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. 2000 Dec;9(12):2294–2301. doi: 10.1110/ps.9.12.2294

Design of a minimal protein oligomerization domain by a structural approach.

P Burkhard 1, M Meier 1, A Lustig 1
PMCID: PMC2144530  PMID: 11206050

Abstract

Because of the simplicity and regularity of the alpha-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly alpha-helical and 100% dimeric tinder physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system.

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Selected References

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  1. Baltzer L., Broo K. S., Nilsson H., Nilsson J. Designed four-helix bundle catalysts--the engineering of reactive sites for hydrolysis and transesterification reactions of p-nitrophenyl esters. Bioorg Med Chem. 1999 Jan;7(1):83–91. doi: 10.1016/s0968-0896(98)00218-1. [DOI] [PubMed] [Google Scholar]
  2. Brünger A. T., Adams P. D., Clore G. M., DeLano W. L., Gros P., Grosse-Kunstleve R. W., Jiang J. S., Kuszewski J., Nilges M., Pannu N. S. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905–921. doi: 10.1107/s0907444998003254. [DOI] [PubMed] [Google Scholar]
  3. Burkhard P., Kammerer R. A., Steinmetz M. O., Bourenkov G. P., Aebi U. The coiled-coil trigger site of the rod domain of cortexillin I unveils a distinct network of interhelical and intrahelical salt bridges. Structure. 2000 Mar 15;8(3):223–230. doi: 10.1016/s0969-2126(00)00100-3. [DOI] [PubMed] [Google Scholar]
  4. Chakrabartty A., Baldwin R. L. Stability of alpha-helices. Adv Protein Chem. 1995;46:141–176. [PubMed] [Google Scholar]
  5. Chao H., Bautista D. L., Litowski J., Irvin R. T., Hodges R. S. Use of a heterodimeric coiled-coil system for biosensor application and affinity purification. J Chromatogr B Biomed Sci Appl. 1998 Sep 11;715(1):307–329. doi: 10.1016/s0378-4347(98)00172-8. [DOI] [PubMed] [Google Scholar]
  6. Cohen C., Parry D. A. Alpha-helical coiled coils and bundles: how to design an alpha-helical protein. Proteins. 1990;7(1):1–15. doi: 10.1002/prot.340070102. [DOI] [PubMed] [Google Scholar]
  7. Doig A. J., Baldwin R. L. N- and C-capping preferences for all 20 amino acids in alpha-helical peptides. Protein Sci. 1995 Jul;4(7):1325–1336. doi: 10.1002/pro.5560040708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fágáin C. O. Understanding and increasing protein stability. Biochim Biophys Acta. 1995 Sep 27;1252(1):1–14. doi: 10.1016/0167-4838(95)00133-f. [DOI] [PubMed] [Google Scholar]
  9. Gong Y., Zhou H. X., Guo M., Kallenbach N. R. Structural analysis of the N- and C-termini in a peptide with consensus sequence. Protein Sci. 1995 Aug;4(8):1446–1456. doi: 10.1002/pro.5560040802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gonzalez L., Jr, Plecs J. J., Alber T. An engineered allosteric switch in leucine-zipper oligomerization. Nat Struct Biol. 1996 Jun;3(6):510–515. doi: 10.1038/nsb0696-510. [DOI] [PubMed] [Google Scholar]
  11. Harbury P. B., Kim P. S., Alber T. Crystal structure of an isoleucine-zipper trimer. Nature. 1994 Sep 1;371(6492):80–83. doi: 10.1038/371080a0. [DOI] [PubMed] [Google Scholar]
  12. Harbury P. B., Plecs J. J., Tidor B., Alber T., Kim P. S. High-resolution protein design with backbone freedom. Science. 1998 Nov 20;282(5393):1462–1467. doi: 10.1126/science.282.5393.1462. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Huyghues-Despointes B. M., Scholtz J. M., Baldwin R. L. Helical peptides with three pairs of Asp-Arg and Glu-Arg residues in different orientations and spacings. Protein Sci. 1993 Jan;2(1):80–85. doi: 10.1002/pro.5560020108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  16. Kammerer R. A., Schulthess T., Landwehr R., Lustig A., Engel J., Aebi U., Steinmetz M. O. An autonomous folding unit mediates the assembly of two-stranded coiled coils. Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13419–13424. doi: 10.1073/pnas.95.23.13419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Kortemme T., Ramírez-Alvarado M., Serrano L. Design of a 20-amino acid, three-stranded beta-sheet protein. Science. 1998 Jul 10;281(5374):253–256. doi: 10.1126/science.281.5374.253. [DOI] [PubMed] [Google Scholar]
  19. Krylov D., Barchi J., Vinson C. Inter-helical interactions in the leucine zipper coiled coil dimer: pH and salt dependence of coupling energy between charged amino acids. J Mol Biol. 1998 Jun 19;279(4):959–972. doi: 10.1006/jmbi.1998.1762. [DOI] [PubMed] [Google Scholar]
  20. Krylov D., Mikhailenko I., Vinson C. A thermodynamic scale for leucine zipper stability and dimerization specificity: e and g interhelical interactions. EMBO J. 1994 Jun 15;13(12):2849–2861. doi: 10.1002/j.1460-2075.1994.tb06579.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lavigne P., Sönnichsen F. D., Kay C. M., Hodges R. S. Interhelical salt bridges, coiled-coil stability, and specificity of dimerization. Science. 1996 Feb 23;271(5252):1136–1138. doi: 10.1126/science.271.5252.1136. [DOI] [PubMed] [Google Scholar]
  22. Lombardi A., Summa C. M., Geremia S., Randaccio L., Pavone V., DeGrado W. F. Retrostructural analysis of metalloproteins: application to the design of a minimal model for diiron proteins. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6298–6305. doi: 10.1073/pnas.97.12.6298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lu M., Shu W., Ji H., Spek E., Wang L., Kallenbach N. R. Helix capping in the GCN4 leucine zipper. J Mol Biol. 1999 May 14;288(4):743–752. doi: 10.1006/jmbi.1999.2707. [DOI] [PubMed] [Google Scholar]
  24. Lumb K. J., Kim P. S. Measurement of interhelical electrostatic interactions in the GCN4 leucine zipper. Science. 1995 Apr 21;268(5209):436–439. doi: 10.1126/science.7716550. [DOI] [PubMed] [Google Scholar]
  25. Lumb K. J., Kim P. S. Response: how much solar radiation do clouds absorb? Science. 1996 Feb 23;271(5252):1137–1138. doi: 10.1126/science.271.5252.1137. [DOI] [PubMed] [Google Scholar]
  26. Lupas A. Coiled coils: new structures and new functions. Trends Biochem Sci. 1996 Oct;21(10):375–382. [PubMed] [Google Scholar]
  27. Merutka G., Stellwagen E. Effect of amino acid ion pairs on peptide helicity. Biochemistry. 1991 Feb 12;30(6):1591–1594. doi: 10.1021/bi00220a021. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Musafia B., Buchner V., Arad D. Complex salt bridges in proteins: statistical analysis of structure and function. J Mol Biol. 1995 Dec 8;254(4):761–770. doi: 10.1006/jmbi.1995.0653. [DOI] [PubMed] [Google Scholar]
  30. Muñoz V., Serrano L. Helix design, prediction and stability. Curr Opin Biotechnol. 1995 Aug;6(4):382–386. doi: 10.1016/0958-1669(95)80066-2. [DOI] [PubMed] [Google Scholar]
  31. Perrakis A., Sixma T. K., Wilson K. S., Lamzin V. S. wARP: improvement and extension of crystallographic phases by weighted averaging of multiple-refined dummy atomic models. Acta Crystallogr D Biol Crystallogr. 1997 Jul 1;53(Pt 4):448–455. doi: 10.1107/S0907444997005696. [DOI] [PubMed] [Google Scholar]
  32. Peters J., Baumeister W., Lupas A. Hyperthermostable surface layer protein tetrabrachion from the archaebacterium Staphylothermus marinus: evidence for the presence of a right-handed coiled coil derived from the primary structure. J Mol Biol. 1996 Apr 19;257(5):1031–1041. doi: 10.1006/jmbi.1996.0221. [DOI] [PubMed] [Google Scholar]
  33. Severin K., Lee D. H., Kennan A. J., Ghadiri M. R. A synthetic peptide ligase. Nature. 1997 Oct 16;389(6652):706–709. doi: 10.1038/39556. [DOI] [PubMed] [Google Scholar]
  34. Spek E. J., Bui A. H., Lu M., Kallenbach N. R. Surface salt bridges stabilize the GCN4 leucine zipper. Protein Sci. 1998 Nov;7(11):2431–2437. doi: 10.1002/pro.5560071121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Steinmetz M. O., Stock A., Schulthess T., Landwehr R., Lustig A., Faix J., Gerisch G., Aebi U., Kammerer R. A. A distinct 14 residue site triggers coiled-coil formation in cortexillin I. EMBO J. 1998 Apr 1;17(7):1883–1891. doi: 10.1093/emboj/17.7.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stetefeld J., Jenny M., Schulthess T., Landwehr R., Engel J., Kammerer R. A. Crystal structure of a naturally occurring parallel right-handed coiled coil tetramer. Nat Struct Biol. 2000 Sep;7(9):772–776. doi: 10.1038/79006. [DOI] [PubMed] [Google Scholar]
  37. Struthers M. D., Cheng R. P., Imperiali B. Design of a monomeric 23-residue polypeptide with defined tertiary structure. Science. 1996 Jan 19;271(5247):342–345. doi: 10.1126/science.271.5247.342. [DOI] [PubMed] [Google Scholar]
  38. Thompson K. S., Vinson C. R., Freire E. Thermodynamic characterization of the structural stability of the coiled-coil region of the bZIP transcription factor GCN4. Biochemistry. 1993 Jun 1;32(21):5491–5496. doi: 10.1021/bi00072a001. [DOI] [PubMed] [Google Scholar]
  39. Trail P. A., Bianchi A. B. Monoclonal antibody drug conjugates in the treatment of cancer. Curr Opin Immunol. 1999 Oct;11(5):584–588. doi: 10.1016/s0952-7915(99)00012-6. [DOI] [PubMed] [Google Scholar]
  40. Tripet B., Wagschal K., Lavigne P., Mant C. T., Hodges R. S. Effects of side-chain characteristics on stability and oligomerization state of a de novo-designed model coiled-coil: 20 amino acid substitutions in position "d". J Mol Biol. 2000 Jul 7;300(2):377–402. doi: 10.1006/jmbi.2000.3866. [DOI] [PubMed] [Google Scholar]
  41. Tripet B., Yu L., Bautista D. L., Wong W. Y., Irvin R. T., Hodges R. S. Engineering a de novo designed coiled-coil heterodimerization domain for the rapid detection, purification and characterization of recombinantly expressed peptides and proteins. Protein Eng. 1997 Mar;10(3):299–299. doi: 10.1093/protein/10.3.299. [DOI] [PubMed] [Google Scholar]
  42. Wang C., Stewart R. J., Kopecek J. Hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains. Nature. 1999 Feb 4;397(6718):417–420. doi: 10.1038/17092. [DOI] [PubMed] [Google Scholar]

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