The quantitative characterization of residue contributions to protein-protein binding across extensive flexible interfaces poses a significant challenge for biophysical computations. It is attributable to the inherent imperfections in the experimental structures themselves, as well as to the lack of reliable computational tools for the evaluation of all types of noncovalent interactions. This study leverages recent advancements in semiempirical quantum-mechanical and implicit solvent approaches embodied in the PM6-D3H4S/COSMO2 method for the development of a hierarchical computational protocols encompassing molecular dynamics, fragmentation, and virtual glycine scan techniques for the investigation of flexible protein-protein interactions. As a model, the binding of insulin to its receptor is selected, a complex and dynamic process that has been extensively studied experimentally. The interaction energies calculated at the PM6-D3H4S/COSMO2 level in ten molecular dynamics snapshots did not correlate with molecular mechanics/generalized Born interaction energies because only the former method is able to describe nonadditive effects. This became evident by the examination of the energetics in small-model dimers featuring all the present types of noncovalent interactions with respect to DFT-D3 calculations. The virtual glycine scan has identified 15 hotspot residues on insulin and 15 on the insulin receptor, and their contributions have been quantified using PM6-D3H4S/COSMO2. The accuracy and credibility of the approach are further supported by the fact that all the insulin hotspots have previously been detected by biochemical and structural methods. The modular nature of the protocol has enabled the formulation of several variants, each tailored to specific accuracy and efficiency requirements. The developed computational strategy is firmly rooted in general biophysical chemistry and is thus offered as a general tool for the quantification of interactions across relevant flexible protein-protein interfaces.

• MeSH
• inzulin metabolismus   chemie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• receptor inzulinu * chemie   metabolismus   MeSH
• simulace molekulární dynamiky * MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inzulin MeSH
• receptor inzulinu * MeSH

Insulin-like growth factors 2 and 1 (IGF2 and IGF1) and insulin are closely related hormones that are responsible for the regulation of metabolic homeostasis, development and growth of the organism. Physiological functions of insulin and IGF1 are relatively well-studied, but information about the role of IGF2 in the body is still sparse. Recent discoveries called attention to emerging functions of IGF2 in the brain, where it could be involved in processes of learning and memory consolidation. It was also proposed that these functions could be mediated by the receptor for IGF2 (IGF2R). Nevertheless, little is known about the mechanism of signal transduction through this receptor. Here we produced His-tagged domain 11 (D11), an IGF2-binding element of IGF2R; we immobilized it on the solid support through a well-defined sandwich, consisting of neutravidin, biotin and synthetic anti-His-tag antibodies. Next, we prepared specifically radiolabeled [125I]-monoiodotyrosyl-Tyr2-IGF2 and optimized a sensitive and robust competitive radioligand binding assay for determination of the nanomolar binding affinities of hormones for D11 of IGF2. The assay will be helpful for the characterization of new IGF2 mutants to study the functions of IGF2R and the development of new compounds for the treatment of neurological disorders.

• MeSH
• insulinu podobný růstový faktor I metabolismus   MeSH
• insulinu podobný růstový faktor II metabolismus   MeSH
• kompetitivní vazba MeSH
• kultivované buňky MeSH
• lidé MeSH
• radioizotopy jodu MeSH
• radioligandová zkouška metody   MeSH
• receptor IGF typ 2 imunologie   ultrastruktura   MeSH
• signální transdukce MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• IGF1 protein, human MeSH Prohlížeč
• IGF2 protein, human MeSH Prohlížeč
• IGF2R protein, human MeSH Prohlížeč
• insulinu podobný růstový faktor I MeSH
• insulinu podobný růstový faktor II MeSH
• Iodine-125 MeSH Prohlížeč
• radioizotopy jodu MeSH
• receptor IGF typ 2 MeSH

The insulin gene mutation c.137G>A (R46Q), which changes an arginine at the B22 position of the mature hormone to glutamine, causes the monogenic diabetes variant maturity-onset diabetes of the young (MODY). In MODY patients, this mutation is heterozygous, and both mutant and wild-type (WT) human insulin are produced simultaneously. However, the patients often depend on administration of exogenous insulin. In this study, we chemically synthesized the MODY mutant [GlnB22]-insulin and characterized its biological and structural properties. The chemical synthesis of this insulin analogue revealed that its folding ability is severely impaired. In vitro and in vivo tests showed that its binding affinity and biological activity are reduced (both approximately 20% that of human insulin). Comparison of the solution structure of [GlnB22]-insulin with the solution structure of native human insulin revealed that the most significant structural effect of the mutation is distortion of the B20-B23 β-turn, leading to liberation of the B chain C-terminus from the protein core. The distortion of the B20-B23 β-turn is caused by the extended conformational freedom of the GlnB22 side chain, which is no longer anchored in a hydrogen bonding network like the native ArgB22. The partially disordered [GlnB22]-insulin structure appears to be one reason for the reduced binding potency of this mutant and may also be responsible for its low folding efficiency in vivo. The altered orientation and flexibility of the B20-B23 β-turn may interfere with the formation of disulfide bonds in proinsulin bearing the R46Q (GlnB22) mutation. This may also have a negative effect on the WT proinsulin simultaneously biosynthesized in β-cells and therefore play a major role in the development of MODY in patients producing [GlnB22]-insulin.

• MeSH
• diabetes mellitus 2. typu farmakoterapie   genetika   metabolismus   MeSH
• glutamin genetika   MeSH
• inzulin chemie   genetika   metabolismus   terapeutické užití   MeSH
• inzulinová rezistence MeSH
• kultivované buňky MeSH
• lidé MeSH
• missense mutace * MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glutamin MeSH
• inzulin MeSH

The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin-insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral φ and ψ main-chain angles. α-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1-B8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the "classical" T-state and that a substantial flexibility of the B1-B8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin-IR interaction.

• MeSH
• cirkulární dichroismus MeSH
• inzulin analogy a deriváty   chemie   MeSH
• krystalografie rentgenová MeSH
• kyseliny aminoisomáselné chemie   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2-aminoisobutyric acid MeSH Prohlížeč
• inzulin MeSH
• kyseliny aminoisomáselné 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