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
. 2000 Jul;9(7):1327–1333. doi: 10.1110/ps.9.7.1327

Soft metal ions, Cd(II) and Hg(II), induce triple-stranded alpha-helical assembly and folding of a de novo designed peptide in their trigonal geometries.

X Li 1, K Suzuki 1, K Kanaori 1, K Tajima 1, A Kashiwada 1, H Hiroaki 1, D Kohda 1, T Tanaka 1
PMCID: PMC2144689  PMID: 10933497

Abstract

We previously reported the de novo design of an amphiphilic peptide [YGG(IEKKIEA)4] that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded alpha-helix structure. The addition of Cd(II), however, resulted in the reconstitution of the triple-stranded alpha-helix bundle, as revealed by circular dichroism (CD) spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant Kd was determined to be 1.5 +/- 0.8 microM for Cd(II). The triple-stranded complex formed by the 113Cd(II) ion showed a single 113Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl- ions. The 113Cd NMR resonance was connected with the betaH protons of the cysteine residue by 1H-113Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg(II) also induced the assembly of the peptide into a triple-stranded alpha-helical bundle below the Hg(II)/peptide ratio of 1/3. With excess Hg(II), however, the alpha-helicity of the peptide was decreased, with the change of the Hg(II) coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.

Full Text

The Full Text of this article is available as a PDF (249.0 KB).

Selected References

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

  1. Ansari A. Z., Bradner J. E., O'Halloran T. V. DNA-bend modulation in a repressor-to-activator switching mechanism. Nature. 1995 Mar 23;374(6520):371–375. doi: 10.1038/374370a0. [DOI] [PubMed] [Google Scholar]
  2. Casas-Finet J. R., Hu S., Hamer D., Karpel R. L. Spectroscopic characterization of the copper(I)-thiolate cluster in the DNA-binding domain of yeast ACE1 transcription factor. FEBS Lett. 1991 Apr 9;281(1-2):205–208. doi: 10.1016/0014-5793(91)80394-i. [DOI] [PubMed] [Google Scholar]
  3. Dieckmann G. R., McRorie D. K., Lear J. D., Sharp K. A., DeGrado W. F., Pecoraro V. L. The role of protonation and metal chelation preferences in defining the properties of mercury-binding coiled coils. J Mol Biol. 1998 Jul 31;280(5):897–912. doi: 10.1006/jmbi.1998.1891. [DOI] [PubMed] [Google Scholar]
  4. Eijkelenboom A. P., van den Ent F. M., Vos A., Doreleijers J. F., Hård K., Tullius T. D., Plasterk R. H., Kaptein R., Boelens R. The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc. Curr Biol. 1997 Oct 1;7(10):739–746. doi: 10.1016/s0960-9822(06)00332-0. [DOI] [PubMed] [Google Scholar]
  5. Fowle D. A., Stillman M. J. Comparison of the structures of the metal-thiolate binding site in Zn(II)-, Cd(II)-, and Hg(II)-metallothioneins using molecular modeling techniques. J Biomol Struct Dyn. 1997 Feb;14(4):393–406. doi: 10.1080/07391102.1997.10508139. [DOI] [PubMed] [Google Scholar]
  6. Frantz B., O'Halloran T. V. DNA distortion accompanies transcriptional activation by the metal-responsive gene-regulatory protein MerR. Biochemistry. 1990 May 22;29(20):4747–4751. doi: 10.1021/bi00472a001. [DOI] [PubMed] [Google Scholar]
  7. Glusker J. P. Structural aspects of metal liganding to functional groups in proteins. Adv Protein Chem. 1991;42:1–76. doi: 10.1016/s0065-3233(08)60534-3. [DOI] [PubMed] [Google Scholar]
  8. Graddis T. J., Myszka D. G., Chaiken I. M. Controlled formation of model homo- and heterodimer coiled coil polypeptides. Biochemistry. 1993 Nov 30;32(47):12664–12671. doi: 10.1021/bi00210a015. [DOI] [PubMed] [Google Scholar]
  9. Harbury P. B., Zhang T., Kim P. S., Alber T. A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science. 1993 Nov 26;262(5138):1401–1407. doi: 10.1126/science.8248779. [DOI] [PubMed] [Google Scholar]
  10. Hodges R. S. Boehringer Mannheim award lecture 1995. La conference Boehringer Mannheim 1995. De novo design of alpha-helical proteins: basic research to medical applications. Biochem Cell Biol. 1996;74(2):133–154. doi: 10.1139/o96-015. [DOI] [PubMed] [Google Scholar]
  11. Kanaori K., Uodome N., Nagai A., Ohta D., Ogawa A., Iwasaki G., Nosaka A. Y. 113Cd nuclear magnetic resonance studies of cabbage histidinol dehydrogenase. Biochemistry. 1996 May 14;35(19):5949–5954. doi: 10.1021/bi951659y. [DOI] [PubMed] [Google Scholar]
  12. Klemba M., Gardner K. H., Marino S., Clarke N. D., Regan L. Novel metal-binding proteins by design. Nat Struct Biol. 1995 May;2(5):368–373. doi: 10.1038/nsb0595-368. [DOI] [PubMed] [Google Scholar]
  13. Kohn W. D., Kay C. M., Hodges R. S. Protein destabilization by electrostatic repulsions in the two-stranded alpha-helical coiled-coil/leucine zipper. Protein Sci. 1995 Feb;4(2):237–250. doi: 10.1002/pro.5560040210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kägi J. H., Kojima Y. Chemistry and biochemistry of metallothionein. Experientia Suppl. 1987;52:25–61. doi: 10.1007/978-3-0348-6784-9_3. [DOI] [PubMed] [Google Scholar]
  15. Kägi J. H., Schäffer A. Biochemistry of metallothionein. Biochemistry. 1988 Nov 15;27(23):8509–8515. doi: 10.1021/bi00423a001. [DOI] [PubMed] [Google Scholar]
  16. Lau S. Y., Taneja A. K., Hodges R. S. Synthesis of a model protein of defined secondary and quaternary structure. Effect of chain length on the stabilization and formation of two-stranded alpha-helical coiled-coils. J Biol Chem. 1984 Nov 10;259(21):13253–13261. [PubMed] [Google Scholar]
  17. Moitra J., Szilák L., Krylov D., Vinson C. Leucine is the most stabilizing aliphatic amino acid in the d position of a dimeric leucine zipper coiled coil. Biochemistry. 1997 Oct 14;36(41):12567–12573. doi: 10.1021/bi971424h. [DOI] [PubMed] [Google Scholar]
  18. Monera O. D., Kay C. M., Hodges R. S. Electrostatic interactions control the parallel and antiparallel orientation of alpha-helical chains in two-stranded alpha-helical coiled-coils. Biochemistry. 1994 Apr 5;33(13):3862–3871. doi: 10.1021/bi00179a010. [DOI] [PubMed] [Google Scholar]
  19. Nielson K. B., Atkin C. L., Winge D. R. Distinct metal-binding configurations in metallothionein. J Biol Chem. 1985 May 10;260(9):5342–5350. [PubMed] [Google Scholar]
  20. O'Neil K. T., DeGrado W. F. A thermodynamic scale for the helix-forming tendencies of the commonly occurring amino acids. Science. 1990 Nov 2;250(4981):646–651. doi: 10.1126/science.2237415. [DOI] [PubMed] [Google Scholar]
  21. Pack P., Plückthun A. Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli. Biochemistry. 1992 Feb 18;31(6):1579–1584. doi: 10.1021/bi00121a001. [DOI] [PubMed] [Google Scholar]
  22. Padmanabhan S., Baldwin R. L. Straight-chain non-polar amino acids are good helix-formers in water. J Mol Biol. 1991 May 20;219(2):135–137. doi: 10.1016/0022-2836(91)90553-i. [DOI] [PubMed] [Google Scholar]
  23. Pessi A., Bianchi E., Crameri A., Venturini S., Tramontano A., Sollazzo M. A designed metal-binding protein with a novel fold. Nature. 1993 Mar 25;362(6418):367–369. doi: 10.1038/362367a0. [DOI] [PubMed] [Google Scholar]
  24. Ralston D. M., O'Halloran T. V. Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex. Proc Natl Acad Sci U S A. 1990 May;87(10):3846–3850. doi: 10.1073/pnas.87.10.3846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Regan L., Clarke N. D. A tetrahedral zinc(II)-binding site introduced into a designed protein. Biochemistry. 1990 Dec 11;29(49):10878–10883. doi: 10.1021/bi00501a003. [DOI] [PubMed] [Google Scholar]
  26. Regan L. Protein design: novel metal-binding sites. Trends Biochem Sci. 1995 Jul;20(7):280–285. doi: 10.1016/s0968-0004(00)89044-1. [DOI] [PubMed] [Google Scholar]
  27. Robbins A. H., McRee D. E., Williamson M., Collett S. A., Xuong N. H., Furey W. F., Wang B. C., Stout C. D. Refined crystal structure of Cd, Zn metallothionein at 2.0 A resolution. J Mol Biol. 1991 Oct 20;221(4):1269–1293. [PubMed] [Google Scholar]
  28. Suzuki K., Hiroaki H., Kohda D., Tanaka T. An isoleucine zipper peptide forms a native-like triple stranded coiled coil in solution. Protein Eng. 1998 Nov;11(11):1051–1055. doi: 10.1093/protein/11.11.1051. [DOI] [PubMed] [Google Scholar]
  29. Suzuki K., Yamada T., Tanaka T. Role of the buried glutamate in the alpha-helical coiled coil domain of the macrophage scavenger receptor. Biochemistry. 1999 Feb 9;38(6):1751–1756. doi: 10.1021/bi9821014. [DOI] [PubMed] [Google Scholar]
  30. Terskikh A. V., Le Doussal J. M., Crameri R., Fisch I., Mach J. P., Kajava A. V. "Peptabody": a new type of high avidity binding protein. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1663–1668. doi: 10.1073/pnas.94.5.1663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Utschig L. M., Bryson J. W., O'Halloran T. V. Mercury-199 NMR of the metal receptor site in MerR and its protein-DNA complex. Science. 1995 Apr 21;268(5209):380–385. doi: 10.1126/science.7716541. [DOI] [PubMed] [Google Scholar]
  32. White A., Ding X., vanderSpek J. C., Murphy J. R., Ringe D. Structure of the metal-ion-activated diphtheria toxin repressor/tox operator complex. Nature. 1998 Jul 30;394(6692):502–506. doi: 10.1038/28893. [DOI] [PubMed] [Google Scholar]
  33. Zeng Q., Stålhandske C., Anderson M. C., Scott R. A., Summers A. O. The core metal-recognition domain of MerR. Biochemistry. 1998 Nov 10;37(45):15885–15895. doi: 10.1021/bi9817562. [DOI] [PubMed] [Google Scholar]
  34. Zhou N. E., Kay C. M., Hodges R. S. The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability. Protein Eng. 1994 Nov;7(11):1365–1372. doi: 10.1093/protein/7.11.1365. [DOI] [PubMed] [Google Scholar]

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

RESOURCES