Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2002 May;82(5):2700–2708. doi: 10.1016/S0006-3495(02)75611-7

Two-dimensional crystal structures of protein kinase C-delta, its regulatory domain, and the enzyme complexed with myelin basic protein.

Alexander S Solodukhin 1, Heather L Caldwell 1, Julianne J Sando 1, Robert H Kretsinger 1
PMCID: PMC1302058  PMID: 11964256

Abstract

Two-dimensional crystals of protein kinase C (PKC) delta, its regulatory domain (RDdelta), and the enzyme complexed with the substrate myelin basic protein have been grown on lipid monolayers composed of phosphatidylcholine: phosphatidylserine: diolein (45:50:5, molar ratio). Images have been reconstructed to 10-A resolution. The unit cells of all three proteins have cell edges a = b and interedge angle gamma = 60 degrees. RDdelta has an edge length of 33 +/- 1 A, and its reconstruction is donut shaped. The three-dimensional reconstructions from the PKCdelta C1b crystal structure () can be accommodated in this two-dimensional projection. Intact PKCdelta has an edge length of 46 +/- 1 A in the presence or absence of a nonhydrolyzable ATP analog, AMP-PnP. Its reconstruction has a similar donut shape, which can accommodate the C1b domain, but the spacing between donuts is greater than that in RDdelta; some additional structure is visible between the donuts. The complex of PKCdelta and myelin basic protein, with or without AMP-PnP, has an edge length of 43 +/- 1 A and a distinct structure. These results indicate that the C1 domains of RDdelta are tightly packed in the plane of the membrane in the two-dimensional crystals, that there is a single molecule of PKCdelta in the unit cell, and that its interaction with myelin basic protein induces a shift in conformation and/or packing of the enzyme.

Full Text

The Full Text of this article is available as a PDF (327.5 KB).

Selected References

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

  1. Carragher B., Smith P. R. Advances in computational image processing for microscopy. J Struct Biol. 1996 Jan-Feb;116(1):2–8. doi: 10.1006/jsbi.1996.0002. [DOI] [PubMed] [Google Scholar]
  2. Conesa-Zamora P., Gómez-Fernández J. C., Corbalán-García S. The C2 domain of protein kinase calpha is directly involved in the diacylglycerol-dependent binding of the C1 domain to the membrane. Biochim Biophys Acta. 2000 Sep 27;1487(2-3):246–254. doi: 10.1016/s1388-1981(00)00099-8. [DOI] [PubMed] [Google Scholar]
  3. Crowther R. A., Henderson R., Smith J. M. MRC image processing programs. J Struct Biol. 1996 Jan-Feb;116(1):9–16. doi: 10.1006/jsbi.1996.0003. [DOI] [PubMed] [Google Scholar]
  4. Frank J., Radermacher M., Penczek P., Zhu J., Li Y., Ladjadj M., Leith A. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol. 1996 Jan-Feb;116(1):190–199. doi: 10.1006/jsbi.1996.0030. [DOI] [PubMed] [Google Scholar]
  5. Frank J., Verschoor A., Boublik M. Computer averaging of electron micrographs of 40S ribosomal subunits. Science. 1981 Dec 18;214(4527):1353–1355. doi: 10.1126/science.7313694. [DOI] [PubMed] [Google Scholar]
  6. García-García J., Gómez-Fernández J. C., Corbalán-García S. Structural characterization of the C2 domain of novel protein kinase Cepsilon. Eur J Biochem. 2001 Feb;268(4):1107–1117. doi: 10.1046/j.1432-1327.2001.2680041107.x. [DOI] [PubMed] [Google Scholar]
  7. Glaeser R. M., Downing K. H. Assessment of resolution in biological electron crystallography. Ultramicroscopy. 1992 Nov;47(1-3):256–265. doi: 10.1016/0304-3991(92)90201-t. [DOI] [PubMed] [Google Scholar]
  8. Goldsmith E. J., Cobb M. H. Protein kinases. Curr Opin Struct Biol. 1994 Dec;4(6):833–840. doi: 10.1016/0959-440x(94)90264-x. [DOI] [PubMed] [Google Scholar]
  9. Hardt S., Wang B., Schmid M. F. A brief description of I.C.E.: the integrated crystallographic environment. J Struct Biol. 1996 Jan-Feb;116(1):68–70. doi: 10.1006/jsbi.1996.0012. [DOI] [PubMed] [Google Scholar]
  10. Henderson R. The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q Rev Biophys. 1995 May;28(2):171–193. doi: 10.1017/s003358350000305x. [DOI] [PubMed] [Google Scholar]
  11. Heymann J. B. Bsoft: image and molecular processing in electron microscopy. J Struct Biol. 2001 Feb-Mar;133(2-3):156–169. doi: 10.1006/jsbi.2001.4339. [DOI] [PubMed] [Google Scholar]
  12. Hommel U., Zurini M., Luyten M. Solution structure of a cysteine rich domain of rat protein kinase C. Nat Struct Biol. 1994 Jun;1(6):383–387. doi: 10.1038/nsb0694-383. [DOI] [PubMed] [Google Scholar]
  13. House C., Kemp B. E. Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science. 1987 Dec 18;238(4834):1726–1728. doi: 10.1126/science.3686012. [DOI] [PubMed] [Google Scholar]
  14. Huang S. M., Leventhal P. S., Wiepz G. J., Bertics P. J. Calcium and phosphatidylserine stimulate the self-association of conventional protein kinase C isoforms. Biochemistry. 1999 Sep 14;38(37):12020–12027. doi: 10.1021/bi990594m. [DOI] [PubMed] [Google Scholar]
  15. Hubbard S. R., Bishop W. R., Kirschmeier P., George S. J., Cramer S. P., Hendrickson W. A. Identification and characterization of zinc binding sites in protein kinase C. Science. 1991 Dec 20;254(5039):1776–1779. doi: 10.1126/science.1763327. [DOI] [PubMed] [Google Scholar]
  16. Hurley J. H., Newton A. C., Parker P. J., Blumberg P. M., Nishizuka Y. Taxonomy and function of C1 protein kinase C homology domains. Protein Sci. 1997 Feb;6(2):477–480. doi: 10.1002/pro.5560060228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Klug A., De Rosier D. J. Optical filtering of electron micrographs: reconstruction of one-sided images. Nature. 1966 Oct 1;212(5057):29–32. doi: 10.1038/212029a0. [DOI] [PubMed] [Google Scholar]
  18. Knighton D. R., Zheng J. H., Ten Eyck L. F., Ashford V. A., Xuong N. H., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. doi: 10.1126/science.1862342. [DOI] [PubMed] [Google Scholar]
  19. Lévy D., Mosser G., Lambert O., Moeck G. S., Bald D., Rigaud J. L. Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins. J Struct Biol. 1999 Aug;127(1):44–52. doi: 10.1006/jsbi.1999.4155. [DOI] [PubMed] [Google Scholar]
  20. Medkova M., Cho W. Interplay of C1 and C2 domains of protein kinase C-alpha in its membrane binding and activation. J Biol Chem. 1999 Jul 9;274(28):19852–19861. doi: 10.1074/jbc.274.28.19852. [DOI] [PubMed] [Google Scholar]
  21. Mellor H., Parker P. J. The extended protein kinase C superfamily. Biochem J. 1998 Jun 1;332(Pt 2):281–292. doi: 10.1042/bj3320281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mosior M., McLaughlin S. Peptides that mimic the pseudosubstrate region of protein kinase C bind to acidic lipids in membranes. Biophys J. 1991 Jul;60(1):149–159. doi: 10.1016/S0006-3495(91)82038-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nalefski E. A., Falke J. J. Location of the membrane-docking face on the Ca2+-activated C2 domain of cytosolic phospholipase A2. Biochemistry. 1998 Dec 22;37(51):17642–17650. doi: 10.1021/bi982372e. [DOI] [PubMed] [Google Scholar]
  24. Nalefski E. A., Falke J. J. The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci. 1996 Dec;5(12):2375–2390. doi: 10.1002/pro.5560051201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Newman R. H., Carpenter E., Freemont P. S., Blundell T. L., Parker P. J. Microcrystals of the beta 1 isoenzyme of protein kinase C: an electron microscopic study. Biochem J. 1994 Mar 1;298(Pt 2):391–393. doi: 10.1042/bj2980391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Oancea E., Meyer T. Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell. 1998 Oct 30;95(3):307–318. doi: 10.1016/s0092-8674(00)81763-8. [DOI] [PubMed] [Google Scholar]
  27. Ochoa W. F., Garcia-Garcia J., Fita I., Corbalan-Garcia S., Verdaguer N., Gomez-Fernandez J. C. Structure of the C2 domain from novel protein kinase Cepsilon. A membrane binding model for Ca(2+)-independent C2 domains. J Mol Biol. 2001 Aug 24;311(4):837–849. doi: 10.1006/jmbi.2001.4910. [DOI] [PubMed] [Google Scholar]
  28. Orlova E. V., Dube P., Harris J. R., Beckman E., Zemlin F., Markl J., van Heel M. Structure of keyhole limpet hemocyanin type 1 (KLH1) at 15 A resolution by electron cryomicroscopy and angular reconstitution. J Mol Biol. 1997 Aug 22;271(3):417–437. doi: 10.1006/jmbi.1997.1182. [DOI] [PubMed] [Google Scholar]
  29. Orr J. W., Newton A. C. Intrapeptide regulation of protein kinase C. J Biol Chem. 1994 Mar 18;269(11):8383–8387. [PubMed] [Google Scholar]
  30. Owens J. M., Kretsinger R. H., Sando J. J., Chertihin O. I. Two-dimensional crystals of protein kinase C. J Struct Biol. 1998 Jan;121(1):61–67. doi: 10.1006/jsbi.1997.3956. [DOI] [PubMed] [Google Scholar]
  31. Pappa H., Murray-Rust J., Dekker L. V., Parker P. J., McDonald N. Q. Crystal structure of the C2 domain from protein kinase C-delta. Structure. 1998 Jul 15;6(7):885–894. doi: 10.1016/s0969-2126(98)00090-2. [DOI] [PubMed] [Google Scholar]
  32. Ron D., Kazanietz M. G. New insights into the regulation of protein kinase C and novel phorbol ester receptors. FASEB J. 1999 Oct;13(13):1658–1676. [PubMed] [Google Scholar]
  33. Sando J. J., Chertihin O. I. Activation of protein kinase C by lysophosphatidic acid: dependence on composition of phospholipid vesicles. Biochem J. 1996 Jul 15;317(Pt 2):583–588. doi: 10.1042/bj3170583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sando J. J., Chertihin O. I., Owens J. M., Kretsinger R. H. Contributions to maxima in protein kinase C activation. J Biol Chem. 1998 Dec 18;273(51):34022–34027. doi: 10.1074/jbc.273.51.34022. [DOI] [PubMed] [Google Scholar]
  35. Sando J. J., Young M. C. Identification of high-affinity phorbol ester receptor in cytosol of EL4 thymoma cells: requirement for calcium, magnesium, and phospholipids. Proc Natl Acad Sci U S A. 1983 May;80(9):2642–2646. doi: 10.1073/pnas.80.9.2642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Saxton W. O., Baumeister W. The correlation averaging of a regularly arranged bacterial cell envelope protein. J Microsc. 1982 Aug;127(Pt 2):127–138. doi: 10.1111/j.1365-2818.1982.tb00405.x. [DOI] [PubMed] [Google Scholar]
  37. Schmid M. F., Dargahi R., Tam M. W. SPECTRA: a system for processing electron images of crystals. Ultramicroscopy. 1993 Mar;48(3):251–264. doi: 10.1016/0304-3991(93)90099-j. [DOI] [PubMed] [Google Scholar]
  38. Shao X., Davletov B. A., Sutton R. B., Südhof T. C., Rizo J. Bipartite Ca2+-binding motif in C2 domains of synaptotagmin and protein kinase C. Science. 1996 Jul 12;273(5272):248–251. doi: 10.1126/science.273.5272.248. [DOI] [PubMed] [Google Scholar]
  39. Smith P. R., Gottesman S. M. The micrograph data processing program. J Struct Biol. 1996 Jan-Feb;116(1):35–40. doi: 10.1006/jsbi.1996.0007. [DOI] [PubMed] [Google Scholar]
  40. Srinivasan N., Bax B., Blundell T. L., Parker P. J. Structural aspects of the functional modules in human protein kinase-C alpha deduced from comparative analyses. Proteins. 1996 Oct;26(2):217–235. doi: 10.1002/(SICI)1097-0134(199610)26:2<217::AID-PROT11>3.0.CO;2-S. [DOI] [PubMed] [Google Scholar]
  41. Valpuesta J. M., Carrascosa J. L., Henderson R. Analysis of electron microscope images and electron diffraction patterns of thin crystals of phi 29 connectors in ice. J Mol Biol. 1994 Jul 22;240(4):281–287. doi: 10.1006/jmbi.1994.1445. [DOI] [PubMed] [Google Scholar]
  42. Wilson K. P., Fitzgibbon M. J., Caron P. R., Griffith J. P., Chen W., McCaffrey P. G., Chambers S. P., Su M. S. Crystal structure of p38 mitogen-activated protein kinase. J Biol Chem. 1996 Nov 1;271(44):27696–27700. doi: 10.1074/jbc.271.44.27696. [DOI] [PubMed] [Google Scholar]
  43. Zhang G., Kazanietz M. G., Blumberg P. M., Hurley J. H. Crystal structure of the cys2 activator-binding domain of protein kinase C delta in complex with phorbol ester. Cell. 1995 Jun 16;81(6):917–924. doi: 10.1016/0092-8674(95)90011-x. [DOI] [PubMed] [Google Scholar]
  44. van Heel M., Harauz G., Orlova E. V., Schmidt R., Schatz M. A new generation of the IMAGIC image processing system. J Struct Biol. 1996 Jan-Feb;116(1):17–24. doi: 10.1006/jsbi.1996.0004. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES