Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1997 Mar;6(3):580–587. doi: 10.1002/pro.5560060307

Inactive conformation of an insulin despite its wild-type sequence.

G Kurapkat 1, E De Wolf 1, J Grötzinger 1, A Wollmer 1
PMCID: PMC2143665  PMID: 9070440

Abstract

The peptide group between residues B24 and B25 of insulin was replaced by an ester bond. This modification only in the backbone was meant to eliminate a structurally important H-bond between the amide proton of B25 and the carbonyl oxygen of A19, and consequently to enhance detachment of the C-terminal B-chain from the body of the molecule, exposing the underlying A-chain. According to a model derived from the effects of side-chain substitutions, main-chain shortening, and crosslinking, this conformational change is prerequisite for receptor binding. Contrary to the expectation that increased flexibility would increase receptor binding and activity, depsi-insulin ([B24-B25 CO-O]insulin) has turned out be only 3-4% potent. In search of an explanation for this observation, the solution structure of depsi-insulin was determined by two-dimensional 1H-NMR spectroscopy. It was found that the loss of the B25-A19 H-bond does not entail detachment of the C-terminal B-chain. On the contrary, it is overcompensated by a gain in hydrophobic interaction achieved by insertion of the Phe B25 side chain into the molecule's core. This is possible because of increased rotational freedom in the backbone owing to the ester bond. Distortion of the B20-B23 turn and an altered direction of the distal B-chain are consequences that also affect self-association. The exceptional position of the B25 side chain is thus the key feature of the depsi-insulin structure. Being buried in the interior, it is not available for guiding the interaction with the receptor, a crucial role attributed to it by the model. This seems to be the main reason why the structure of depsi-insulin represents an inactive conformation.

Full Text

The Full Text of this article is available as a PDF (5.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker E. N., Blundell T. L., Cutfield J. F., Cutfield S. M., Dodson E. J., Dodson G. G., Hodgkin D. M., Hubbard R. E., Isaacs N. W., Reynolds C. D. The structure of 2Zn pig insulin crystals at 1.5 A resolution. Philos Trans R Soc Lond B Biol Sci. 1988 Jul 6;319(1195):369–456. doi: 10.1098/rstb.1988.0058. [DOI] [PubMed] [Google Scholar]
  2. Brandenburg D. Preparation of N A1 ,N B29 -adipoylinsulin, an intramolecularly crosslinked derivative of beef insulin. Hoppe Seylers Z Physiol Chem. 1972 Jun;353(6):869–873. doi: 10.1515/bchm2.1972.353.1.869. [DOI] [PubMed] [Google Scholar]
  3. Brandenburg D., Schermutzki W., Zahn H. NalphaA1-N-epsilon B-29-crosslinked diaminosuberoylinsulin, a potential intermediate for the chemical synthesis of insulin. Hoppe Seylers Z Physiol Chem. 1973 Oct-Nov;354(10-11):1521–1524. [PubMed] [Google Scholar]
  4. Brandenburg D., Wollmer A. The effect of a non-peptide interchain crosslink on the reoxidation of reduced insulin. Hoppe Seylers Z Physiol Chem. 1973 Jun;354(6):613–627. doi: 10.1515/bchm2.1973.354.1.613. [DOI] [PubMed] [Google Scholar]
  5. Brems D. N., Brown P. L., Nakagawa S. H., Tager H. S. The conformational stability and flexibility of insulin with an additional intramolecular cross-link. J Biol Chem. 1991 Jan 25;266(3):1611–1615. [PubMed] [Google Scholar]
  6. Casaretto M., Spoden M., Diaconescu C., Gattner H. G., Zahn H., Brandenburg D., Wollmer A. Shortened insulin with enhanced in vitro potency. Biol Chem Hoppe Seyler. 1987 Jun;368(6):709–716. doi: 10.1515/bchm3.1987.368.1.709. [DOI] [PubMed] [Google Scholar]
  7. Cutfield J., Cutfield S., Dodson E., Dodson G., Hodgkin D., Reynolds C. Evidence concerning insulin activity from the structure of a cross-linked derivative. Hoppe Seylers Z Physiol Chem. 1981 Jun;362(6):755–761. doi: 10.1515/bchm2.1981.362.1.755. [DOI] [PubMed] [Google Scholar]
  8. Derewenda U., Derewenda Z., Dodson E. J., Dodson G. G., Bing X., Markussen J. X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue. J Mol Biol. 1991 Jul 20;220(2):425–433. doi: 10.1016/0022-2836(91)90022-x. [DOI] [PubMed] [Google Scholar]
  9. Fischer W. H., Saunders D., Brandenburg D., Wollmer A., Zahn H. A shortened insulin with full in vitro potency. Biol Chem Hoppe Seyler. 1985 May;366(5):521–525. doi: 10.1515/bchm3.1985.366.1.521. [DOI] [PubMed] [Google Scholar]
  10. Gattner H. G. B-Kettenverkürzung von polymergebundenem Insulin mit Pepsin, I. Darstellung und Eigenschaften von Des-Pentapeptid (B26-30)-Rinderinsulin. Hoppe Seylers Z Physiol Chem. 1975 Sep;356(9):1397–1404. [PubMed] [Google Scholar]
  11. Gliemann J., Gammeltoft S. The biological activity and the binding affinity of modified insulins determined on isolated rat fat cells. Diabetologia. 1974 Apr;10(2):105–113. doi: 10.1007/BF01219665. [DOI] [PubMed] [Google Scholar]
  12. Hua Q. X., Shoelson S. E., Inouye K., Weiss M. A. Paradoxical structure and function in a mutant human insulin associated with diabetes mellitus. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):582–586. doi: 10.1073/pnas.90.2.582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hua Q. X., Shoelson S. E., Kochoyan M., Weiss M. A. Receptor binding redefined by a structural switch in a mutant human insulin. Nature. 1991 Nov 21;354(6350):238–241. doi: 10.1038/354238a0. [DOI] [PubMed] [Google Scholar]
  14. Hua Q. X., Shoelson S. E., Weiss M. A. 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. Biochemistry. 1992 Dec 1;31(47):11940–11951. doi: 10.1021/bi00162a037. [DOI] [PubMed] [Google Scholar]
  15. Hua Q. X., Weiss M. A. Toward the solution structure of human insulin: sequential 2D 1H NMR assignment of a des-pentapeptide analogue and comparison with crystal structure. Biochemistry. 1990 Nov 20;29(46):10545–10555. doi: 10.1021/bi00498a018. [DOI] [PubMed] [Google Scholar]
  16. Jones R. H., Dron D. I., Ellis M. J., Sönksen P. H., Brandenburg D. Biological properties of chemically modified insulins. I. Biological activity of proinsulin and insulin modified at A1-glycine and B29-lysine. Diabetologia. 1976 Dec;12(6):601–608. doi: 10.1007/BF01220637. [DOI] [PubMed] [Google Scholar]
  17. Kaptein R., Boelens R., Scheek R. M., van Gunsteren W. F. Protein structures from NMR. Biochemistry. 1988 Jul 26;27(15):5389–5395. doi: 10.1021/bi00415a001. [DOI] [PubMed] [Google Scholar]
  18. Kobayashi M., Sasaoka T., Sugibayashi M., Iwanishi M., Shigeta Y. Receptor binding and biologic activity of biosynthetic human insulin and mini-proinsulin produced by recombinant gene technology. Diabetes Res Clin Pract. 1989 Jun 20;7(1):25–28. doi: 10.1016/0168-8227(89)90040-5. [DOI] [PubMed] [Google Scholar]
  19. Krüger P., Hahnen J., Wollmer A. Comparative studies on the dynamics of crosslinked insulin. Eur Biophys J. 1994;23(3):177–187. doi: 10.1007/BF01007609. [DOI] [PubMed] [Google Scholar]
  20. Kurose T., Pashmforoush M., Yoshimasa Y., Carroll R., Schwartz G. P., Burke G. T., Katsoyannis P. G., Steiner D. F. Cross-linking of a B25 azidophenylalanine insulin derivative to the carboxyl-terminal region of the alpha-subunit of the insulin receptor. Identification of a new insulin-binding domain in the insulin receptor. J Biol Chem. 1994 Nov 18;269(46):29190–29197. [PubMed] [Google Scholar]
  21. Leyer S., Gattner H. G., Leithäuser M., Brandenburg D., Wollmer A., Höcker H. The role of the C-terminus of the insulin B-chain in modulating structural and functional properties of the hormone. Int J Pept Protein Res. 1995 Nov;46(5):397–407. doi: 10.1111/j.1399-3011.1995.tb01074.x. [DOI] [PubMed] [Google Scholar]
  22. Lindsay D. G. Intramolecular cross-linked insulin. FEBS Lett. 1972 Mar;21(1):105–108. doi: 10.1016/0014-5793(72)80175-3. [DOI] [PubMed] [Google Scholar]
  23. Ludvigsen S., Roy M., Thøgersen H., Kaarsholm N. C. High-resolution structure of an engineered biologically potent insulin monomer, B16 Tyr-->His, as determined by nuclear magnetic resonance spectroscopy. Biochemistry. 1994 Jul 5;33(26):7998–8006. doi: 10.1021/bi00192a003. [DOI] [PubMed] [Google Scholar]
  24. Markussen J., Jørgensen K. H., Sørensen A. R., Thim L. Single chain des-(B30) insulin. Intramolecular crosslinking of insulin by trypsin catalyzed transpeptidation. Int J Pept Protein Res. 1985 Jul;26(1):70–77. [PubMed] [Google Scholar]
  25. Mirmira R. G., Nakagawa S. H., Tager H. S. Importance of the character and configuration of residues B24, B25, and B26 in insulin-receptor interactions. J Biol Chem. 1991 Jan 25;266(3):1428–1436. [PubMed] [Google Scholar]
  26. Mirmira R. G., Tager H. S. Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor. Importance of main chain conformation. J Biol Chem. 1989 Apr 15;264(11):6349–6354. [PubMed] [Google Scholar]
  27. Murray-Rust J., McLeod A. N., Blundell T. L., Wood S. P. Structure and evolution of insulins: implications for receptor binding. Bioessays. 1992 May;14(5):325–331. doi: 10.1002/bies.950140507. [DOI] [PubMed] [Google Scholar]
  28. Nakagawa S. H., Johansen N. L., Madsen K., Schwartz T. W., Tager H. S. Implications of replacing peptide bonds in the COOH-terminal B chain domain of insulin by the psi (CH2-NH) linker. Int J Pept Protein Res. 1993 Dec;42(6):578–584. doi: 10.1111/j.1399-3011.1993.tb00367.x. [DOI] [PubMed] [Google Scholar]
  29. Nakagawa S. H., Tager H. S. Importance of aliphatic side-chain structure at positions 2 and 3 of the insulin A chain in insulin-receptor interactions. Biochemistry. 1992 Mar 31;31(12):3204–3214. doi: 10.1021/bi00127a023. [DOI] [PubMed] [Google Scholar]
  30. Nakagawa S. H., Tager H. S. Perturbation of insulin-receptor interactions by intramolecular hormone cross-linking. Analysis of relative movement among residues A1, B1, and B29. J Biol Chem. 1989 Jan 5;264(1):272–279. [PubMed] [Google Scholar]
  31. Nakagawa S. H., Tager H. S. Role of the COOH-terminal B-chain domain in insulin-receptor interactions. Identification of perturbations involving the insulin mainchain. J Biol Chem. 1987 Sep 5;262(25):12054–12058. [PubMed] [Google Scholar]
  32. Nakagawa S. H., Tager H. S. Role of the phenylalanine B25 side chain in directing insulin interaction with its receptor. Steric and conformational effects. J Biol Chem. 1986 Jun 5;261(16):7332–7341. [PubMed] [Google Scholar]
  33. Pullen R. A., Lindsay D. G., Wood S. P., Tickle I. J., Blundell T. L., Wollmer A., Krail G., Brandenburg D., Zahn H., Gliemann J. Receptor-binding region of insulin. Nature. 1976 Feb 5;259(5542):369–373. doi: 10.1038/259369a0. [DOI] [PubMed] [Google Scholar]
  34. Scheek R. M., van Gunsteren W. F., Kaptein R. Molecular dynamics simulation techniques for determination of molecular structures from nuclear magnetic resonance data. Methods Enzymol. 1989;177:204–218. doi: 10.1016/0076-6879(89)77012-9. [DOI] [PubMed] [Google Scholar]
  35. Srinivasa B. R., Carpenter F. H. Intramolecular cross-linking of insulin. Preparation and properties of oxalyl- and malonyl-bis(methionyl) insulin. Int J Pept Protein Res. 1983 Aug;22(2):214–222. doi: 10.1111/j.1399-3011.1983.tb02088.x. [DOI] [PubMed] [Google Scholar]
  36. Wishart D. S., Sykes B. D., Richards F. M. The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry. 1992 Feb 18;31(6):1647–1651. doi: 10.1021/bi00121a010. [DOI] [PubMed] [Google Scholar]
  37. Wollmer A., Brandenburg D., Vogt H. P., Schermutzki W. Reduction/reoxidation studies with cross-linked insulin derivatives. Hoppe Seylers Z Physiol Chem. 1974 Nov;355(11):1471–1476. [PubMed] [Google Scholar]
  38. Wollmer A., Gilge G., Brandenburg D., Gattner H. G. An insulin with the native sequence but virtually no activity. Biol Chem Hoppe Seyler. 1994 Mar;375(3):219–222. [PubMed] [Google Scholar]
  39. Wood S. P., Blundell T. L., Wollmer A., Lazarus N. R., Neville R. W. The relation of conformation and association of insulin to receptor binding; x-ray and circular-dichroism studies on bovine and hystricomorph insulins. Eur J Biochem. 1975 Jul 15;55(3):531–542. doi: 10.1111/j.1432-1033.1975.tb02190.x. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES