 Cells , Humans , Insulin-Secreting Cells/metabolism , MCF-7 Cells , Proinsulin/metabolism , Protein Binding/physiology , Protein Folding , Rats , Receptor, Insulin/metabolism , Structure-Activity RelationshipABSTRACT
Long-acting insulin analogues represent the most prescribed class of therapeutic proteins. An innovative design strategy was recently proposed: diselenide substitution of an external disulfide bridge. This approach exploited the distinctive physicochemical properties of selenocysteine (U). Relative to wild type (WT), Se-insulin[C7UA , C7UB ] was reported to be protected from proteolysis by insulin-degrading enzyme (IDE), predicting prolonged activity. Because of this strategy's novelty and potential clinical importance, we sought to validate these findings and test their therapeutic utility in an animal model of diabetes mellitus. Surprisingly, the analogue did not exhibit enhanced stability, and its susceptibility to cleavage by either IDE or a canonical serine protease (glutamyl endopeptidase Glu-C) was similar to WT. Moreover, the analogue's pharmacodynamic profile in rats was not prolonged relative to a rapid-acting clinical analogue (insulin lispro). Although [C7UA , C7UB ] does not confer protracted action, nonetheless its comparison to internal diselenide bridges promises to provide broad biophysical insight.
ABSTRACT
Domain-minimized insulin receptors (IRs) have enabled crystallographic analysis of insulin-bound "micro-receptors." In such structures, the C-terminal segment of the insulin B chain inserts between conserved IR domains, unmasking an invariant receptor-binding surface that spans both insulin A and B chains. This "open" conformation not only rationalizes the inactivity of single-chain insulin (SCI) analogs (in which the A and B chains are directly linked), but also suggests that connecting (C) domains of sufficient length will bind the IR. Here, we report the high-resolution solution structure and dynamics of such an active SCI. The hormone's closed-to-open transition is foreshadowed by segmental flexibility in the native state as probed by heteronuclear NMR spectroscopy and multiple conformer simulations of crystallographic protomers as described in the companion article. We propose a model of the SCI's IR-bound state based on molecular-dynamics simulations of a micro-receptor complex. In this model, a loop defined by the SCI's B and C domains encircles the C-terminal segment of the IR α-subunit. This binding mode predicts a conformational transition between an ultra-stable closed state (in the free hormone) and an active open state (on receptor binding). Optimization of this switch within an ultra-stable SCI promises to circumvent insulin's complex global cold chain. The analog's biphasic activity, which serendipitously resembles current premixed formulations of soluble insulin and microcrystalline suspension, may be of particular utility in the developing world.
Subject(s)
Hypoglycemic Agents/chemistry , Hypoglycemic Agents/pharmacology , Insulin/analogs & derivatives , Insulin/pharmacology , Receptor, Insulin/metabolism , Amino Acid Sequence , Animals , Diabetes Mellitus, Experimental/drug therapy , Humans , Hypoglycemic Agents/therapeutic use , Insulin/genetics , Insulin/therapeutic use , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Protein Denaturation , Protein Engineering , Protein Stability , Rats , Swine , ThermodynamicsABSTRACT
Thermal degradation of insulin complicates its delivery and use. Previous efforts to engineer ultra-stable analogs were confounded by prolonged cellular signaling in vivo, of unclear safety and complicating mealtime therapy. We therefore sought an ultra-stable analog whose potency and duration of action on intravenous bolus injection in diabetic rats are indistinguishable from wild-type (WT) insulin. Here, we describe the structure, function, and stability of such an analog, a 57-residue single-chain insulin (SCI) with multiple acidic substitutions. Cell-based studies revealed native-like signaling properties with negligible mitogenic activity. Its crystal structure, determined as a novel zinc-free hexamer at 2.8 Å, revealed a native insulin fold with incomplete or absent electron density in the C domain; complementary NMR studies are described in the accompanying article. The stability of the analog (ΔGU 5.0(±0.1) kcal/mol at 25 °C) was greater than that of WT insulin (3.3(±0.1) kcal/mol). On gentle agitation, the SCI retained full activity for >140 days at 45 °C and >48 h at 75 °C. These findings indicate that marked resistance to thermal inactivation in vitro is compatible with native duration of activity in vivo Further, whereas WT insulin forms large and heterogeneous aggregates above the standard 0.6 mm pharmaceutical strength, perturbing the pharmacokinetic properties of concentrated formulations, dynamic light scattering, and size-exclusion chromatography revealed only limited SCI self-assembly and aggregation in the concentration range 1-7 mm Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain.
Subject(s)
Hypoglycemic Agents/chemistry , Insulin/analogs & derivatives , Amino Acid Sequence , Amino Acid Substitution , Animals , Humans , Hypoglycemic Agents/metabolism , Insulin/genetics , Insulin/metabolism , Models, Molecular , Protein Aggregates , Protein Conformation , Protein Engineering , Protein Multimerization , Protein Stability , Solubility , Swine , TemperatureABSTRACT
Insulin, a protein critical for metabolic homeostasis, provides a classical model for protein design with application to human health. Recent efforts to improve its pharmaceutical formulation demonstrated that iodination of a conserved tyrosine (TyrB26) enhances key properties of a rapid-acting clinical analog. Moreover, the broad utility of halogens in medicinal chemistry has motivated the use of hybrid quantum- and molecular-mechanical methods to study proteins. Here, we (i) undertook quantitative atomistic simulations of 3-[iodo-TyrB26]insulin to predict its structural features, and (ii) tested these predictions by X-ray crystallography. Using an electrostatic model of the modified aromatic ring based on quantum chemistry, the calculations suggested that the analog, as a dimer and hexamer, exhibits subtle differences in aromatic-aromatic interactions at the dimer interface. Aromatic rings (TyrB16, PheB24, PheB25, 3-I-TyrB26, and their symmetry-related mates) at this interface adjust to enable packing of the hydrophobic iodine atoms within the core of each monomer. Strikingly, these features were observed in the crystal structure of a 3-[iodo-TyrB26]insulin analog (determined as an R6 zinc hexamer). Given that residues B24-B30 detach from the core on receptor binding, the environment of 3-I-TyrB26 in a receptor complex must differ from that in the free hormone. Based on the recent structure of a "micro-receptor" complex, we predict that 3-I-TyrB26 engages the receptor via directional halogen bonding and halogen-directed hydrogen bonding as follows: favorable electrostatic interactions exploiting, respectively, the halogen's electron-deficient σ-hole and electronegative equatorial band. Inspired by quantum chemistry and molecular dynamics, such "halogen engineering" promises to extend principles of medicinal chemistry to proteins.
Subject(s)
Chemistry, Pharmaceutical , Insulin/analogs & derivatives , Receptor, Insulin/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Halogens , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Insulin/chemistry , Insulin/genetics , Insulin/metabolism , Models, Molecular , Phenylalanine/chemistry , Phenylalanine/genetics , Phenylalanine/metabolism , Protein Binding , Receptor, Insulin/chemistry , Structure-Activity Relationship , Tyrosine/chemistry , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent ("default") cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity.
Subject(s)
Disorder of Sex Development, 46,XY/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Sertoli Cells/metabolism , Sex-Determining Region Y Protein/metabolism , Ubiquitination , Animals , Disorder of Sex Development, 46,XY/genetics , Female , Humans , Male , Proteasome Endopeptidase Complex/genetics , Rats , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Sex-Determining Region Y Protein/geneticsABSTRACT
Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. We investigated the role of Tyr(B26), a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, non-aromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakest-binding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the Glu(B26) analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although Tyr(B26) is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stability and self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences.
Subject(s)
Insulin/chemistry , Amino Acid Sequence , Conserved Sequence , Humans , Hydrogen Bonding , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Stability , Protein Structure, Secondary , Protein Subunits/chemistry , Receptor, Insulin/chemistry , Structure-Activity Relationship , Tyrosine/chemistryABSTRACT
Crystallographic studies of insulin bound to fragments of the insulin receptor have recently defined the topography of the primary hormone-receptor interface. Here, we have investigated the role of Phe(B24), an invariant aromatic anchor at this interface and site of a human mutation causing diabetes mellitus. An extensive set of B24 substitutions has been constructed and tested for effects on receptor binding. Although aromaticity has long been considered a key requirement at this position, Met(B24) was found to confer essentially native affinity and bioactivity. Molecular modeling suggests that this linear side chain can serve as an alternative hydrophobic anchor at the hormone-receptor interface. These findings motivated further substitution of Phe(B24) by cyclohexanylalanine (Cha), which contains a nonplanar aliphatic ring. Contrary to expectations, [Cha(B24)]insulin likewise exhibited high activity. Furthermore, its resistance to fibrillation and the rapid rate of hexamer disassembly, properties of potential therapeutic advantage, were enhanced. The crystal structure of the Cha(B24) analog, determined as an R6 zinc-stabilized hexamer at a resolution of 1.5 Å, closely resembles that of wild-type insulin. The nonplanar aliphatic ring exhibits two chair conformations with partial occupancies, each recapitulating the role of Phe(B24) at the dimer interface. Together, these studies have defined structural requirements of an anchor residue within the B24-binding pocket of the insulin receptor; similar molecular principles are likely to pertain to insulin-related growth factors. Our results highlight in particular the utility of nonaromatic side chains as probes of the B24 pocket and suggest that the nonstandard Cha side chain may have therapeutic utility.
Subject(s)
Drug Design , Insulin/chemistry , Insulin/metabolism , Phenylalanine , Receptor, Insulin/metabolism , Amino Acid Sequence , Animals , Conserved Sequence , Humans , Hydrophobic and Hydrophilic Interactions , Insulin/genetics , Kinetics , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Folding , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Receptor, IGF Type 1/chemistry , Receptor, IGF Type 1/metabolism , Receptor, Insulin/chemistryABSTRACT
Human testis determination is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in SRY cause 46 XY gonadal dysgenesis with female somatic phenotype (Swyer syndrome) and confer a high risk of malignancy (gonadoblastoma). Such mutations cluster in the SRY high mobility group (HMG) box, a conserved motif of specific DNA binding and bending. To explore structure-function relationships, we constructed all possible substitutions at a site of clinical mutation (W70L). Our studies thus focused on a core aromatic residue (position 15 of the consensus HMG box) that is invariant among SRY-related HMG box transcription factors (the SOX family) and conserved as aromatic (Phe or Tyr) among other sequence-specific boxes. In a yeast one-hybrid system sensitive to specific SRY-DNA binding, the variant domains exhibited reduced (Phe and Tyr) or absent activity (the remaining 17 substitutions). Representative nonpolar variants with partial or absent activity (Tyr, Phe, Leu, and Ala in order of decreasing side-chain volume) were chosen for study in vitro and in mammalian cell culture. The clinical mutation (Leu) was found to markedly impair multiple biochemical and cellular activities as respectively probed through the following: (i) in vitro assays of specific DNA binding and protein stability, and (ii) cell culture-based assays of proteosomal degradation, nuclear import, enhancer DNA occupancy, and SRY-dependent transcriptional activation. Surprisingly, however, DNA bending is robust to this or the related Ala substitution that profoundly impairs box stability. Together, our findings demonstrate that the folding, trafficking, and gene-regulatory function of SRY requires an invariant aromatic "buttress" beneath its specific DNA-bending surface.
Subject(s)
DNA-Binding Proteins/chemistry , DNA/chemistry , Nucleic Acid Conformation , Protein Structure, Tertiary , Sex-Determining Region Y Protein/chemistry , Amino Acid Sequence , Amino Acids, Aromatic/chemistry , Amino Acids, Aromatic/genetics , Amino Acids, Aromatic/metabolism , Animals , Binding Sites/genetics , Blotting, Western , Cell Line , Circular Dichroism , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fluorescence Resonance Energy Transfer , Humans , Male , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding/genetics , Protein Folding , Sequence Homology, Amino Acid , Sex-Determining Region Y Protein/genetics , Sex-Determining Region Y Protein/metabolism , Structure-Activity Relationship , Thermodynamics , Transcriptional ActivationABSTRACT
Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in ß cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 ß-turn, coupling reorientation of Phe(B24) to a 60° rotation of the B25-B28 ß-strand away from the hormone core to lie antiparallel to the receptor's L1-ß2 sheet. Opening of this hinge enables conserved nonpolar side chains (Ile(A2), Val(A3), Val(B12), Phe(B24), and Phe(B25)) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.
Subject(s)
Insulin/metabolism , Receptor, Insulin/metabolism , Crystallography, X-Ray , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein BindingABSTRACT
Insulin provides a model for the therapeutic application of protein engineering. A paradigm in molecular pharmacology was defined by design of rapid-acting insulin analogs for the prandial control of glycemia. Such analogs, a cornerstone of current diabetes regimens, exhibit accelerated subcutaneous absorption due to more rapid disassembly of oligomeric species relative to wild-type insulin. This strategy is limited by a molecular trade-off between accelerated disassembly and enhanced susceptibility to degradation. Here, we demonstrate that this trade-off may be circumvented by nonstandard mutagenesis. Our studies employed Lys(B28), Pro(B29)-insulin ("lispro") as a model prandial analog that is less thermodynamically stable and more susceptible to fibrillation than is wild-type insulin. We have discovered that substitution of an invariant tyrosine adjoining the engineered sites in lispro (Tyr(B26)) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔGu 0.5 ± 0.2 kcal/mol), (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C was prolonged by 4-fold), (iii) enhances affinity for the insulin receptor (1.5 ± 0.1-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. (1)H NMR studies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-Tyr(B26) modification stabilizes an oligomeric form of insulin pertinent to pharmaceutical formulation (the R6 zinc hexamer) but preserves rapid disassembly of the oligomeric form pertinent to subcutaneous absorption (T6 hexamer). By exploiting this allosteric switch, 3-iodo-Tyr(B26)-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages.
Subject(s)
Insulin/analogs & derivatives , Mutagenesis , Animals , Biophysical Phenomena , Circular Dichroism , Insulin/chemistry , Insulin/genetics , Insulin/pharmacokinetics , Male , Nuclear Magnetic Resonance, Biomolecular , Protein Engineering , Rats , Rats, Inbred Lew , Receptor, Insulin/metabolism , Spectrophotometry, UltravioletABSTRACT
Human testis determination is initiated by SRY (sex determining region on Y chromosome). Mutations in SRY cause gonadal dysgenesis with female somatic phenotype. Two subtle variants (V60L and I90M in the high-mobility group box) define inherited alleles shared by an XY sterile daughter and fertile father. Whereas specific DNA binding and bending are unaffected in a rat embryonic pre-Sertoli cell line, the variants exhibited selective defects in nucleocytoplasmic shuttling due to impaired nuclear import (V60L; mediated by Exportin-4) or export (I90M; mediated by chromosome region maintenance 1). Decreased shuttling limits nuclear accumulation of phosphorylated (activated) SRY, in turn reducing occupancy of DNA sites regulating Sertoli-cell differentiation [the testis-specific SRY-box 9 (Sox9) enhancer]. Despite distinct patterns of biochemical and cell-biological perturbations, V60L and I90M each attenuated Sox9 expression in transient transfection assays by twofold. Such attenuation was also observed in studies of V60A, a clinical variant associated with ovotestes and hence ambiguity between divergent cell fates. This shared twofold threshold is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9. Our results demonstrate that nucleocytoplasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and import-dependent phosphorylation are compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects in the nucleocytoplasmic shuttling of SRY suggests that its transcriptional activity lies near the edge of developmental ambiguity.
Subject(s)
Cell Nucleus/metabolism , Gene Expression Regulation, Developmental/genetics , Gonadal Dysgenesis, 46,XY/genetics , SOX9 Transcription Factor/metabolism , Sex-Determining Region Y Protein/genetics , Sex-Determining Region Y Protein/metabolism , Testis/growth & development , Active Transport, Cell Nucleus/physiology , Blotting, Western , Cell Differentiation/physiology , Cell Line , Chromatin Immunoprecipitation , Female , Humans , Immunohistochemistry , Male , Models, Molecular , Protein Conformation , SOX9 Transcription Factor/genetics , Sertoli Cells/cytologyABSTRACT
The male program of therian mammals is determined by Sry, a transcription factor encoded by the Y chromosome. Specific DNA binding is mediated by a high mobility group (HMG) box. Expression of Sry in the gonadal ridge activates a Sox9-dependent gene regulatory network leading to testis formation. A subset of Sry alleles in superfamily Muroidea (order Rodentia) is remarkable for insertion of an unstable DNA microsatellite, most commonly encoding (as in mice) a CAG repeat-associated glutamine-rich domain. We provide evidence, based on an embryonic pre-Sertoli cell line, that this domain functions at a threshold length as a genetic capacitor to facilitate accumulation of variation elsewhere in the protein, including the HMG box. The glutamine-rich domain compensates for otherwise deleterious substitutions in the box and absence of nonbox phosphorylation sites to ensure occupancy of DNA target sites. Such compensation enables activation of a male transcriptional program despite perturbations to the box. Whereas human SRY requires nucleocytoplasmic shuttling and coupled phosphorylation, mouse Sry contains a defective nuclear export signal analogous to a variant human SRY associated with inherited sex reversal. We propose that the rodent glutamine-rich domain has (i) fostered accumulation of cryptic intragenic variation and (ii) enabled unmasking of such variation due to DNA replicative slippage. This model highlights genomic contingency as a source of protein novelty at the edge of developmental ambiguity and may underlie emergence of non-Sry-dependent sex determination in the radiation of Muroidea.
Subject(s)
Biological Evolution , DNA/metabolism , Gene Expression Regulation/physiology , Gene Regulatory Networks/genetics , Rodentia/genetics , Sex Determination Processes/genetics , Sex-Determining Region Y Protein/genetics , Animals , Blotting, Western , Cell Line , Chromatin Immunoprecipitation , Circular Dichroism , DNA/genetics , Fluorescence Resonance Energy Transfer , Gene Expression Regulation/genetics , HMG-Box Domains/genetics , Humans , Immunohistochemistry , Male , Mice , Microsatellite Repeats/genetics , Protein Structure, Tertiary/genetics , Rats , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sex-Determining Region Y Protein/chemistry , Sex-Determining Region Y Protein/metabolism , Spectrometry, Fluorescence , Trinucleotide Repeats/geneticsABSTRACT
Efficient total synthesis of insulin is important to enable the application of medicinal chemistry to the optimization of the properties of this important protein molecule. Recently we described "ester insulin"--a novel form of insulin in which the function of the 35 residue C-peptide of proinsulin is replaced by a single covalent bond--as a key intermediate for the efficient total synthesis of insulin. Here we describe a fully convergent synthetic route to the ester insulin molecule from three unprotected peptide segments of approximately equal size. The synthetic ester insulin polypeptide chain folded much more rapidly than proinsulin, and at physiological pH. Both the D-protein and L-protein enantiomers of monomeric DKP ester insulin (i.e., [Asp(B10), Lys(B28), Pro(B29)]ester insulin) were prepared by total chemical synthesis. The atomic structure of the synthetic ester insulin molecule was determined by racemic protein X-ray crystallography to a resolution of 1.6 Å. Diffraction quality crystals were readily obtained from the racemic mixture of {D-DKP ester insulin + L-DKP ester insulin}, whereas crystals were not obtained from the L-ester insulin alone even after extensive trials. Both the D-protein and L-protein enantiomers of monomeric DKP ester insulin were assayed for receptor binding and in diabetic rats, before and after conversion by saponification to the corresponding DKP insulin enantiomers. L-DKP ester insulin bound weakly to the insulin receptor, while synthetic L-DKP insulin derived from the L-DKP ester insulin intermediate was fully active in binding to the insulin receptor. The D- and L-DKP ester insulins and D-DKP insulin were inactive in lowering blood glucose in diabetic rats, while synthetic L-DKP insulin was fully active in this biological assay. The structural basis of the lack of biological activity of ester insulin is discussed.
Subject(s)
Crystallography, X-Ray , Esters/chemistry , Insulin/chemical synthesis , Proteins/chemistry , Amino Acid Sequence , Insulin/chemistry , Models, Molecular , Molecular Sequence DataABSTRACT
The primary hormone-binding surface of the insulin receptor spans one face of the N-terminal ß-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 ß-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 ß-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 ß-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.
Subject(s)
Protein-Tyrosine Kinases/chemistry , Receptor, Insulin/metabolism , Amino Acyl-tRNA Synthetases/metabolism , Bacillus/metabolism , Binding Sites , Cell Membrane/metabolism , Cross-Linking Reagents/pharmacology , Crystallography, X-Ray/methods , Escherichia coli/metabolism , Hormones/metabolism , Models, Biological , Molecular Conformation , Mutagenesis , Mutation , Protein Structure, Secondary , Protein Structure, Tertiary , Signal Transduction , Tyrosine/chemistryABSTRACT
Insulin is susceptible to thermal fibrillation, a misfolding process that leads to nonnative cross-ß 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-ß 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.
Subject(s)
Hot Temperature , Hypoglycemic Agents/chemistry , Insulin Infusion Systems , Proinsulin/chemistry , Animals , Blood Glucose/drug effects , Blood Glucose/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Chemistry, Pharmaceutical , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/drug therapy , Dose-Response Relationship, Drug , Drug Stability , Hypoglycemic Agents/administration & dosage , Hypoglycemic Agents/pharmacokinetics , Male , Models, Molecular , Proinsulin/administration & dosage , Proinsulin/pharmacokinetics , Protein Conformation , Protein Denaturation , Protein Folding , Protein Stability , Rats , Rats, Inbred Lew , Receptor, IGF Type 1/metabolism , Receptor, Insulin/metabolismABSTRACT
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.
Subject(s)
Amino Acid Substitution , DNA/chemistry , Gonadal Dysgenesis, 46,XY , Mutation, Missense , Nucleic Acid Conformation , Protein Folding , Sex-Determining Region Y Protein/chemistry , Animals , Cell Line , DNA/metabolism , Humans , Magnetic Resonance Spectroscopy , Male , Protein Structure, Tertiary , Rodentia , SOX9 Transcription Factor/chemistry , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Sex-Determining Region Y Protein/genetics , Sex-Determining Region Y Protein/metabolism , Structure-Activity Relationship , Transcription, Genetic/geneticsABSTRACT
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.