Insulin and insulin-like growth factor 1 (IGF-1) are closely related hormones involved in the regulation of metabolism and growth. They elicit their functions through activation of tyrosine kinase-type receptors: insulin receptors (IR-A and IR-B) and IGF-1 receptor (IGF-1R). Despite similarity in primary and three-dimensional structures, insulin and IGF-1 bind the noncognate receptor with substantially reduced affinity. We prepared [d-HisB24, GlyB31, TyrB32]-insulin, which binds all three receptors with high affinity (251 or 338% binding affinity to IR-A respectively to IR-B relative to insulin and 12.4% binding affinity to IGF-1R relative to IGF-1). We prepared other modified insulins with the aim of explaining the versatility of [d-HisB24, GlyB31, TyrB32]-insulin. Through structural, activity, and kinetic studies of these insulin analogs, we concluded that the ability of [d-HisB24, GlyB31, TyrB32]-insulin to stimulate all three receptors is provided by structural changes caused by a reversed chirality at the B24 combined with the extension of the C terminus of the B chain by two extra residues. We assume that the structural changes allow the directing of the B chain C terminus to some extra interactions with the receptors. These unusual interactions lead to a decrease of dissociation rate from the IR and conversely enable easier association with IGF-1R. All of the structural changes were made at the hormones' Site 1, which is thought to interact with the Site 1 of the receptors. The results of the study suggest that merely modifications of Site 1 of the hormone are sufficient to change the receptor specificity of insulin.

• Klíčová slova
• Site 1, binding, insulin, insulin receptor, insulin-like growth factor (IGF), kinetics, protein design, structure-function,
• MeSH
• insulinu podobný růstový faktor I chemie   genetika   metabolismus   MeSH
• inzulin agonisté   metabolismus   MeSH
• kinetika MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• receptor IGF typ 1 MeSH
• receptor inzulinu chemie   genetika   metabolismus   MeSH
• receptory somatomedinů chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• IGF1R protein, human MeSH Prohlížeč
• insulinu podobný růstový faktor I MeSH
• inzulin MeSH
• receptor IGF typ 1 MeSH
• receptor inzulinu MeSH
• receptory somatomedinů MeSH

Insulin is a key hormone of human metabolism with major therapeutic importance for both types of diabetes. New insulin analogues with more physiological profiles and better glycemic control are needed, especially analogues that preferentially bind to the metabolic B-isoform of insulin receptor (IR-B). Here, we aimed to stabilize and modulate the receptor-compatible conformation of insulin by covalent intra-chain crosslinking within its B22-B30 segment, using the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of azides and alkynes. This approach resulted in 14 new, systematically crosslinked insulin analogues whose structures and functions were extensively characterized and correlated. One of the analogues, containing a B26-B29 triazole bridge, was highly active in binding to both IR isoforms, with a significant preference for IR-B. Our results demonstrate the potential of chemistry-driven modulation of insulin function, also shedding new light on the functional importance of hormone's B-chain C-terminus for its IR-B specificity.

• MeSH
• alkyny chemie   MeSH
• azidy chemie   MeSH
• cykloadiční reakce MeSH
• inzulin chemie   metabolismus   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• protein - isoformy MeSH
• receptor IGF typ 1 chemie   metabolismus   MeSH
• receptor inzulinu chemie   metabolismus   MeSH
• stabilita proteinů MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkyny MeSH
• azidy MeSH
• inzulin MeSH
• protein - isoformy MeSH
• receptor IGF typ 1 MeSH
• receptor inzulinu MeSH

Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved. Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant Phe(B24), one of the key residues of the hormone, in this process remains unclear. For example, the B24 site functionally tolerates substitutions to D-amino acids but not to L-amino acids. Here, we prepared and characterized a series of B24-modified insulin analogues, also determining the structures of [D-HisB24]-insulin and [HisB24]-insulin. The inactive [HisB24]-insulin molecule is remarkably rigid due to a tight accommodation of the L-His side chain in the B24 binding pocket that results in the stronger tethering of B25-B28 residues to the protein core. In contrast, the highly active [D-HisB24]-insulin is more flexible, and the reverse chirality of the B24C(α) atom swayed the D-His(B24) side chain into the solvent. Furthermore, the pocket vacated by Phe(B24) is filled by Phe(B25), which mimics the Phe(B24) side and main chains. The B25→B24 downshift results in a subsequent downshift of Tyr(B26) into the B25 site and the departure of B26-B30 residues away from the insulin core. Our data indicate the importance of the aromatic L-amino acid at the B24 site and the structural invariance/integrity of this position for an effective binding of insulin to its receptor. Moreover, they also suggest limited, B25-B30 only, unfolding of the C terminus of the B-chain upon insulin activation.

• MeSH
• inzulin chemie   genetika   metabolismus   MeSH
• lidé MeSH
• receptor inzulinu chemie   genetika   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• vazba proteinů fyziologie   MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inzulin MeSH
• receptor inzulinu MeSH

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.

• MeSH
• inzulin prasečí chemie   MeSH
• krystalografie rentgenová MeSH
• kvarterní struktura proteinů MeSH
• metylace MeSH
• multimerizace proteinu * MeSH
• prasata MeSH
• sekundární struktura proteinů MeSH
• stabilita proteinů MeSH
• vodíková vazba MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inzulin prasečí MeSH

Insulin is a key protein hormone that regulates blood glucose levels and, thus, has widespread impact on lipid and protein metabolism. Insulin action is manifested through binding of its monomeric form to the Insulin Receptor (IR). At present, however, our knowledge about the structural behavior of insulin is based upon inactive, multimeric, and storage-like states. The active monomeric structure, when in complex with the receptor, must be different as the residues crucial for the interactions are buried within the multimeric forms. Although the exact nature of the insulin's induced-fit is unknown, there is strong evidence that the C-terminal part of the B-chain is a dynamic element in insulin activation and receptor binding. Here, we present the design and analysis of highly active (200-500%) insulin analogues that are truncated at residue 26 of the B-chain (B(26)). They show a structural convergence in the form of a new beta-turn at B(24)-B(26). We propose that the key element in insulin's transition, from an inactive to an active state, may be the formation of the beta-turn at B(24)-B(26) associated with a trans to cis isomerisation at the B(25)-B(26) peptide bond. Here, this turn is achieved with N-methylated L-amino acids adjacent to the trans to cis switch at the B(25)-B(26) peptide bond or by the insertion of certain D-amino acids at B(26). The resultant conformational changes unmask previously buried amino acids that are implicated in IR binding and provide structural details for new approaches in rational design of ligands effective in combating diabetes.

• MeSH
• CD antigeny metabolismus   MeSH
• inzulin analogy a deriváty   chemie   metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• podjednotky proteinů MeSH
• receptor inzulinu metabolismus   MeSH
• sekundární struktura proteinů MeSH
• statická elektřina MeSH
• techniky in vitro MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CD antigeny MeSH
• INSR protein, human MeSH Prohlížeč
• inzulin MeSH
• podjednotky proteinů MeSH
• receptor inzulinu MeSH