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. 1998 Apr 1;17(7):1883–1891. doi: 10.1093/emboj/17.7.1883

A distinct 14 residue site triggers coiled-coil formation in cortexillin I.

M O Steinmetz 1, A Stock 1, T Schulthess 1, R Landwehr 1, A Lustig 1, J Faix 1, G Gerisch 1, U Aebi 1, R A Kammerer 1
PMCID: PMC1170535  PMID: 9524112

Abstract

We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed, two-stranded, parallel coiled-coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation and electron microscopy. Deletion mapping identified a distinct 14 residue site within the Ir coiled coil, Arg311-Asp324, which was absolutely necessary for dimer formation, indicating that heptad repeats alone are not sufficient for stable coiled-coil formation. Moreover, deletion of the six N-terminal heptad repeats of Ir led to the formation of a four- rather than a two-helix structure, suggesting that the full-length cortexillin I coiled-coil domain behaves as a cooperative folding unit. Most interestingly, a 16 residue peptide containing the distinct coiled-coil 'trigger' site Arg311-Asp324 yielded approximately 30% helix formation as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptide's helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311-Asp324 behaves as an autonomous helical folding unit and that this distinct Ir segment controls the process of coiled-coil formation of cortexillin I.

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Selected References

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  1. Beck K., Gambee J. E., Kamawal A., Bächinger H. P. A single amino acid can switch the oligomerization state of the alpha-helical coiled-coil domain of cartilage matrix protein. EMBO J. 1997 Jul 1;16(13):3767–3777. doi: 10.1093/emboj/16.13.3767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Betz S. F., Bryson J. W., DeGrado W. F. Native-like and structurally characterized designed alpha-helical bundles. Curr Opin Struct Biol. 1995 Aug;5(4):457–463. doi: 10.1016/0959-440x(95)80029-8. [DOI] [PubMed] [Google Scholar]
  3. Brandenberger R., Kammerer R. A., Engel J., Chiquet M. Native chick laminin-4 containing the beta 2 chain (s-laminin) promotes motor axon growth. J Cell Biol. 1996 Dec;135(6 Pt 1):1583–1592. doi: 10.1083/jcb.135.6.1583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown J. H., Cohen C., Parry D. A. Heptad breaks in alpha-helical coiled coils: stutters and stammers. Proteins. 1996 Oct;26(2):134–145. doi: 10.1002/(SICI)1097-0134(199610)26:2<134::AID-PROT3>3.0.CO;2-G. [DOI] [PubMed] [Google Scholar]
  5. Chen Y. H., Yang J. T., Chau K. H. Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. Biochemistry. 1974 Jul 30;13(16):3350–3359. doi: 10.1021/bi00713a027. [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. Conway J. F., Parry D. A. Three-stranded alpha-fibrous proteins: the heptad repeat and its implications for structure. Int J Biol Macromol. 1991 Feb;13(1):14–16. doi: 10.1016/0141-8130(91)90004-e. [DOI] [PubMed] [Google Scholar]
  8. Eaton W. A., Muñoz V., Thompson P. A., Chan C. K., Hofrichter J. Submillisecond kinetics of protein folding. Curr Opin Struct Biol. 1997 Feb;7(1):10–14. doi: 10.1016/s0959-440x(97)80003-6. [DOI] [PubMed] [Google Scholar]
  9. Engel J. Electron microscopy of extracellular matrix components. Methods Enzymol. 1994;245:469–488. doi: 10.1016/0076-6879(94)45024-2. [DOI] [PubMed] [Google Scholar]
  10. FRASER R. D., MACRAE T. P., MILLER A. THE COILED-COIL MODEL OF ALPHA-KERATIN STRUCTURE. J Mol Biol. 1964 Oct;10:147–156. doi: 10.1016/s0022-2836(64)80034-6. [DOI] [PubMed] [Google Scholar]
  11. Fairman R., Chao H. G., Mueller L., Lavoie T. B., Shen L., Novotny J., Matsueda G. R. Characterization of a new four-chain coiled-coil: influence of chain length on stability. Protein Sci. 1995 Aug;4(8):1457–1469. doi: 10.1002/pro.5560040803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Faix J., Steinmetz M., Boves H., Kammerer R. A., Lottspeich F., Mintert U., Murphy J., Stock A., Aebi U., Gerisch G. Cortexillins, major determinants of cell shape and size, are actin-bundling proteins with a parallel coiled-coil tail. Cell. 1996 Aug 23;86(4):631–642. doi: 10.1016/s0092-8674(00)80136-1. [DOI] [PubMed] [Google Scholar]
  13. Gilmanshin R., Williams S., Callender R. H., Woodruff W. H., Dyer R. B. Fast events in protein folding: relaxation dynamics of secondary and tertiary structure in native apomyoglobin. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3709–3713. doi: 10.1073/pnas.94.8.3709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Jelesarov I., Bosshard H. R. Thermodynamic characterization of the coupled folding and association of heterodimeric coiled coils (leucine zippers). J Mol Biol. 1996 Oct 25;263(2):344–358. doi: 10.1006/jmbi.1996.0579. [DOI] [PubMed] [Google Scholar]
  18. Kammerer R. A. Alpha-helical coiled-coil oligomerization domains in extracellular proteins. Matrix Biol. 1997 Mar;15(8-9):555–568. doi: 10.1016/s0945-053x(97)90031-7. [DOI] [PubMed] [Google Scholar]
  19. Kammerer R. A., Antonsson P., Schulthess T., Fauser C., Engel J. Selective chain recognition in the C-terminal alpha-helical coiled-coil region of laminin. J Mol Biol. 1995 Jun 30;250(1):64–73. doi: 10.1006/jmbi.1995.0358. [DOI] [PubMed] [Google Scholar]
  20. King L., Seidel J. C., Lehrer S. S. Unfolding domains in smooth muscle myosin rod. Biochemistry. 1995 May 23;34(20):6770–6774. doi: 10.1021/bi00020a023. [DOI] [PubMed] [Google Scholar]
  21. Kohn W. D., Mant C. T., Hodges R. S. Alpha-helical protein assembly motifs. J Biol Chem. 1997 Jan 31;272(5):2583–2586. doi: 10.1074/jbc.272.5.2583. [DOI] [PubMed] [Google Scholar]
  22. Lehrer S. S. Effects of an interchain disulfide bond on tropomyosin structure: intrinsic fluorescence and circular dichroism studies. J Mol Biol. 1978 Jan 15;118(2):209–226. doi: 10.1016/0022-2836(78)90413-8. [DOI] [PubMed] [Google Scholar]
  23. Lumb K. J., Carr C. M., Kim P. S. Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4. Biochemistry. 1994 Jun 14;33(23):7361–7367. doi: 10.1021/bi00189a042. [DOI] [PubMed] [Google Scholar]
  24. Lupas A. Coiled coils: new structures and new functions. Trends Biochem Sci. 1996 Oct;21(10):375–382. [PubMed] [Google Scholar]
  25. Lupas A., Van Dyke M., Stock J. Predicting coiled coils from protein sequences. Science. 1991 May 24;252(5009):1162–1164. doi: 10.1126/science.252.5009.1162. [DOI] [PubMed] [Google Scholar]
  26. Malashkevich V. N., Kammerer R. A., Efimov V. P., Schulthess T., Engel J. The crystal structure of a five-stranded coiled coil in COMP: a prototype ion channel? Science. 1996 Nov 1;274(5288):761–765. doi: 10.1126/science.274.5288.761. [DOI] [PubMed] [Google Scholar]
  27. Marqusee S., Baldwin R. L. Helix stabilization by Glu-...Lys+ salt bridges in short peptides of de novo design. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8898–8902. doi: 10.1073/pnas.84.24.8898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Matsudaira P. Modular organization of actin crosslinking proteins. Trends Biochem Sci. 1991 Mar;16(3):87–92. doi: 10.1016/0968-0004(91)90039-x. [DOI] [PubMed] [Google Scholar]
  29. McLachlan A. D., Stewart M. Tropomyosin coiled-coil interactions: evidence for an unstaggered structure. J Mol Biol. 1975 Oct 25;98(2):293–304. doi: 10.1016/s0022-2836(75)80119-7. [DOI] [PubMed] [Google Scholar]
  30. Mo J. M., Holtzer M. E., Holtzer A. Kinetics of self-assembly of alpha alpha-tropomyosin coiled coils from unfolded chains. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):916–920. doi: 10.1073/pnas.88.3.916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mo J., Holtzer M. E., Holtzer A. Kinetics of folding and unfolding of beta beta-tropomyosin. Biopolymers. 1992 Nov;32(11):1581–1587. doi: 10.1002/bip.360321115. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. O'Shea E. K., Klemm J. D., Kim P. S., Alber T. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science. 1991 Oct 25;254(5031):539–544. doi: 10.1126/science.1948029. [DOI] [PubMed] [Google Scholar]
  34. O'Shea E. K., Rutkowski R., Kim P. S. Mechanism of specificity in the Fos-Jun oncoprotein heterodimer. Cell. 1992 Feb 21;68(4):699–708. doi: 10.1016/0092-8674(92)90145-3. [DOI] [PubMed] [Google Scholar]
  35. O'Shea E. K., Rutkowski R., Stafford W. F., 3rd, Kim P. S. Preferential heterodimer formation by isolated leucine zippers from fos and jun. Science. 1989 Aug 11;245(4918):646–648. doi: 10.1126/science.2503872. [DOI] [PubMed] [Google Scholar]
  36. Parthasarathy R., Chaturvedi S., Go K. Design of alpha-helical peptides: their role in protein folding and molecular biology. Prog Biophys Mol Biol. 1995;64(1):1–54. doi: 10.1016/0079-6107(95)00009-7. [DOI] [PubMed] [Google Scholar]
  37. Scholtz J. M., Baldwin R. L. The mechanism of alpha-helix formation by peptides. Annu Rev Biophys Biomol Struct. 1992;21:95–118. doi: 10.1146/annurev.bb.21.060192.000523. [DOI] [PubMed] [Google Scholar]
  38. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  39. Shoemaker K. R., Fairman R., Kim P. S., York E. J., Stewart J. M., Baldwin R. L. The C-peptide helix from ribonuclease A considered as an autonomous folding unit. Cold Spring Harb Symp Quant Biol. 1987;52:391–398. doi: 10.1101/sqb.1987.052.01.045. [DOI] [PubMed] [Google Scholar]
  40. Sodek J., Hodges R. S., Smillie L. B., Jurasek L. Amino-acid sequence of rabbit skeletal tropomyosin and its coiled-coil structure. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3800–3804. doi: 10.1073/pnas.69.12.3800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sosnick T. R., Jackson S., Wilk R. R., Englander S. W., DeGrado W. F. The role of helix formation in the folding of a fully alpha-helical coiled coil. Proteins. 1996 Apr;24(4):427–432. doi: 10.1002/(SICI)1097-0134(199604)24:4<427::AID-PROT2>3.0.CO;2-B. [DOI] [PubMed] [Google Scholar]
  42. Stryer L. The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. J Mol Biol. 1965 Sep;13(2):482–495. doi: 10.1016/s0022-2836(65)80111-5. [DOI] [PubMed] [Google Scholar]
  43. Su J. Y., Hodges R. S., Kay C. M. Effect of chain length on the formation and stability of synthetic alpha-helical coiled coils. Biochemistry. 1994 Dec 27;33(51):15501–15510. doi: 10.1021/bi00255a032. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Tripet B., Vale R. D., Hodges R. S. Demonstration of coiled-coil interactions within the kinesin neck region using synthetic peptides. Implications for motor activity. J Biol Chem. 1997 Apr 4;272(14):8946–8956. doi: 10.1074/jbc.272.14.8946. [DOI] [PubMed] [Google Scholar]
  46. Trybus K. M., Freyzon Y., Faust L. Z., Sweeney H. L. Spare the rod, spoil the regulation: necessity for a myosin rod. Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):48–52. doi: 10.1073/pnas.94.1.48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wendt H., Berger C., Baici A., Thomas R. M., Bosshard H. R. Kinetics of folding of leucine zipper domains. Biochemistry. 1995 Mar 28;34(12):4097–4107. doi: 10.1021/bi00012a028. [DOI] [PubMed] [Google Scholar]
  48. Wendt H., Leder L., Härmä H., Jelesarov I., Baici A., Bosshard H. R. Very rapid, ionic strength-dependent association and folding of a heterodimeric leucine zipper. Biochemistry. 1997 Jan 7;36(1):204–213. doi: 10.1021/bi961672y. [DOI] [PubMed] [Google Scholar]
  49. Williams S., Causgrove T. P., Gilmanshin R., Fang K. S., Callender R. H., Woodruff W. H., Dyer R. B. Fast events in protein folding: helix melting and formation in a small peptide. Biochemistry. 1996 Jan 23;35(3):691–697. doi: 10.1021/bi952217p. [DOI] [PubMed] [Google Scholar]
  50. Wrigley N. G. The lattice spacing of crystalline catalase as an internal standard of length in electron microscopy. J Ultrastruct Res. 1968 Sep;24(5):454–464. doi: 10.1016/s0022-5320(68)80048-6. [DOI] [PubMed] [Google Scholar]
  51. Zhou N. E., Kay C. M., Hodges R. S. Synthetic model proteins: the relative contribution of leucine residues at the nonequivalent positions of the 3-4 hydrophobic repeat to the stability of the two-stranded alpha-helical coiled-coil. Biochemistry. 1992 Jun 30;31(25):5739–5746. doi: 10.1021/bi00140a008. [DOI] [PubMed] [Google Scholar]
  52. Zhou N. E., Kay C. M., Hodges R. S. The role of interhelical ionic interactions in controlling protein folding and stability. De novo designed synthetic two-stranded alpha-helical coiled-coils. J Mol Biol. 1994 Apr 8;237(4):500–512. doi: 10.1006/jmbi.1994.1250. [DOI] [PubMed] [Google Scholar]
  53. Zitzewitz J. A., Bilsel O., Luo J., Jones B. E., Matthews C. R. Probing the folding mechanism of a leucine zipper peptide by stopped-flow circular dichroism spectroscopy. Biochemistry. 1995 Oct 3;34(39):12812–12819. doi: 10.1021/bi00039a042. [DOI] [PubMed] [Google Scholar]

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