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1.
Ming Zhao Zhu-li Wan Linda Whittaker Bin Xu Nelson B. Phillips Panayotis G. Katsoyannis Faramarz Ismail-Beigi Jonathan Whittaker Michael A. Weiss 《The Journal of biological chemistry》2009,284(46):32178-32187
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. 相似文献
2.
Vijay Pandyarajan Nelson B. Phillips Gabriela P. Cox Yanwu Yang Jonathan Whittaker Faramarz Ismail-Beigi Michael A. Weiss 《The Journal of biological chemistry》2014,289(34):23367-23381
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 LysB28, ProB29-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 (TyrB26) 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. 1H NMR studies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-TyrB26 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-TyrB26-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages. 相似文献
3.
Wan Z Xu B Huang K Chu YC Li B Nakagawa SH Qu Y Hu SQ Katsoyannis PG Weiss MA 《Biochemistry》2004,43(51):16119-16133
The receptor-binding surface of insulin is broadly conserved, reflecting its evolutionary optimization. Neighboring positions nevertheless offer an opportunity to enhance activity, through either transmitted structural changes or introduction of novel contacts. Nonconserved residue A8 is of particular interest as Thr(A8) --> His substitution (a species variant in birds and fish) augments the potency of human insulin. Diverse A8 substitutions are well tolerated, suggesting that the hormone-receptor interface is not tightly packed at this site. To resolve whether enhanced activity is directly or indirectly mediated by the variant A8 side chain, we have determined the crystal structure of His(A8)-insulin and investigated the photo-cross-linking properties of an A8 analogue containing p-azidophenylalanine. The structure, characterized as a T(3)R(3)(f) zinc hexamer at 1.8 A resolution, is essentially identical to that of native insulin. The photoactivatable analogue exhibits efficient cross-linking to the insulin receptor. The site of cross-linking lies within a 14 kDa C-terminal domain of the alpha-subunit. This contact, to our knowledge the first to be demonstrated from the A chain, is inconsistent with a recent model of the hormone-receptor complex derived from electron microscopy. Optimizing the binding interaction of a nonconserved side chain on the surface of insulin may thus enhance its activity. 相似文献
4.
Ming Liu Qing-xin Hua Shi-Quan Hu Wenhua Jia Yanwu Yang Sunil Evan Saith Jonathan Whittaker Peter Arvan Michael A. Weiss 《The Journal of biological chemistry》2010,285(40):30989-31001
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. 相似文献
5.
Michal Avital-Shmilovici Jonathan Whittaker Michael A. Weiss Stephen B. H. Kent 《The Journal of biological chemistry》2014,289(34):23683-23692
Misfolding of proinsulin variants in the pancreatic β-cell, a monogenic cause of permanent neonatal-onset diabetes mellitus, provides a model for a disease of protein toxicity. A hot spot for such clinical mutations is found at position B8, conserved as glycine within the vertebrate insulin superfamily. We set out to investigate the molecular basis of the aberrant properties of a proinsulin clinical mutant in which residue GlyB8 is replaced by SerB8. Modular total chemical synthesis was used to prepare the wild-type [GlyB8]proinsulin molecule and three analogs: [d-AlaB8]proinsulin, [l-AlaB8]proinsulin, and the clinical mutant [l-SerB8]proinsulin. The protein diastereomer [d-AlaB8]proinsulin produced higher folding yields at all pH values compared with the wild-type proinsulin and the other two analogs, but showed only very weak binding to the insulin receptor. The clinical mutant [l-SerB8]proinsulin impaired folding at pH 7.5 even in the presence of protein-disulfide isomerase. Surprisingly, although [l-SerB8]proinsulin did not fold well under the physiological conditions investigated, once folded the [l-SerB8]proinsulin protein molecule bound to the insulin receptor more effectively than wild-type proinsulin. Such paradoxical gain of function (not pertinent in vivo due to impaired secretion of the mutant insulin) presumably reflects induced fit in the native mechanism of hormone-receptor engagement. This work provides insight into the molecular mechanism of a clinical mutation in the insulin gene associated with diabetes mellitus. These results dramatically illustrate the power of total protein synthesis, as enabled by modern chemical ligation methods, for the investigation of protein folding and misfolding. 相似文献
6.
Christophe Blanchetot Natalie De Jonge Aline Desmyter Nico Ongenae Erik Hofman Alex Klarenbeek Ava Sadi Anna Hultberg Anke Kretz-Rommel Silvia Spinelli Remy Loris Christian Cambillau Hans de Haard 《The Journal of biological chemistry》2016,291(26):13846-13854
Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe229 and Phe279 of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe279. Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe279. In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe279, whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe229. 相似文献
7.
Marta Del Cadia Francesca De Rienzo David A. Weston Andrew J. Thompson Maria Cristina Menziani Sarah C.R. Lummis 《Bioorganic & medicinal chemistry》2013,21(23):7523-7528
Palonosetron (Aloxi) is a potent second generation 5-HT3 receptor antagonist whose mechanism of action is not yet fully understood. Palonosetron acts at the 5-HT3 receptor binding site but recent computational studies indicated other possible sites of action in the extracellular domain. To test this hypothesis we mutated a series of residues in the 5-HT3A receptor subunit (Tyr73, Phe130, Ser163, and Asp165) and in the 5-HT3B receptor subunit (His73, Phe130, Glu170, and Tyr143) that were previously predicted by in silico docking studies to interact with palonosetron. Homomeric (5-HT3A) and heteromeric (5-HT3AB) receptors were then expressed in HEK293 cells to determine the potency of palonosetron using both fluorimetric and radioligand methods to test function and ligand binding, respectively. The data show that the substitutions have little or no effect on palonosetron inhibition of 5-HT-evoked responses or binding. In contrast, substitutions in the orthosteric binding site abolish palonosetron binding. Overall, the data support a binding site for palonosetron at the classic orthosteric binding pocket between two 5-HT3A receptor subunits but not at allosteric sites previously identified by in silico modelling and docking. 相似文献
8.
Yingkui Yang Vinod Mishra Chiquito J. Crasto Min Chen Reed Dimmitt Carroll M. Harmon 《The Journal of biological chemistry》2015,290(12):7685-7692
The ACTH receptor, known as the melanocortin-2 receptor (MC2R), plays an important role in regulating and maintaining adrenocortical function. MC2R is a subtype of the melanocortin receptor (MCR) family and has unique characteristics among MCRs. Endogenous ACTH is the only endogenous agonist for MC2R, whereas the melanocortin peptides α-, β-, and γ-melanocyte-stimulating hormone and ACTH are full agonists for all other MCRs. In this study, we examined the molecular basis of MC2R responsible for ligand selectivity using ACTH analogs and MC2R mutagenesis. Our results indicate that substitution of Phe7 with d-Phe or d-naphthylalanine (d-Nal(2′)) in ACTH(1–24) caused a significant decrease in ligand binding affinity and potency. Substitution of Phe7 with d-Nal(2′) in ACTH(1–24) did not switch the ligand from agonist to antagonist at MC2R, which was observed in MC3R and MC4R. Substitution of Phe7 with d-Phe7 in ACTH(1–17) resulted in the loss of ligand binding and activity. Molecular analysis of MC2R indicated that only mutation of the third transmembrane domain of MC2R resulted in a decrease in d-Phe ACTH binding affinity and potency. Our results suggest that Phe7 in ACTH plays an important role in ligand selectivity and that the third transmembrane domain of MC2R is crucial for ACTH selectivity and potency. 相似文献
9.
Masashi Matsue Eiji Majima Eiji Ichishima 《Bioscience, biotechnology, and biochemistry》2013,77(10):2485-2490
The specificity of highly purified alkaline proteinase B (EC 3.4.21.14) from thermophilic Streptomyces rectus var. proteolyticus was investigated with an oxidized insulin B chain. Hydrolysis of the oxidized insulin B chain in a 4-hr incubation was observed mainly at three peptide bonds (Phe24-Phe25, Leu15-Tyr16 and Leu11-Val12) and additionally at six others (Leu6-CySO3H7, Gln4-His5, Leu17-Val18, His5-Leu6, Glu13-Ala14, Asn3-Gln4).Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr4-Ile5 bond. Hydrolysis of proangiotension (formerly designated angiotensin I) was observed at the Tyr4-Ile5 and Phe8-His9 bonds. 相似文献
10.
《Journal of receptor and signal transduction research》2013,33(1):69-81
AbstractMelanin‐concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food‐intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH‐R1 and MCH‐R2, are thought to mediate mainly the central effects of MCH, the MCH‐R on pigment cells has not yet been identified, although in some studies MCH‐R1 was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure‐activity study in which 12 MCH peptides were tested on rat MCH‐R1 and mouse B16 melanoma cell MCH‐R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK‐293 cells expressing rat MCH‐R1 (SLC‐1), the radioligand was [125I]–[Tyr13]‐MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH‐R, the analog [125I]–[D‐Phe13, Tyr19]‐MCH served as radioligand. The bioassay used for MCH‐R1 was intracellular Ca2+ mobilization quantified with the FLIPR instrument, whereas for B16 MCH‐R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrase of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side‐chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N‐terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5‐ to 10‐fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH‐R1 and B16 MCH‐R was however observed with modifications at position 13 of MCH: whereas L‐Phe13 in [Phe13, Tyr19]‐MCH was well tolerated by both MCH‐R1 and B16 MCH‐R, change of configuration to D‐Phe13 in [D‐Phe13, Tyr19]‐MCH or [D‐Phe13]‐MCH led to a complete loss of biological activity and to a 5‐ to 10‐fold lower binding activity with MCH‐R1. By contrast, the D‐Phe13 residue increased the affinity of [D‐Phe13, Tyr19]‐MCH to B16 MCH‐R about 10‐fold and elicited MAP kinase activation as observed with [Phe13, Tyr19]‐MCH or MCH. These data demonstrate that ligand recognition by B16 MCH‐R differs from that of MCH‐R1 in several respects, indicating that the B16 MCH‐R represents an MCH‐R subtype different from MCH‐R1. 相似文献
11.
The P-glycoprotein (P-gp) drug pump (ABCB1) has two transmembrane domains and two nucleotide-binding domains (NBDs). Coupling of the drug-binding sites in the transmembrane domains to the NBDs occurs through interaction of the intracellular helices (IHs) with residues in the NBDs (IH1/IH4/NBD1 and IH2/IH3/NBD2). We showed previously that cross-linking of cysteines in IH3 and IH1 with a short cross-linker mimicked drug binding as it activated P-gp ATPase activity. Here we show that residue A259C(IH2) could be directly cross-linked to W803C(IH3). Cross-linking was inhibited by the presence of ATP and adenosine 5′-(β,γ-imino)triphosphate but not by ADP. Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol. Because these residues are close to the ball-and-socket joint A266C(IH2)/Phe1086(NBD2), we mutated the adjacent Tyr1087(NBD2) close to IH3. Mutants Y1087A and Y1087L, but not Y1087F, were misprocessed, and all inhibited ATPase activity. Mutation of hydrophobic residues (F793A, L797A, L814A, and L818A) flanking IH3 also inhibited maturation. The results suggest that these residues, together with Trp803 and Phe804, form a large hydrophobic pocket. The results show that there is an important hydrophobic network at the IH2/IH3/NBD2 transmission interface that is critical for folding and activity of P-gp. 相似文献
12.
Antolíková E Žáková L Turkenburg JP Watson CJ Hančlová I Šanda M Cooper A Kraus T Brzozowski AM Jiráček J 《The Journal of biological chemistry》2011,286(42):36968-36977
Apart from its role in insulin receptor (IR) activation, the C terminus of the B-chain of insulin is also responsible for the formation of insulin dimers. The dimerization of insulin plays an important role in the endogenous delivery of the hormone and in the administration of insulin to patients. Here, we investigated insulin analogues with selective N-methylations of peptide bond amides at positions B24, B25, or B26 to delineate their structural and functional contribution to the dimer interface. All N-methylated analogues showed impaired binding affinities to IR, which suggests a direct IR-interacting role for the respective amide hydrogens. The dimerization capabilities of analogues were investigated by isothermal microcalorimetry. Selective N-methylations of B24, B25, or B26 amides resulted in reduced dimerization abilities compared with native insulin (K(d) = 8.8 μM). Interestingly, although the N-methylation in [NMeTyrB26]-insulin or [NMePheB24]-insulin resulted in K(d) values of 142 and 587 μM, respectively, the [NMePheB25]-insulin did not form dimers even at high concentrations. This effect may be attributed to the loss of intramolecular hydrogen bonding between NHB25 and COA19, which connects the B-chain β-strand to the core of the molecule. The release of the B-chain β-strand from this hydrogen bond lock may result in its higher mobility, thereby shifting solution equilibrium toward the monomeric state of the hormone. The study was complemented by analyses of two novel analogue crystal structures. All examined analogues crystallized only in the most stable R(6) form of insulin oligomers (even if the dimer interface was totally disrupted), confirming the role of R(6)-specific intra/intermolecular interactions for hexamer stability. 相似文献
13.
The crystal structure of an inactive chiral analogue of insulin containing nonstandard substitution allo-Ile(A2) is described at 2.0 A resolution. In native insulin, the invariant Ile(A2) side chain anchors the N-terminal alpha-helix of the A-chain to the hydrophobic core. The structure of the variant protein was determined by molecular replacement as a T(3)R(3) zinc hexamer. Whereas respective T- and R-state main-chain structures are similar to those of native insulin (main-chain root-mean-square deviations (RMSD) of 0.45 and 0.54 A, respectively), differences in core packing are observed near the variant side chain. The R-state core resembles that of the native R-state with a local inversion of A2 orientation (core side chain RMSD 0.75 A excluding A2); in the T-state, allo-Ile(A2) exhibits an altered conformation in association with the reorganization of the surrounding side chains (RMSD 0.98 A). Surprisingly, the core of the R-state is similar to that observed in solution nuclear magnetic resonance (NMR) studies of an engineered T-like monomer containing the same chiral substitution (allo-Ile(A2)-DKP-insulin; Xu, B., Hua, Q. X., Nakagawa, S. H., Jia, W., Chu, Y. C., Katsoyannis, P. G., and Weiss, M. A. (2002) J. Mol. Biol. 316, 435-441). Simulation of NOESY spectra based on crystallographic protomers enables the analysis of similarities and differences in solution. The different responses of the T- and R-state cores to chiral perturbation illustrates both their intrinsic plasticity and constraints imposed by hexamer assembly. Although variant T- and R-protomers retain nativelike protein surfaces, the receptor-binding activity of allo-Ile(A2)-insulin is low (2% relative to native insulin). This seeming paradox suggests that insulin undergoes a change in conformation to expose Ile(A2) at the hormone-receptor interface. 相似文献
14.
Xavier Iturrioz Romain Gerbier Vincent Leroux Rodrigo Alvear-Perez Bernard Maigret Catherine Llorens-Cortes 《The Journal of biological chemistry》2010,285(42):32627-32637
Apelin is the endogenous ligand of the orphan seven-transmembrane domain (TM) G protein-coupled receptor APJ. Apelin is involved in the regulation of body fluid homeostasis and cardiovascular functions. We previously showed the importance of the C-terminal Phe of apelin 17 (K17F) in the hypotensive activity of this peptide. Here, we show either by deleting the Phe residue (K16P) or by substituting it by an Ala (K17A), that it plays a crucial role in apelin receptor internalization but not in apelin binding or in Gαi-protein coupling. Then we built a homology three-dimensional model of the human apelin receptor using the cholecystokinin receptor-1 model as a template, and we subsequently docked K17F into the binding site. We visualized a hydrophobic cavity at the bottom of the binding pocket in which the C-terminal Phe of K17F was embedded by Trp152 in TMIV and Trp259 and Phe255 in TMVI. Using molecular modeling and site-directed mutagenesis studies, we further showed that Phe255 and Trp259 are key residues in triggering receptor internalization without playing a role in apelin binding or in Gαi-protein coupling. These findings bring new insights into apelin receptor activation and show that Phe255 and Trp259, by interacting with the C-terminal Phe of the pyroglutamyl form of apelin 13 (pE13F) or K17F, are crucial for apelin receptor internalization. 相似文献
15.
Walrati Limapichat Henry A. Lester Dennis A. Dougherty 《The Journal of biological chemistry》2010,285(12):8976-8984
The functions of two conserved residues, Phe135 and Pro136, located at the apex of the Cys loop of the nicotinic acetylcholine receptor are investigated. Both residues were substituted with natural and unnatural amino acids, focusing on the role of aromaticity at Phe135, backbone conformation at Pro136, side chain polarity and volume, and the specific interaction between the aromatic side chain and the proline. NMR spectroscopy studies of model peptides containing proline and unnatural proline analogues following a Phe show a consistent increase in the population of the cis conformer relative to peptides lacking the Phe. In the receptor, a strong interaction between the Phe and Pro residues is evident, as is a strong preference for aromaticity and hydrophobicity at the Phe site. A similar influence of hydrophobicity is observed at the proline site. In addition, across a simple homologous series of proline analogues, the results reveal a correlation between receptor function and cis bias at the proline backbone. This could suggest a significant role for the cis proline conformer at this site in receptor function. 相似文献
16.
Semisynthetic analogues of insulin. The use of N-substituted derivatives of methionine as acid-stable protecting groups 总被引:2,自引:2,他引:0
1. We describe the use of benzyloxycarbonylmethionine and ethoxycarbonylmethionine for the selective protection of the amino groups of glycine-A1 and lysine-B29 of pig insulin. We have used the Edman method to remove residues from the N-terminal and of the B-chain of the NA1NB29-di-protected derivatives. The benzyloxycarbonyl group shows slight but noticeable lability in the acid-cleavage step, but the ethoxycarbonyl group remained intact even after five cycles of degradation. 2. We have prepared the following truncated forms of insulin via the di(ethoxycarbonylmethionyl) derivative: des-PheB1-insulin;des-(PheB1-ValB2)-insulin; des-(PheB1-ValB2-AsnB3)-insulin;des- (PheB1-ValB2-AsnB3-GlnB4)-insulin; des-(PheB1-ValB2-AsnB3 -GlnB4-HisB5)-insulin. 3. Insulin was re-synthesized from the di-protected des-PheB1-insulin by reaction with an active ester of t-butoxycarbonyl-l-phenylalanine. The product after deprotection crystallized, and the immunoreactivity of the crystalline material was identical with that of the native protein. 4. We have prepared the following analogues of insulin in a similar manner: [l-AlaB1]insulin; [l-ValB1]insulin; [l-TyrB1]insulin; [m-F-l-PheB1]insulin; [o-F-l-PheB1]-insulin; [o-F-l-PheB2]des-PheB1-insulin. All had between 34 and 62% of the activity of insulin in the fat-cell test. 5. We have also investigated the use of the benzyol, toluene-p-sulphonyl, p-nitrobenzyloxycarbonyl and 2,4-dinitrophenyl groups for the N-protection of the methionine active esters. Each should have had some particular advantage over the benzyloxycarbonyl and ethoxycarbonyl groups, but all proved in practice to have disadvantages that more than outweighed anything in their favour. 相似文献
17.
Qing-xin Hua Bin Xu Kun Huang Shi-Quan Hu Satoe Nakagawa Wenhua Jia Shuhua Wang Jonathan Whittaker Panayotis G. Katsoyannis Michael A. Weiss 《The Journal of biological chemistry》2009,284(21):14586-14596
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.How insulin binds to the insulin receptor
(IR)2 is not well
understood despite decades of investigation. The hormone is a globular protein
containing two chains, A (21 residues) and B (30 residues)
(Fig. 1A). In
pancreatic β-cells, insulin is stored as Zn2+-stabilized
hexamers (Fig. 1B),
which form microcrystal-line arrays within specialized secretory granules
(1). The hexamers dissociate
upon secretion into the portal circulation, enabling the hormone to function
as a zinc-free monomer. The monomer is proposed to undergo a change in
conformation upon receptor binding
(2). In this study, we
investigated a site of conformational change in the B-chain
(PheB24) (arrow in Fig.
1A). In classical crystal structures, this invariant
aromatic side chain (tawny in Fig.
1B) anchors an antiparallel β-sheet at the dimer
interface (blue in Fig.
1C). Total chemical synthesis is exploited to enable
comparison of corresponding d- and l-amino acid
substitutions at this site, an approach designated “chiral
mutagenesis”
(3-5).
In the accompanying article, the consequences of this conformational change
are investigated by photomapping of the receptor-binding surface
(6). Together, these studies
redefine the interrelation of structure and activity in a protein central to
the hormonal control of metabolism.Open in a separate windowFIGURE 1.Sequence and structure of insulin. A, sequences of the
B-chain (upper) and A-chain (lower) with disulfide bridges
as indicated. The arrow indicates invariant PheB24. The
B24-B28 β-strand is highlighted in blue. B, crystal structure of
the T6 zinc insulin hexamer (Protein Data Bank code 4INS): ribbon
model (left) and space-filling model (right). The B24-B28
β-strand is shown in blue, and the side chain of
PheB24 is highlighted in tawny. The B-chain is otherwise
dark gray; the A-chain, light gray; and zinc ions,
magenta. Also shown at the left are the side chains of
HisB10 at the axial zinc-binding sites. C, cylinder model
of the insulin dimer showing the B24-B26 antiparallel β-sheet
(blue) anchored by the B24 side chain (tawny circle). The A-
and B-chains are shown in light and dark gray, respectively.
The protomer at the left is shown in the R-state, in which the central
α-helix of the B-chain is elongated (B3-B19 in the frayed Rf
protomer of T3Rf3 hexamers and B1-B19 in the
R protomer of R6 hexamers). The three types of zinc insulin
hexamers share similar B24-B26 antiparallel β-sheets as conserved
dimerization elements.The structure of an insulin monomer in solution resembles a
crystallographic protomer (Fig.
2A)
(7-9).
The A-chain contains an N-terminal α-helix, non-canonical turn, and
second helix; the B-chain contains an N-terminal segment, central
α-helix, and C-terminal β-strand. The β-strand is maintained
in an isolated monomer wherein the side chain of PheB24
(tawny in Fig.
2A), packing against the central α-helix of the
B-chain, provides a “plug” to seal a crevice in the hydrophobic
core (Fig. 2B).
Anomalies encountered in previous studies of insulin analogs suggest that
PheB24 functions as a conformational switch
(4,
7,
10-14).
Whereas l-amino acid substitutions at B24 generally impair activity
(even by such similar residues as l-Tyr)
(15), a seeming paradox is
posed by the enhanced activities of nonstandard analogs containing
d-amino acids (10-12).
Open in a separate windowaAffinities are given relative to wild-type insulin (100%).bLymphocytes are human, and hepatocytes are rat; CHO designates Chinese
hamster ovary.cStandard deviations are not provided in this reference.Open in a separate windowFIGURE 2.Role of PheB24 in an insulin monomer. A, shown
is a cylinder model of insulin as a T-state protomer. The C-terminal B-chain
β-strand is shown in blue, and the PheB24 side chain
is shown in tawny. The black portion of the N-terminal
A-chain α-helix (labeled buried) indicates a hidden
receptor-binding surface (IleA2 and ValA3). B,
the schematic representation of insulin highlights the proposed role of the
PheB24 side chain as a plug that inserts into a crevice at the edge
of the hydrophobic core. C and D, whereas substitution of
PheB24 by l-Ala (C) would only partially fill
the B24-related crevice, its substitution by d-Ala (D)
would be associated with a marked packing defect. An alternative conformation,
designated the R-state, is observed in zinc insulin hexamers at high ionic
strength (74) and upon binding
of small cyclic alcohols (75)
but has not been observed in an insulin monomer.Why do d-amino acid substitutions at B24 enhance the activity of
insulin? In this study, we describe the structure and function of insulin
analogs containing l-Ala or d-Ala at B24
(Fig. 2, C and
D). Our studies were conducted within an engineered
monomer (DKP-insulin, an insulin analog containing three substitutions in the
B-chain: AspB10, LysB28, and ProB29) to
circumvent effects of self-assembly
(16). Whereas the inactive
l-analog retains a native-like structure, the active
d-analog exhibits segmental unfolding of the B-chain. Studies of
corresponding analogs containing either l- or
d-photoactivable probes
(l-para-azido-PheB24 or
d-para-azido-PheB24 (l- or
d-PapB24), obtained from photostable
para-amino-Phe (Pmp) precursors
(17)) demonstrate specific
cross-linking to the IR. Although photo-contacts map in each case to the
N-terminal domain of the receptor α-subunit (the L1 β-helix),
higher cross-linking efficiency is achieved by the d-probe.
Together, this and the following study
(6) provide evidence that
insulin deploys a detachable arm that inserts between domains of the IR.Induced fit of insulin illuminates by its scope general principles at the
intersection of protein structure and cell biology. Protein evolution is
enjoined by multiple layers of biological selection. The pathway of insulin
biosynthesis, for example, successively requires (a) specific
disulfide pairing (in the endoplasmic reticulum), (b) subcellular
targeting and prohormone processing (in the trans-Golgi network),
(c) zinc-mediated protein assembly and microcrystallization (in
secretory granules), and (d) exocytosis and rapid disassembly of
insulin hexamers (in the portal circulation), in turn enabling binding of the
monomeric hormone to target tissues
(1). Each step imposes
structural constraints, which may be at odds. This study demonstrates that
stereospecific pre-detachment of a receptor-binding arm enhances biological
activity but impairs disulfide pairing and renders the hormone susceptible to
aggregation-coupled misfolding
(18). Whereas the classical
globular structure of insulin and its self-assembly prevent proteotoxicity
(3,
19), partial unfolding enables
receptor engagement. We envisage that a choreography of conformational change
has evolved as an adaptative response to the universal threat of toxic protein
misfolding. 相似文献
TABLE 1
Previous studies of insulin analogsAnalog | Affinitya | Assayb | Ref. |
---|---|---|---|
% | |||
d-PheB24-insulin | 180 | Lymphocytes | 10 |
l-AlaB24-insulin | 1 | Hepatocytes | 68 |
l-AlaB24-insulin | 3 | Lymphocytes | 69 |
d-PheB24-insulin | 140 ± 9 | Hepatocytes | 11 |
l-AlaB24-insulin | 1.0 ± 0.1 | Hepatocytes | 11 |
d-AlaB24-insulin | 150 ± 9 | Hepatocytes | 11 |
GlyB24-insulin | 78 ± 11 | Hepatocytes | 11 |
DKP-insulin | 200c | CHO cells | 12 |
d-PheB24-DKP-insulin | 180 | CHO cells | 12 |
l-AlaB24-DKP-insulin | 7 | CHO cells | 12 |
GlyB24-DKP-insulin | 50 | CHO cells | 12 |
18.
Zhaomin Li Hui Peng Li Qin Jing Qi Xiaobing Zuo Jing-Yuan Liu Jian-Ting Zhang 《The Journal of biological chemistry》2013,288(44):31447-31457
Many proteins exist and function as homodimers. Understanding the detailed mechanism driving the homodimerization is important and will impact future studies targeting the “undruggable” oncogenic protein dimers. In this study, we used 14-3-3σ as a model homodimeric protein and performed a systematic investigation of the potential roles of amino acid residues in the interface for homodimerization. Unlike other members of the conserved 14-3-3 protein family, 14-3-3σ prefers to form a homodimer with two subareas in the dimeric interface that has 180° symmetry. We found that both subareas of the dimeric interface are required to maintain full dimerization activity. Although the interfacial hydrophobic core residues Leu12 and Tyr84 play important roles in 14-3-3σ dimerization, the non-core residue Phe25 appears to be more important in controlling 14-3-3σ dimerization activity. Interestingly, a similar non-core residue (Val81) is less important than Phe25 in contributing to 14-3-3σ dimerization. Furthermore, dissociating dimeric 14-3-3σ into monomers by mutating the Leu12, Phe25, or Tyr84 dimerization residue individually diminished the function of 14-3-3σ in resisting drug-induced apoptosis and in arresting cells at G2/M phase in response to DNA-damaging treatment. Thus, dimerization appears to be required for the function of 14-3-3σ. 相似文献
19.
Schlein M Havelund S Kristensen C Dunn MF Kaarsholm NC 《Journal of molecular biology》2000,303(2):161-169
Within the class of insulin and insulin-like growth factor receptors, detailed information about the molecular recognition event at the hormone-receptor interface is limited by the absence of suitable co-crystals. We describe the use of a biologically active insulin derivative labeled with the NBD fluorophore (B29NBD-insulin) to characterize the mechanism of reversible 1:1 complex formation with a fragment of the insulin receptor ectodomain. The accompanying 40 % increase in the fluorescence quantum yield of the label provides the basis for a dynamic study of the hormone-receptor binding event. Stopped-flow fluorescence experiments show that the kinetics of complex formation are biphasic comprising a bimolecular binding event followed by a conformational change. Displacement with excess unlabeled insulin gave monophasic kinetics of dissociation. The rate data are rationalized in terms of available experiments on mutant receptors and the X-ray structure of a non-binding fragment of the receptor of the homologous insulin-like growth factor (IGF-1). 相似文献
20.
Mutational analysis of invariant valine B12 in insulin: implications for receptor binding 总被引:1,自引:0,他引:1
An invariant residue, valine B12, is part of the insulin B-chain central alpha-helix (B9-B19), and its aliphatic side chain lies at the surface of the hydrophobic core of the insulin monomer in close contact with the neighboring aromatic side chains of phenylalanines (B24 and B25) and tyrosines (B26 and B16). This surface contributes to the dimerization of insulin, maintains the active conformation of the insulin monomer, and has been suspected to be directly involved in receptor recognition. To investigate in detail the role of the B12 residue in insulin-receptor interactions, we have synthesized nine analogues bearing natural or unnatural amino acid replacements for valine B12 by chemical synthesis of modified insulin B-chains and the subsequent combination of each synthetic B-chain with natural insulin A-chain. The receptor binding potencies of the synthetic B12 analogues relative to porcine insulin were determined by use of isolated canine hepatocytes, and the following results were obtained: isoleucine, 13%; allo-isoleucine, 77%; tert-leucine, 107%; cyclopropylglycine, 43%; threonine, 5.4%; D-valine, 3.4%; alpha-amino-n-butyric acid, 14%; alanine, 1.0%; and glycine, 0.32%. Selected analogues were also analyzed by far-UV circular dichroic spectroscopy and by absorption spectroscopy of their complexes with Co(2+). Our results indicate that beta-branched aliphatic amino acids are generally tolerated at the B12 position with specific steric preferences and that the receptor binding potencies of these analogues correlate with their abilities to form dimers. Furthermore, the structure-activity relationships of valine B12 are quite similar to those of valine A3, suggesting that valine residues at both A3 and B12 contribute to the insulin-receptor interactions in a similar manner. 相似文献