Solution Structure of the Superactive Monomeric Des-[Phe(B25)] Human Insulin Mutant: Elucidation of the Structural Basis for the Monomerization of Des-[Phe(B25)] Insulin and the Dimerization of Native Insulin

Jørgensen, Andreas; Olsen, Henrik Baare; Balschmidt, Per; Led, Jens J.

doi:10.1006/jmbi.1996.0194

Cited by 49 publications

(67 citation statements)

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“…In particular, there are questions about the conformation of the C-terminal region of the B-chain, which in insulin forms a ␤-strand that hydrogen bonds in an intermolecular ␤-sheet, whereas in H2 relaxin, this region appears to be disordered. To obtain structural data on the insulin monomer and to investigate the determinants of the aggregation, several mutants have been studied, most involving alteration of the C terminus of the B-chain to remove hydrophobic interactions in the dimer (33,34). Fig.…”

Section: Discussionmentioning

confidence: 99%

“…In particular, an insulin mutant in which the fold was stabilized by the addition of a peptide linker between the N terminus of the A-chain and the C terminus of the B-chain was found to be biologically inactive, despite adopting a native fold (35). In contrast, the des-Phe B25 and PT insulin mutants have also been studied by NMR and shown to have significantly destabilized structures (34,36). These conclusions were based on increased amide exchange rates (34); and in the extreme case of PT insulin, the co-solvent trifluoroethanol, known to stabilize helical structures, had to be added to achieve a well defined structure in solution (36).…”

Section: B19mentioning

confidence: 99%

“…In contrast, the des-Phe B25 and PT insulin mutants have also been studied by NMR and shown to have significantly destabilized structures (34,36). These conclusions were based on increased amide exchange rates (34); and in the extreme case of PT insulin, the co-solvent trifluoroethanol, known to stabilize helical structures, had to be added to achieve a well defined structure in solution (36). Strikingly, both these molecules have increased biological activity relative to native insulin (33,37).…”

Section: B19mentioning

confidence: 99%

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Solution Structure and Novel Insights into the Determinants of the Receptor Specificity of Human Relaxin-3

Rosengren

Lin

Bathgate

et al. 2006

Journal of Biological Chemistry

117

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Relaxin-3 is the most recently discovered member of the relaxin family of peptide hormones. In contrast to relaxin-1 and -2, whose main functions are associated with pregnancy, relaxin-3 is involved in neuropeptide signaling in the brain. Here, we report the solution structure of human relaxin-3, the first structure of a relaxin family member to be solved by NMR methods. Overall, relaxin-3 adopts an insulin-like fold, but the structure differs crucially from the crystal structure of human relaxin-2 near the B-chain terminus. In particular, the B-chain C terminus folds back, allowing Trp B27 to interact with the hydrophobic core. This interaction partly blocks the conserved RXXXRXXI motif identified as a determinant for the interaction with the relaxin receptor LGR7 and may account for the lower affinity of relaxin-3 relative to relaxin for this receptor. This structural feature is likely important for the activation of its endogenous receptor, GPCR135.Recent developments have caused considerable excitement in the relaxin field, including the discovery of a new member of the relaxin family, relaxin-3 (1), and the identification of several long sought after relaxin receptors (2). Prior to the discovery of the relaxin-3 gene (RLN3), only one relaxin gene had been characterized in most mammals, with the exception of humans and higher primates, in which two separate genes, RLN1 and RLN2, were known (3, 4). The product of the human RLN2 gene, human relaxin-2 (H2 relaxin), is the functional ortholog of the RLN1 gene product from non-primate species, which is termed relaxin. The function of the product of the human RLN1 gene, human relaxin-1 (H1 relaxin), is unknown; and indeed, a native H1 relaxin peptide has not been isolated (5). Hence, throughout this work, "relaxin" will refer to the pregnancy hormones H2 relaxin and non-primate relaxin. Relaxin-3 is the ancestor of the entire relaxin peptide family (5), and RLN3 genes have been identified in all mammalian genomes as well as in the genomes of chicken, frog, and various fish species. In contrast, the RLN1 gene is found only in mammals. Fig. 1 shows a sequence comparison of the product of the human RLN3 gene, human relaxin-3 (H3 relaxin); H2 and H1 relaxins; and relaxin-3 orthologs from other species. Interestingly, the relaxin-3 sequences are highly conserved between species, in contrast to relaxin, which shows considerable sequence variation.Relaxin has long been regarded as a hormone mainly associated with pregnancy. Produced in the corpus luteum and/or placenta of most mammals, it has numerous pregnancy-specific actions, including the remodeling of the reproductive tract and preparation of the mammary apparatus for lactation (6). However, relaxin also has important physiological roles outside of pregnancy. It inhibits collagen biosynthesis and promotes collagen breakdown (7-9) and causes vasodilatation in various tissues (10). Interestingly, the expression pattern of relaxin-3 differs significantly from that of relaxin, suggesting a distinctly different physiolog...

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Section: Discussionmentioning

confidence: 99%

Section: B19mentioning

confidence: 99%

Section: B19mentioning

confidence: 99%

See 1 more Smart Citation

Solution Structure and Novel Insights into the Determinants of the Receptor Specificity of Human Relaxin-3

Rosengren

Lin

Bathgate

et al. 2006

Journal of Biological Chemistry

117

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“…Subsequently distance geometry simulated annealing calculations and refinement by restrained energy minimization were carried out as described previously. 23 The force constants of the NOE and dihedral angle terms were 50 kcal mol 1Å 2 and 200 kcal mol 1 rad 2 , respectively.…”

Section: Structure Calculation Of Glp-1mentioning

confidence: 99%

Structure and folding of glucagon‐like peptide‐1‐(7–36)‐amide in aqueous trifluoroethanol studied by NMR spectroscopy

Chang

Keller

Bjørn

et al. 2001

Magnetic Reson in Chemistry

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The conformational changes of free, monomeric glucagon-like peptide-1-(7-36)-amide (GLP-1) in aqueous solution with increasing concentrations of 2,2,2-trifluoroethanol (TFE) were monitored by NMR spectroscopy. It was found that GLP-1 gradually assumes a stable, single-stranded helical structure in water solution when the TFE concentration is increased from 0 to 35% (v/v). No further structural changes were observed at higher TFE concentrations. The structure of GLP-1 in 35% TFE was determined from 292 distance restraints and 44 angle restraints by distance geometry, simulating annealing and restrained energy minimization. The helical structure extends from T7 to K28, with a less well-defined region around G16 and a disordered six-residue N-terminal domain. The folding process of GLP-1 from random coil (in water) to helix (in 35% TFE) is initiated by the formation of the C-terminal segment of the helix that is extended gradually towards the N-terminus of the peptide with increasing concentration of TFE. The exchange rates of the slow exchanging amide protons indicate that the C-terminal part of the helix is more stable than the N-terminal part.

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“…The results show that the hydrophobic interaction between B28 Pro and the other monomer in a dimer is the dominant reason of dimer formation and stabilization. Jrgensen et al [7] also indicated that the monomerization of Des-[B25 Phe] insulin in solution was caused by the lack of hydrophobic interaction with the other molecule when B28 Pro moved to B27. All studies show that B28 Pro is crucial for insulin association.…”

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confidence: 99%

Crystal structural studies of destripeptide (B28-B30) insulin

Jun

Mao

Gui

et al. 2000

Sc. China Ser. B-Chem.

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Single crystals of destripeptide (B28-B30) insulin (DTRI) in three forms were obtained by hanging-drop vapor diffusion method. Form 1 belongs to P2 1 space group with cell parameters a=4.77 nm b=6.19 nm c=6.12 nm β=110.3 o . Form 2 belongs to P4 1 22 or P4 3 22 space group with cell parameters a= 6.45 nm, c=12.07 nm. Form 3 belongs to P2 1 2 1 2 1 space group with cell parameters a=4.98 nm b=5.16 nm c=10.06 nm. The structure of form 1 crystal was determined by molecular replacement method and refined at 0.23 nm resolution. The R-factor of the final model is 18.8% with r.m.s. deviations of 0.001 5 nm and 3.3 o for the bond lengths and the bond angles, respectively. Studies on the crystal structure show that the removal of B28 Pro has brought DTRI structural changes which made it dissociate more easily than native insulin although DTRI can still form a hexamer.Insulin has been used successfully in the treatment of diabetes, but its self-association property is always a disturbing problem. Subcutaneously injected insulin exists as oligomers [1] and it takes a long time for insulin to migrate into the bloodstream, consequently hindering its effective absorption of insulin. It is important to find a fast-acting insulin that exists as monomer or dissociates to monomer rapidly in treating diabetes.The interactions between the dimerizing monomers are crucial in insulin association [2] . B28 Pro is located in the dimerization surface and it is very important for dimer formation and stabilization. Brems et al. [3] found that the association ability of mutants at B28 Pro were all significantly reduced whenever B28 Pro was substituted by acid amino acid Asp, neutral Ala or basic Lys. Their results also showed that the removal of B30 and B29 were not critical for insulin association but the additional removal of B28 Pro reduced the dimerization constant by a factor of 300. Lys B28 Pro B29 insulin (Humalog®) is a commercially available monomer insulin analogue [4] . It is obtained by simple amino acid reversion of B28 and B29. The crystal structures of B28 Pro Asp insulin [5] and Lys B28 Pro B29 insulin [6] have been solved. The results show that the hydrophobic interaction between B28 Pro and the other monomer in a dimer is the dominant reason of dimer formation and stabilization. Jrgensen et al. [7] also indicated that the monomerization of Des-[B25 Phe] insulin in solution was caused by the lack of hydrophobic interaction with the other molecule when B28 Pro moved to B27. All studies show that B28 Pro is crucial for insulin association.

show abstract

Solution Structure of the Superactive Monomeric Des-[Phe(B25)] Human Insulin Mutant: Elucidation of the Structural Basis for the Monomerization of Des-[Phe(B25)] Insulin and the Dimerization of Native Insulin

Cited by 49 publications

References 0 publications

Solution Structure and Novel Insights into the Determinants of the Receptor Specificity of Human Relaxin-3

Solution Structure and Novel Insights into the Determinants of the Receptor Specificity of Human Relaxin-3

Structure and folding of glucagon‐like peptide‐1‐(7–36)‐amide in aqueous trifluoroethanol studied by NMR spectroscopy

Crystal structural studies of destripeptide (B28-B30) insulin

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