Importance of aliphatic side-chain structure at positions 2 and 3 of the insulin A chain in insulin-receptor interactions.

In order to evaluate the cause of the greatly decreased receptor-binding potency of the naturally occurring mutant human insulin Insulin Wakayama ([LeuA3]insulin, 0.2% relative potency), we examined (by the semisynthesis of insulin analogues based on N alpha-PheB1,N epsilon-LysB29-bisacetyl-insulin) the importance of aliphatic side chain structure at positions A2 and A3 (Ile and Val, respectively) in directing the interaction of insulin with its receptor. Analogues bearing glycine, alanine, alpha-amino-n-butyric acid, norvaline, norleucine, valine, isoleucine, allo-isoleucine, threonine, tert-leucine, or leucine at positions A2 or A3 were assayed for their potencies in competing for the binding of 125I-labeled insulin to isolated canine hepatocytes, as were analogues bearing deletions from the A-chain amino terminus or the B-chain carboxyl terminus. Selected analogues were also analyzed by far-UV CD and absorption spectroscopy of Co2+ complexes. Our results identify that (a) Ile and Val serve well at position A2, whereas residues with other side chains (including those with straight chains, alternatively configured beta-branches, or a gamma-branch) exhibit relative receptor-binding potencies in the range 1-5%; (b) greater flexibility is allowed side-chain structure at position A3, with Ile, allo-Ile, alpha-amino-n-butyric acid, and tert-Leu exhibiting relative receptor-binding potencies in the range 11-36%; and (c) simultaneous replacements at positions A2 and A3, and deletions of the COOH-terminal domain of the insulin B chain in related analogues, yield cumulative effects. These findings are discussed with respect to a model for insulin-receptor interactions that involves a structure-orienting role for residue A2, the direct interaction of residue A3 with receptor, and multiple separately defined elements of structure and of conformational adjustment.     

Importance of the character and configuration of residues B24, B25, and B26 in insulin-receptor interactions

By use of isolated canine hepatocytes and insulin analogs prepared by trypsin-catalyzed semisynthesis, we have investigated the importance of the aromatic triplet PheB24-PheB25-TyrB26 of the COOH-terminal B-chain domain of insulin in directing the affinity of insulin-receptor interactions. Analysis of the receptor binding potencies of analogs bearing transpositions or replacements (by Tyr, D-Tyr or their corresponding 3,5-diiodo derivatives) in this region demonstrates a wide divergence in the acceptance both of configurational change (with [D-TyrB24,PheB26]insulin and [D-TyrB25,PheB26]insulin exhibiting 160 and 0.1% of the receptor binding potency of insulin, respectively) and of detailed side chain structure (with [TyrB24,PheB26]insulin and [TyrB25,PheB26]insulin exhibiting 2 and 80% of the receptor binding potency of insulin, respectively). Additional experiments addressed the solvent accessibilities of the 4 tyrosine residues of insulin and the insulin analogs at selected peptide concentrations by use of analytical radioiodination. Whereas two analogs ([TyrB25,PheB26]insulin and [D-TyrB24,PheB26]insulin) were found to undergo self aggregation, no strict correlation was found between the ability of an analog to aggregate and its potency for interaction with the insulin receptor. Related findings are discussed in terms of the interplay between side chain and main chain structure in the COOH-terminal domain of the insulin B-chain and the structural attributes of insulin that determine the affinity of insulin-receptor interactions.     

Role of the COOH-terminal B-chain domain in insulin-receptor interactions. Identification of perturbations involving the insulin mainchain

Previous studies have suggested that the COOH-terminal pentapeptide of the insulin B-chain can play a negative role in ligand-receptor interactions involving insulin analogs having amino acid replacements at position B25 (Nakagawa, S. H., and Tager, H. S. (1986) J. Biol. Chem. 261, 7332-7341). We undertook by the current investigations to identify the molecular site in insulin that induces this negative effect and to explore further the importance of conformational changes that might occur during insulin-receptor interactions. By use of semisynthetic insulin analogs containing amino acid replacements or deletions and of isolated canine hepatocytes, we show here that (a) the markedly decreased affinity of receptor for insulin analogs in which PheB25 is replaced by Ser is apparent for analogs in which up to 3 residues of the insulin B-chain have been deleted, but is progressively reversed in the corresponding des-tetrapeptide and des-pentapeptide analogs, and (b) unlike the case for deletion of TyrB26 and ThrB27, replacement of residue TyrB26 or ThrB27 has no effect to reverse the decreased affinity of full length analogs containing Ser for Phe substitutions at position B25. Additional experiments demonstrated that introduction of a cross-link between Lys epsilon B29 and Gly alpha A1 of insulin decreases the affinity of ligand-receptor interactions whether or not PheB25 is replaced by Ser. We conclude that the negative effect of the COOH-terminal B-chain domain on insulin-receptor interactions arises in greatest part from the insulin mainchain near the site of the TyrB26-ThrB27 peptide bond and that multiple conformational perturbations may be necessary to induce a high-affinity state of receptor-bound insulin.     

Implications of invariant residue LeuB6 in insulin-receptor interactions

By the chemical synthesis of modified insulin B chains and the combination of the synthetic B chains with natural insulin A chains, we have prepared insulin analogs with natural and unnatural amino acid replacements of invariant residue LeuB6. Analogs have been investigated by reference to their potencies for interaction with the insulin receptor (as assessed by competition for 125I-labeled binding to isolated canine hepatocytes) and to their abilities to undergo the structural transitions that are characteristic of insulin self-aggregation (as assessed by the spectroscopic analysis of analog complexes with cobalt). Our results identify that (a) replacement of LeuB6 by glycine has nearly the equivalent effect as deletion of residues B1-B6 in decreasing receptor binding potency of the analog to only about 0.05% of that of insulin; (b) relative to the GlyB6 derivative, replacements that increase the relative hydrophobicity of the residue B6 side chain also increase the relative receptor binding potencies of the resulting analogs; (c) negative steric effects resulting from substitutions by valine, phenylalanine, and gamma-ethylnorleucine limit the potential for enhancing potency as the result of increased hydrophobicity; and (d) two analogs with disparate potency for receptor interaction (those with alanine and gamma-ethylnorleucine at position B6, analogs exhibiting about 1 and 48% of the potency of insulin, respectively) undergo the T6----R6 structural transition in the presence of Co2+ and phenol which is typical of insulin but result in hexameric complexes with greatly reduced stability. We conclude that leucine provides a closely determined best fit at insulin position B6, and we discuss our findings in terms of insulin conformations that may apply to the receptor-bound state of the hormone.     

Systematic evaluation of the metabolic to mitogenic potency ratio for B10-substituted insulin analogues

Background

Insulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate. However, B10 is still an attractive position for amino acid substitutions given its important role in hexamer formation. The aim of this study was to investigate the relationships between the receptor binding properties as well as the metabolic and mitogenic potencies of a series of insulin analogues with different amino acid substitutions at position B10 and to identify a B10-substituted insulin analogue without an increased mitogenic to metabolic potency ratio.

Methodology/Principal Findings

A panel of ten singly-substituted B10 insulin analogues with different amino acid side chain characteristics were prepared and insulin receptor (both isoforms) and IGF-I receptor binding affinities using purified receptors, insulin receptor dissociation rates using BHK cells over-expressing the human insulin receptor, metabolic potencies by lipogenesis in isolated rat adipocytes, and mitogenic potencies using two different cell types predominantly expressing either the insulin or the IGF-I receptor were systematically investigated. Only analogues B10D and B10E with significantly increased insulin and IGF-I receptor affinities as well as decreased insulin receptor dissociation rates displayed enhanced mitogenic potencies in both cell types employed. For the remaining analogues with less pronounced changes in receptor affinities and insulin receptor dissociation rates, no apparent correlation between insulin receptor occupancy time and mitogenicity was observed.

Conclusions/Significance

Several B10-substituted insulin analogues devoid of disproportionate increases in mitogenic compared to metabolic potencies were identified. In the present study, receptor binding affinity rather than insulin receptor off-rate appears to be the major determinant of both metabolic and mitogenic potency. Our results also suggest that the increased mitogenic potency is attributable to both insulin and IGF-I receptor activation.     

Disposition of the phenylalanine B25 side chain during insulin-receptor and insulin-insulin interactions

By the semisynthesis of both full-length insulin analogues and their des-pentapeptide-(B26-B30)-alpha-carboxamide counterparts, we have examined the importance of the electronic character and bulk of the position B25 side chain both in directing insulin interaction with its receptor on isolated canine hepatocytes and in determining the ability of insulin to self-associate in solution. Analogues include those in which PheB25 was replaced by cyclohexyl-Ala; Tyr; p-nitro-, p-fluoro-, p-iodo-, or p-amino-Phe; or p-amino-Phe in which the aromatic amino function had been acylated by the acetyl, hexanoyl, decanoyl, or 1-adamantanoyl group. Our findings identify that (a) the beta-aromatic side chain at position B25 is indeed critical for high-affinity ligand-receptor interactions, (b) neither electron withdrawal from nor electron donation to the beta-aromatic ring perturbs ligand-receptor interactions in major ways, (c) considerable latitude is allowed the placement of linear or polycyclic apolar mass at the para position in p-amino-PheB25-substituted analogues with respect both to receptor binding affinity and to biological activity in vivo, and (d) para apolar mass at position B25 is readily accommodated during the self-association of insulin monomers, as assessed by analytical tyrosine radioiodination and spectroscopic analysis of analogue complexes with Co2+ and Co3+. These findings are discussed in terms of a model for insulin-receptor interactions at the cell membrane in which the position B25 side chain defines the edge of intermolecular contact.     

Role of the phenylalanine B25 side chain in directing insulin interaction with its receptor. Steric and conformational effects

To gain an understanding of the causes of decreased biological activity in insulins bearing amino acid substitutions at position B25 and the importance of the PheB25 side chain in directing hormone-receptor interactions, we have prepared a variety of insulin analogs and have studied both their interactions with isolated canine hepatocytes and their abilities to stimulate glucose oxidation by isolated rat adipocytes. The semisynthetic analogs fall into three structural classes: (a) analogs in which the COOH-terminal 5, 6, or 7 residues of the insulin B-chain have been deleted, but in which the COOH-terminal residue of the B-chain has been derivatized by alpha-carboxamidation; (b) analogs in which PheB25 has been replaced by unnatural aromatic or natural L-amino acids; and (c) analogs in which the COOH-terminal 5 residues of the insulin B-chain have been deleted and in which residue B25 has been replaced by selected alpha-carboxamidated amino acids. Our results showed that (a) insulin residues B26-B30 can be deleted without decrease in biological potency, whereas deletion of residues B25-B30 and B24-B30 causes a marked and cumulative decrease in potency; (b) replacement of PheB25 in insulin by Leu or Ser results in analogs with biological potency even less than that observed when residues B25-B30 are deleted; (c) the side chain bulk of naphthyl(1)-alanine or naphthyl(2)-alanine at position B25 is well tolerated during insulin interactions with receptor, whereas that of homophenylalanine is not; and (d) the decreased biological potency attending substitution of insulin PheB25 by Ala, Ser, Leu, or homophenylalanine is reversed, in part or in total, by deletion of COOH-terminal residues B26-B30. Additional experiments showed that the rate of dissociation of receptor-bound 125I-labeled insulin from isolated hepatocytes is enhanced by incubating cells with insulin or [naphthyl(2)-alanineB25]insulin, but not with analogs in which PheB25 is replaced by serine, leucine, or homophenylalanine; deletion of residues B26-B30, however, results in analogs that enhance the rate of dissociation of receptor-bound insulin in all cases studied. We conclude that (a) steric hindrance involving the COOH-terminal domain of the B chain plays a major role in directing the interaction of insulin with its receptor; (b) the initial negative effect of this domain is reversed upon the filling of a site reflecting interaction of the receptor and the beta-aromatic ring of the PheB25 side chain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Shortened insulin analogues: marked changes in biological activity resulting from replacement of TyrB26 and N-methylation of peptide bonds in the C-terminus of the B-chain

The role of three highly conserved insulin residues PheB24, PheB25, and TyrB26 was studied to better understand the subtleties of the structure-function relationship between insulin and its receptor. Ten shortened insulin analogues with modifications in the beta-strand of the B-chain were synthesized by trypsin-catalyzed coupling of des-octapeptide (B23-B30)-insulin with synthetic peptides. Insulin analogues with a single amino acid substitution in the position B26 and/or single N-methylation of the peptide bond at various positions were all shortened in the C-terminus of the B-chain by four amino acids. The effect of modifications was followed by two types of in vitro assays, i.e., by the binding to the receptor of rat adipose plasma membranes and by the stimulation of the glucose transport into the isolated rat adipocytes. From our results, we can deduce several conclusions: (i) the replacement of tyrosine in the position B26 by phenylalanine has no significant effect on the binding affinity and the stimulation of the glucose transport of shortened analogues, whereas the replacement of TyrB26 by histidine affects the potency highly positively; [HisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide and [NMeHisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide show binding affinity 529 and 5250%, respectively, of that of human insulin; (ii) N-methylation of the B24-B25 peptide bond exhibits a disruptive effect on the potency of analogues in both in vitro studies regardless the presence of amino acid in the position B26; (iii) N-methylation of the B23-B24 peptide bond markedly reduces the binding affinity and the glucose transport of respective analogue [NMePheB24]-des-tetrapeptide (B27-B30)-insulin-B26-amide.     

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

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

Insulin analogues with modifications in the β-turn of the B-chain

The β-turn formed by the amino acid residues 20–23 of the B-chain of insulin has been implicated as an important structural feature of the molecule. In other biologically active peptides, stabilization of β-turns has resulted in increases in activity. We have synthesized three insulin analogues containing modifications which would be expected to increase the stability of the β-turn. In two analogues, we have substituted α-aminoisobutyric acid (Aib) for the Glu residue normally present in position B21 or for the Arg residue normally present in position B22; in a third compound, we have replaced the Glu residue with its D-isomer. Biological evaluation of these compounds showed that [B21 Aib]insulin displays a potencyca. one-fourth that of natural insulin, while [B22 Aib]insulin is less than 10% as potent. In contrast, [B21 D-Glu]insulin is equipotent with natural insulin. We conclude that the β-turn region of the insulin molecule normally possesses considerable flexibility, which may be necessary for it to assume a conformation commensurate with high biological activity. If this is the case, [B21 D-Glu]insulin may exhibit a stabilized geometry similar to that of natural insulin when bound to the insulin receptor.     

Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor. Importance of main chain conformation

We have investigated (by use of semisynthetic insulin analogs and isolated canine hepatocytes) the role of invariant residue PheB24 in determining the affinity of insulin-receptor interactions. Our results confirm that replacement of PheB24 by D-Phe is not detrimental to ligand binding to receptor, show that D-Ala is well tolerated at position B24 (whereas Ala is not), and demonstrate that [GlyB24]insulin retains as much as 78% of the receptor binding potency of native insulin. Additional findings show that replacement of PheB24 by D-Pro or by alpha-aminoisobutyric acid results in analogs with severely decreased binding potency, and that the COOH-terminal domain containing residues B26-B30 plays a positive role in determining receptor binding potency in GlyB24-substituted insulin (whereas it plays a negative role in determining the receptor binding potency of its GlyB25-substituted counterpart). We interpret our results as identifying (a) a critical role for the insulin main chain near residue B24 in determining the affinity of receptor for ligand, (b) the importance of main chain flexibility in achieving a high affinity state of receptor-bound hormone, and (c) a potential interaction of the PheB24 side chain with receptor which initiates main chain structural changes in the natural hormone, but which does not itself confer affinity to ligand-receptor interactions.     

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

How insulin binds to its receptor is unknown despite decades of investigation. Here, we employ chiral mutagenesis-comparison of corresponding d and l amino acid substitutions in the hormone-to define a structural switch between folding-competent and active conformations. Our strategy is motivated by the T --> R transition, an allosteric feature of zinc-hexamer assembly in which an invariant glycine in the B chain changes conformations. In the classical T state, Gly(B8) lies within a beta-turn and exhibits a positive phi angle (like a d amino acid); in the alternative R state, Gly(B8) is part of an alpha-helix and exhibits a negative phi angle (like an l amino acid). Respective B chain libraries containing mixtures of d or l substitutions at B8 exhibit a stereospecific perturbation of insulin chain combination: l amino acids impede native disulfide pairing, whereas diverse d substitutions are well-tolerated. Strikingly, d substitutions at B8 enhance both synthetic yield and thermodynamic stability but markedly impair biological activity. The NMR structure of such an inactive analogue (as an engineered T-like monomer) is essentially identical to that of native insulin. By contrast, l analogues exhibit impaired folding and stability. Although synthetic yields are very low, such analogues can be highly active. Despite the profound differences between the foldabilities of d and l analogues, crystallization trials suggest that on protein assembly substitutions of either class can be accommodated within classical T or R states. Comparison between such diastereomeric analogues thus implies that the T state represents an inactive but folding-competent conformation. We propose that within folding intermediates the sign of the B8 phi angle exerts kinetic control in a rugged landscape to distinguish between trajectories associated with productive disulfide pairing (positive T-like values) or off-pathway events (negative R-like values). We further propose that the crystallographic T -->R transition in part recapitulates how the conformation of an insulin monomer changes on receptor binding. At the very least the ostensibly unrelated processes of disulfide pairing, allosteric assembly, and receptor binding appear to utilize the same residue as a structural switch; an "ambidextrous" glycine unhindered by the chiral restrictions of the Ramachandran plane. We speculate that this switch operates to protect insulin-and the beta-cell-from protein misfolding.     

Nonlocal structural perturbations in a mutant human insulin: sequential resonance assignment and 13C-isotope-aided 2D-NMR studies of [PheB24-->Gly]insulin with implications for receptor recognition.

Insulin's mechanism of receptor binding is not well understood despite extensive study by mutagenesis and X-ray crystallography. Of particular interest are "anomalous" analogues whose bioactivities are not readily rationalized by crystal structures. Here the structure and dynamics of one such analogue (GlyB24-insulin) are investigated by circular dichroism (CD) and isotope-aided 2D-NMR spectroscopy. The mutant insulin retains near-native receptor-binding affinity despite a nonconservative substitution (PheB24-->Gly) in the receptor-binding surface. Relative to native insulin, GlyB24-insulin exhibits reduced dimerization; the monomer (the active species) exhibits partial loss of ordered structure, as indicated by CD studies and motional narrowing of selected 1H-NMR resonance. 2D-NMR studies demonstrate that the B-chain beta-turn (residues B20-23) and beta-strand (residues B24-B28) are destabilized; essentially native alpha-helical secondary structure (residues A3-A8, A13-A18, and B9-B19) is otherwise maintained. 13C-Isotope-edited NOESY studies demonstrate that long-range contacts observed between the B-chain beta-strand and the alpha-helical core in native insulin are absent in the mutant. Implications for the mechanism of insulin's interaction with its receptor are discussed.     

Synthesis and biological activity of seventeen analogues of human insulin

We synthesized seventeen analogues of human insulin, applying the principle of stepwise, selective formation of the disulphide bonds. Most of these analogues only differ from human insulin in the replacement of a single amino acid in positions 2, 5, 6, 7, 8 and 11 of the A chain and 5, 7, 13 and 16 of the B-chain. The influence of these modifications on the physicochemical properties of the analogues is discussed. Eight analogues could be crystallized. All the analogues produce the same biological effects as insulin, but differ markedly in their potency. In isolated fat cells in vitro, [HisA8]insulin showed a relative potency of 2.46 in stimulating glucose oxidation (human insulin = 1), whereas [D-CysA6,A11]insulin had a potency of only 0.00027. Very low potency was observed when IleA2 or the half-cystines A6, A7, A11 or B7 were modified. Replacement of the invariant GlnA5 by alanine only reduced potency slightly. All the analogues are full agonists. The effects of the analogues on glucose oxidation and lipolysis are correlated, supporting the view that they are mediated by a common receptor on the fat-cell membrane. Hypoglycaemic potencies in the rat were similar to potencies in vitro. As expected, no correlation was demonstrable between antiserum binding--measured in the radioimmunoassay--and biological activity. Several results of this investigation are difficult to reconcile with the current view regarding the structure-activity relationship of insulin which appears to require further refinement.     

Characterization of Rarobacter faecitabidus protease I, a yeast-lytic serine protease having mannose-binding activity.

Rarobacter faecitabidus protease I, a yeast-lytic serine protease, was characterized in order to elucidate the mechanism of lysis of yeast cells by this enzyme. The N-terminal amino acid sequence of the enzyme was found to be homologous to those of Lysobacter enzymogenes alpha-lytic protease and Streptomyces griseus proteases A and B around the catalytic His residue, showing that it is a mammalian type serine protease. In a study of its substrate specificity, it preferentially hydrolyzed the ester of alanine among amino acid p-nitrophenylesters. It also efficiently hydrolyzed succinyl Ala-Pro-Ala p-nitroanilide, the specific synthetic substrate for pancreatic elastase. With oxidized insulin B-chain, it hydrolyzed almost exclusively the peptide bond between valine 18 and cysteic acid 19 in the early step of the reaction, and thereafter it partially hydrolyzed Val12-Glu13, Ala14-Leu15, and Leu15-Tyr16. These results indicate that Rarobacter protease I is elastase-like in its substrate specificity, preferentially hydrolyzing the peptide bond of aliphatic amino acids. Its affinity for yeast cells was also investigated, and while Rarobacter protease I was adsorbed by yeast cells, pancreatic elastase was not. This difference was thought to account for the failure of pancreatic elastase to lyse yeast cells, even though its specificity is similar to that of the yeast-lytic enzyme. Rarobacter protease I was adsorbed by a mannose-agarose column and specifically eluted from the column with a buffer containing D-mannose or D-glucose. These monosaccharides also inhibited its yeast-lytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of N-methylation of selected peptide bonds on the biological activity of insulin. [2-N-methylisoleucine-A]insulin and [3-N-methylvaline-A]insulin

Hydrogen bonding involving peptide bonds of the backbone of the insulin molecule may play an important role in insulin-receptor interaction. Our previous work suggested that the A2-A8 helical segment of the hormone molecule participates in this interaction. To investigate the possible involvement of peptide bonds of this segment in insulin-receptor interaction the [2-N-methylisoleucine-A]insulin and [3-N-methylvaline-A]insulin ([MeIle2-A]- and [MeVal3-A]insulins) were synthesized. The circular dichroic spectra of the analogues were obtained and their properties were examined in several biological assays. The circular dichroic spectra suggested that the analogues remained monomeric at concentrations at which insulin is predominantly dimeric, and that their A2-A8 helical segments are distorted. The in vitro biological activity and the receptor binding affinity of these analogues were compared with that of natural insulin. Both analogues are weak full agonists. [MeIle2-A]insulin displayed a potency of 5.4 +/- 0.3% in stimulating lipogenesis and 4.6 +/- 2.3% in receptor binding affinity in rat fat cells and rat liver plasma membranes respectively. [MeVal3-A]insulin displayed a potency of 2.1 +/- 0.2% in lipogenesis and 1.0 +/- 0.3% in receptor binding assays. In radioimmunoassays [MeIle2-A]- and [MeVal3-A]insulins exhibited potencies of 13% and 11% respectively relative to the natural hormone. The substantially decreased biological activity and receptor binding affinity of these analogues may be attributed partly to the change of conformation and partly to the loss of hydrogen bonding capacity of the A2-A8 segment brought about by N-methylation of the A1-A2 or A2-A3 peptide bonds.     

Relaxin-like bioactivity of ovine Insulin 3 (INSL3) analogues.

Relaxin is an insulin-like peptide consisting of two separate chains (A and B) joined by two inter- and one intrachain disulfide bonds. Binding to its receptor requires an Arg-X-X-X-Arg-X-X-Ile motif in the B-chain. A related member of the insulin superfamily, INSL3, has a tertiary structure that is predicted to be similar to relaxin. It also possesses an Arg-X-X-X-Arg motif within its B-chain, although this is displaced by four amino acids towards the C-terminus from the corresponding position within relaxin. We have previously shown that synthetic INSL3 itself does not display relaxin-like activity although analogue (Analogue A) with an introduced arginine residue in the B-chain giving it an Arg cassette in the exact relaxin position does possess weak activity. In order to identify further the structural features that impart relaxin function, solid phase peptide synthesis was used to prepare three additional analogues for bioassay. Each of these contained point substitutions within the arginine cassette. Analogue D contained the full human relaxin binding cassette, Analogue G consisted of the native INSL3 sequence containing an Arg to Ala substitution, and Analogue E was a further modification of Analogue A, with the same substitution. Each analogue was fully chemically characterized by a number of criteria. Detailed circular dichroism spectroscopy analyses showed that the changes caused little alteration of secondary structure and, hence, overall conformation. However, each analogue displayed only weak relaxin-like activity. These results indicate that while the arginine cassette is vital for relaxin-like activity, there are additional, as yet unidentified structural requirements for relaxin binding.     

Importance of the solvent-exposed residues of the insulin B chain alpha-helix for receptor binding

Conjointly, the solvent-exposed residues of the central alpha-helix of the B chain form a well-defined ridge, which is flanked and partly overlapped by the two described insulin receptor binding surfaces on either side of the insulin molecule. To evaluate the importance of this interface in insulin receptor binding, we developed a new powerful method that allows us to introduce all the naturally occurring amino acids into a given position and subsequently determine the receptor binding affinities of the resulting insulin analogues. The total amino acid scanning mutagenesis was performed at positions B9, B10, B12, B13, B16, and B17, and the vast majority of the insulin analogue precursors were expressed and secreted in amounts close to that of the wild-type (human insulin) precursor. The analogue binding data revealed that positions B12 and B16 were the two positions most affected by the amino acid substitutions. Interestingly, the receptor binding affinities of the B13 analogues were also markedly affected by the amino acid substitutions, suggesting that GluB13 indeed is a part of insulin's binding surface. The B10 library screen generated analogues covering a wide range of (20-340%) of relative binding affinities, and the results indicated that a structural stabilization of the central alpha-helix and thereby a more rigid presentation of the binding epitope at the insulin receptor is important for receptor recognition. In conclusion, systematic amino acid scanning mutagenesis allowed us to confirm the importance of the B chain alpha-helix as a central recognition element serving as a linker of a continual binding surface.     

TyrB26位点改变的人类胰岛素类似物的化学合成与体外活性

摘要：为了研究人类胰岛素B链第26位的酪氨酸对胰岛素和受体之间的结合的影响,包括单独的氨基酸替换或化合物替换的不同的胰岛素类似物被合成,其中化合物替代的类似物的B链C末端都减少了4个氨基酸。在对它们与胰岛素受体的亲和力进行研究中,结果发现它们与胰岛素受体的亲和力没有丢失, HisB26类似物和N-MeHisB26类似物的结合能力与胰岛素相比改变不大,分别是胰岛素的72 %和107 %。N-MeGluB26类似物,AadB26类似物和Phe (4-carboxy) B26类似物的结合能力有很大的提高,分别是130 %, 234 %和160 %。     

Insulin analogues with permuted A chain N-terminus (author's transl)]

By partial synthesis insulin analogues were prepared in which the amino acid in position 1 of the A chain was permuted. Glycine in position A 1 was exchanged for leucine, tert.- butyloxycarbonylvaline, valine, proline, lysine as well as glutamic acid. Two pathways of partial synthesis were followed: Firstly, des-1-glycine-A-chain S-sulfonate was reacted with active esters of tert.-butyloxycarbonylamino acids. The ensuing modified A-chains were combined with natural B-chain to give A1-permuted insulins. In the second procedure, the preparation of tris-Boc-[A1-leucine]insulin was accomplished by reaction of Boc-leucine N-hydroxysuccinimide ester with NalphaB1,NepsilonB29-bis(tert.-butyloxycarbonyl)-des-A1-glycine-insulin. The protected insulin derivative had been prepared by combination of des-glycine-A-chain with Nalpha1,Nepsilon29-bis(tert.-butyloxycarbonyl)-B-chain. The deprotected analogues differed considerably in their CD-spectra from insulin and possessed low in vitro biological activities of 2.5-17%. Crystallization attempts failed. Thus, the introduction of side chains in position A1 distorts the conformation sterically and decreases the biological activity.