Skip to main content
NCBI home page
Protein & Cell logoLink to Protein & Cell
. 2011 Nov 6;2(10):814–826. doi: 10.1007/s13238-011-1090-6

Crystal structure of human Gadd45 reveals an active dimer

Wenzheng Zhang 1,2,3, Sheng Fu 1, Xuefeng Liu 4, Xuelian Zhao 4, Wenchi Zhang 1, Wei Peng 1, Congying Wu 3, Yuanyuan Li 3, Xuemei Li 1, Mark Bartlam 1,2, Zong-Hao Zeng 1,✉, Qimin Zhan 4,✉, Zihe Rao 1,2,3
PMCID: PMC4875293  PMID: 22058036

Abstract

The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45, revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45 identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45 is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.

Electronic Supplementary Material

Supplementary material is available for this article at 10.1007/s13238-011-1090-6 and is accessible for authorized users.

Keywords: crystal structure, Gadd45, dimer, DNA repair, growth inhibition, apoptosis

Electronic supplementary material

13238_2011_1090_MOESM1_ESM.pdf (870.3KB, pdf)

Supplementary material, approximately 870 KB.

Footnotes

These authors contributed equally to the work

Electronic Supplementary Material

Supplementary material is available for this article at 10.1007/s13238-011-1090-6 and is accessible for authorized users.

An erratum to this article can be found at http://dx.doi.org/10.1007/s13238-012-2038-1.

Contributor Information

Zong-Hao Zeng, Email: zzh@ibp.ac.cn.

Qimin Zhan, Email: zhanqimin@pumc.edu.cn.

References

  1. Azam N., Vairapandi M., Zhang W., Hoffman B., Liebermann D.A. Interaction of CR6 (GADD45gammax) with proliferating cell nuclear antigen impedes negative growth control. J Biol Chem. 2001;276:2766–2774. doi: 10.1074/jbc.M005626200. [DOI] [PubMed] [Google Scholar]
  2. Barreto G., Schäfer A., Marhold J., Stach D., Swaminathan S.K., Handa V., Döderlein G., Maltry N., Wu W., Lyko F., et al. Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation. Nature. 2007;445:671–675. doi: 10.1038/nature05515. [DOI] [PubMed] [Google Scholar]
  3. 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., et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr. 1998;54:905–921. doi: 10.1107/S0907444998003254. [DOI] [PubMed] [Google Scholar]
  4. Bulavin D.V., Kovalsky O., Hollander M.C., Fornace A.J., Jr. Loss of oncogenic H-ras-induced cell cycle arrest and p38 mitogen-activated protein kinase activation by disruption of Gadd45a. Mol Cell Biol. 2003;23:3859–3871. doi: 10.1128/MCB.23.11.3859-3871.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carrier F., Georgel P.T., Pourquier P., Blake M., Kontny H.U., Antinore M.J., Gariboldi M., Myers T.G., Weinstein J.N., Pommier Y., et al. Gadd45, a p53-responsive stress protein, modifies DNA accessibility on damaged chromatin. Mol Cell Biol. 1999;19:1673–1685. doi: 10.1128/MCB.19.3.1673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chung H.K., Yi Y.W., Jung N.C., Kim D., Suh J.M., Kim H., Park K. C., Song J.H., Kim D.W., Hwang E.S., et al. CR6-interacting factor 1 interacts with Gadd45 family proteins and modulates the cell cycle. J Biol Chem. 2003;278:28079–28088. doi: 10.1074/jbc.M212835200. [DOI] [PubMed] [Google Scholar]
  7. Emsley P., Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004;60:2126–2132. doi: 10.1107/S0907444904019158. [DOI] [PubMed] [Google Scholar]
  8. Fan W., Richter G., Cereseto A., Beadling C., Smith K.A. Cytokine response gene 6 induces p21 and regulates both cell growth and arrest. Oncogene. 1999;18:6573–6582. doi: 10.1038/sj.onc.1203054. [DOI] [PubMed] [Google Scholar]
  9. Furukawa-Hibi Y., Yoshida-Araki K., Ohta T., Ikeda K., Motoyama N. FOXO forkhead transcription factors induce G(2)-M checkpoint in response to oxidative stress. J Biol Chem. 2002;277:26729–26732. doi: 10.1074/jbc.C200256200. [DOI] [PubMed] [Google Scholar]
  10. Gao H., Jin S., Song Y., Fu M., Wang M., Liu Z., Wu M., Zhan Q. B23 regulates GADD45a nuclear translocation and contributes to GADD45a-induced cell cycle G2-M arrest. J Biol Chem. 2005;280:10988–10996. doi: 10.1074/jbc.M412720200. [DOI] [PubMed] [Google Scholar]
  11. Harkin D.P., Bean J.M., Miklos D., Song Y.H., Truong V.B., Englert C., Christians F.C., Ellisen L.W., Maheswaran S., Oliner J.D., et al. Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1. Cell. 1999;97:575–586. doi: 10.1016/S0092-8674(00)80769-2. [DOI] [PubMed] [Google Scholar]
  12. Jin S., Antinore M.J., Lung F.D., Dong X., Zhao H., Fan F., Colchagie A.B., Blanck P., Roller P.P., Fornace A.J., Jr, et al. The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression. J Biol Chem. 2000;275:16602–16608. doi: 10.1074/jbc.M000284200. [DOI] [PubMed] [Google Scholar]
  13. Jin S., Tong T., Fan W., Fan F., Antinore M.J., Zhu X., Mazzacurati L., Li X., Petrik K.L., Rajasekaran B., et al. GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity. Oncogene. 2002;21:8696–8704. doi: 10.1038/sj.onc.1206034. [DOI] [PubMed] [Google Scholar]
  14. Jung N., Yi Y.W., Kim D., Shong M., Hong S.S., Lee H.S., Bae I. Regulation of Gadd45gamma expression by C/EBP. Eur J Biochem. 2000;267:6180–6187. doi: 10.1046/j.1432-1327.2000.01692.x. [DOI] [PubMed] [Google Scholar]
  15. Kastan M.B., Zhan Q., el-Deiry W.S., Carrier F., Jacks T., Walsh W.V., Plunkett B.S., Vogelstein B., Fornace A.J., Jr. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 1992;71:587–597. doi: 10.1016/0092-8674(92)90593-2. [DOI] [PubMed] [Google Scholar]
  16. Kearsey J.M., Coates P.J., Prescott A.R., Warbrick E., Hall P. A. Gadd45 is a nuclear cell cycle regulated protein which interacts with p21Cip1. Oncogene. 1995;11:1675–1683. [PubMed] [Google Scholar]
  17. Kovalsky O., Lung F.D., Roller P.P., Fornace A.J., Jr. Oligomerization of human Gadd45a protein. J Biol Chem. 2001;276:39330–39339. doi: 10.1074/jbc.M105115200. [DOI] [PubMed] [Google Scholar]
  18. Kraulis P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Cryst. 1991;24:946–950. doi: 10.1107/S0021889891004399. [DOI] [Google Scholar]
  19. Laue T., Shaw B.D., Ridgeway T.M., Pelletier S.L. Computer-aided Interpretation of Analytical Sedimentation Data For Proteins. In: Harding S.E., Rowe A.J., Horton J.C., editors. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Cambridge, UK: Royal Soc Chem; 1991. pp. 90–125. [Google Scholar]
  20. Lefort K., Rouault J.P., Tondereau L., Magaud J.P., Doré J.F. The specific activation of gadd45 following UVB radiation requires the POU family gene product N-oct3 in human melanoma cells. Oncogene. 2001;20:7375–7385. doi: 10.1038/sj.onc.1204923. [DOI] [PubMed] [Google Scholar]
  21. Liebermann D.A., Hoffman B. Gadd45 in the response of hematopoietic cells to genotoxic stress. Blood Cells Mol Dis. 2007;39:329–335. doi: 10.1016/j.bcmd.2007.06.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mak S.K., Kültz D. Gadd45 proteins induce G2/M arrest and modulate apoptosis in kidney cells exposed to hyperosmotic stress. J Biol Chem. 2004;279:39075–39084. doi: 10.1074/jbc.M406643200. [DOI] [PubMed] [Google Scholar]
  23. Merritt E.A., Bacon D.J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 1997;277:505–524. doi: 10.1016/S0076-6879(97)77028-9. [DOI] [PubMed] [Google Scholar]
  24. Murshudov G.N., Vagin A.A., Dodson E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997;53:240–255. doi: 10.1107/S0907444996012255. [DOI] [PubMed] [Google Scholar]
  25. Nakayama K., Hara T., Hibi M., Hirano T., Miyajima A. A novel oncostatin M-inducible gene OIG37 forms a gene family with MyD118 and GADD45 and negatively regulates cell growth. J Biol Chem. 1999;274:24766–24772. doi: 10.1074/jbc.274.35.24766. [DOI] [PubMed] [Google Scholar]
  26. Otwinowski Z., Minor W. Processing of X-ray Diffraction Data Collected in Oscillation Mode. In: Carter C.W. Jr., Sweet R.M., editors. Macromolecular Crystallography. New York: Academic; 1997. pp. 307–326. [DOI] [PubMed] [Google Scholar]
  27. Papa S., Zazzeroni F., Bubici C., Jayawardena S., Alvarez K., Matsuda S., Nguyen D.U., Pham C.G., Nelsbach A.H., Melis T., et al. Gadd45 beta mediates the NF-kappa B suppression of JNK signalling by targeting MKK7/JNKK2. Nat Cell Biol. 2004;6:146–153. doi: 10.1038/ncb1093. [DOI] [PubMed] [Google Scholar]
  28. Schrag J.D., Jiralerspong S., Banville M., Jaramillo M.L., O’Connor-McCourt M.D. The crystal structure and dimerization interface of GADD45gamma. Proc Natl Acad Sci U S A. 2008;105:6566–6571. doi: 10.1073/pnas.0800086105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schuck P., Perugini M.A., Gonzales N.R., Howlett G.J., Schubert D. Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems. Biophys J. 2002;82:1096–1111. doi: 10.1016/S0006-3495(02)75469-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shao S., Wang Y., Jin S., Song Y., Wang X., Fan W., Zhao Z., Fu M., Tong T., Dong L., et al. Gadd45a interacts with aurora-A and inhibits its kinase activity. J Biol Chem. 2006;281:28943–28950. doi: 10.1074/jbc.M600235200. [DOI] [PubMed] [Google Scholar]
  31. Smith G.B., Mocarski E.S. Contribution of GADD45 family members to cell death suppression by cellular Bcl-xL and cytomegalovirus vMIA. J Virol. 2005;79:14923–14932. doi: 10.1128/JVI.79.23.14923-14932.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Smith M.L., Chen I.T., Zhan Q., Bae I., Chen C.Y., Gilmer T.M., Kastan M.B., O’Connor P.M., Fornace A.J., Jr. Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science. 1994;266:1376–1380. doi: 10.1126/science.7973727. [DOI] [PubMed] [Google Scholar]
  33. Smith M.L., Ford J.M., Hollander M.C., Bortnick R.A., Amundson S.A., Seo Y.R., Deng C.X., Hanawalt P.C., Fornace A.J., Jr. p53-mediated DNA repair responses to UV radiation: studies of mouse cells lacking p53, p21, and/or gadd45 genes. Mol Cell Biol. 2000;20:3705–3714. doi: 10.1128/MCB.20.10.3705-3714.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sun L., Gong R., Wan B., Huang X., Wu C., Zhang X., Zhao S., Yu L. GADD45gamma, down-regulated in 65% hepatocellular carcinoma (HCC) from 23 chinese patients, inhibits cell growth and induces cell cycle G2/M arrest for hepatoma Hep-G2 cell lines. Mol Biol Rep. 2003;30:249–253. doi: 10.1023/A:1026370726763. [DOI] [PubMed] [Google Scholar]
  35. Takekawa M., Saito H. A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell. 1998;95:521–530. doi: 10.1016/S0092-8674(00)81619-0. [DOI] [PubMed] [Google Scholar]
  36. Thyss R., Virolle V., Imbert V., Peyron J.F., Aberdam D., Virolle T. NF-kappaB/Egr-1/Gadd45 are sequentially activated upon UVB irradiation to mediate epidermal cell death. EMBO J. 2005;24:128–137. doi: 10.1038/sj.emboj.7600501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tornatore L., Marasco D., Dathan N., Vitale R.M., Benedetti E., Papa S., Franzoso G., Ruvo M., Monti S.M. Gadd45 beta forms a homodimeric complex that binds tightly to MKK7. J Mol Biol. 2008;378:97–111. doi: 10.1016/j.jmb.2008.01.074. [DOI] [PubMed] [Google Scholar]
  38. Vairapandi M., Azam N., Balliet A.G., Hoffman B., Liebermann D.A. Characterization of MyD118, Gadd45, and proliferating cell nuclear antigen (PCNA) interacting domains. PCNA impedes MyD118 AND Gadd45-mediated negative growth control. J Biol Chem. 2000;275:16810–16819. doi: 10.1074/jbc.275.22.16810. [DOI] [PubMed] [Google Scholar]
  39. Vairapandi M., Balliet A.G., Fornace A.J., Jr, Hoffman B., Liebermann D.A. The differentiation primary response gene MyD118, related to GADD45, encodes for a nuclear protein which interacts with PCNA and p21WAF1/CIP1. Oncogene. 1996;12:2579–2594. [PubMed] [Google Scholar]
  40. Vairapandi M., Balliet A.G., Hoffman B., Liebermann D.A. GADD45b and GADD45g are cdc2/cyclinB1 kinase inhibitors with a role in S and G2/M cell cycle checkpoints induced by genotoxic stress. J Cell Physiol. 2002;192:327–338. doi: 10.1002/jcp.10140. [DOI] [PubMed] [Google Scholar]
  41. Wang X.W., Zhan Q., Coursen J.D., Khan M.A., Kontny H.U., Yu L., Hollander M.C., O’Connor P.M., Fornace A.J., Jr, Harris C.C. GADD45 induction of a G2/M cell cycle checkpoint. Proc Natl Acad Sci U S A. 1999;96:3706–3711. doi: 10.1073/pnas.96.7.3706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zhan Q., Antinore M.J., Wang X.W., Carrier F., Smith M.L., Harris C.C., Fornace A.J. Jr. Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53-regulated protein Gadd45. Oncogene. 1999;18:2892–2900. doi: 10.1038/sj.onc.1202667. [DOI] [PubMed] [Google Scholar]
  43. Zhao H., Jin S., Antinore M.J., Lung F.D., Fan F., Blanck P., Roller P., Fornace A.J., Jr, Zhan Q. The central region of Gadd45 is required for its interaction with p21/WAF1. Exp Cell Res. 2000;258:92–100. doi: 10.1006/excr.2000.4906. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

13238_2011_1090_MOESM1_ESM.pdf (870.3KB, pdf)

Supplementary material, approximately 870 KB.

Articles from Protein & Cell are provided here courtesy of Oxford University Press

RESOURCES