Crystal structure of human Gadd45 reveals an active dimer

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.

Key words： crystal structure; Gadd45; dimer; DNA repair; growth inhibition; apoptosis

References

[1] Azam, N., Vairapandi, M., Zhang, W., Hoffman, B., and Liebermann, D.A. (2001). Interaction of CR6 (GADD45gamma ) with proliferating cell nuclear antigen impedes negative growth control. J Biol Chem 276, 2766-2774 11022036.
[2] Barreto, G., Sch?fer, A., Marhold, J., Stach, D., Swaminathan, S.K., Handa, V., D?derlein, G., Maltry, N., Wu, W., Lyko, F., (2007). Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation. Nature 445, 671-675 17268471.
[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., (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54, 905-921 9757107.
[4] Bulavin, D.V., Kovalsky, O., Hollander, M.C., and Fornace, A.J. Jr. (2003). Loss of oncogenic H-ras-induced cell cycle arrest and p38 mitogen-activated protein kinase activation by disruption of Gadd45a. Mol Cell Biol 23, 3859-3871 12748288.
[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., (1999). Gadd45, a p53-responsive stress protein, modifies DNA accessibility on damaged chromatin. Mol Cell Biol 19, 1673-1685 10022855.
[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., (2003). CR6-interacting factor 1 interacts with Gadd45 family proteins and modulates the cell cycle. J Biol Chem 278, 28079-28088 12716909.
[7] Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126-2132 15572765.
[8] Fan, W., Richter, G., Cereseto, A., Beadling, C., and Smith, K.A. (1999). Cytokine response gene 6 induces p21 and regulates both cell growth and arrest. Oncogene 18, 6573-6582 10597261.
[9] Furukawa-Hibi, Y., Yoshida-Araki, K., Ohta, T., Ikeda, K., and Motoyama, N. (2002). FOXO forkhead transcription factors induce G(2)-M checkpoint in response to oxidative stress. J Biol Chem 277, 26729-26732 12048180.
[10] Gao, H., Jin, S., Song, Y., Fu, M., Wang, M., Liu, Z., Wu, M., and Zhan, Q. (2005). B23 regulates GADD45a nuclear translocation and contributes to GADD45a-induced cell cycle G2-M arrest. J Biol Chem 280, 10988-10996 15644315.
[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., (1999). Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1. Cell 97, 575-586 10367887.
[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, (2000). The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression. J Biol Chem 275, 16602-16608 10747892.
[13] Jin, S., Tong, T., Fan, W., Fan, F., Antinore, M.J., Zhu, X., Mazzacurati, L., Li, X., Petrik, K.L., Rajasekaran, B., (2002). GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity. Oncogene 21, 8696-8704 12483522.
[14] Jung, N., Yi, Y.W., Kim, D., Shong, M., Hong, S.S., Lee, H.S., and Bae, I. (2000). Regulation of Gadd45gamma expression by C/EBP. Eur J Biochem 267, 6180-6187 11012671.
[15] Kastan, M.B., Zhan, Q., el-Deiry, W.S., Carrier, F., Jacks, T., Walsh, W.V., Plunkett, B.S., Vogelstein, B., and Fornace, A.J. Jr. (1992). A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71, 587-597 1423616.
[16] Kearsey, J.M., Coates, P.J., Prescott, A.R., Warbrick, E., and Hall, P.A. (1995). Gadd45 is a nuclear cell cycle regulated protein which interacts with p21Cip1. Oncogene 11, 1675-1683 7478594.
[17] Kovalsky, O., Lung, F.D., Roller, P.P., and Fornace, A.J. Jr. (2001). Oligomerization of human Gadd45a protein. J Biol Chem 276, 39330-39339 11498536.
[18] Kraulis, P.J. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Cryst 24, 946-950 .
[19] Laue, T., Shaw, B.D., Ridgeway, T.M., and Pelletier, S.L. (1991). Computer-aided Interpretation of Analytical Sedimentation Data For Proteins. In Analytical Ultracentrifugation in Biochemistry and Polymer Science , Harding S.E. , Rowe A.J. , and Horton J.C., eds. (Cambridge, UK, Royal Soc. Chem.), pp. 90-125 .
[20] Lefort, K., Rouault, J.P., Tondereau, L., Magaud, J.P., and Doré, J.F. (2001). The specific activation of gadd45 following UVB radiation requires the POU family gene product N-oct3 in human melanoma cells. Oncogene 20, 7375-7385 11704867.
[21] Liebermann, D.A., and Hoffman, B. (2007). Gadd45 in the response of hematopoietic cells to genotoxic stress. Blood Cells Mol Dis 39, 329-335 17659913.
[22] Mak, S.K., and Kültz, D. (2004). Gadd45 proteins induce G2/M arrest and modulate apoptosis in kidney cells exposed to hyperosmotic stress. J Biol Chem 279, 39075-39084 15262964.
[23] Merritt, E.A., and Bacon, D.J. (1997). Raster3D: photorealistic molecular graphics. Methods Enzymol 277, 505-524 18488322.
[24] Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53, 240-255 15299926.
[25] Nakayama, K., Hara, T., Hibi, M., Hirano, T., and Miyajima, A. (1999). A novel oncostatin M-inducible gene OIG37 forms a gene family with MyD118 and GADD45 and negatively regulates cell growth. J Biol Chem 274, 24766-24772 10455148.
[26] Otwinowski, Z., and Minor, W. (1997). Processing of X-ray Diffraction Data Collected in Oscillation Mode. In Macromolecular Crystallography , Carter C.W. Jr., and Sweet R.M., eds. (New York, Academic), pp. 307-326 .
[27] Papa, S., Zazzeroni, F., Bubici, C., Jayawardena, S., Alvarez, K., Matsuda, S., Nguyen, D.U., Pham, C.G., Nelsbach, A.H., Melis, T., (2004). Gadd45 beta mediates the NF-kappa B suppression of JNK signalling by targeting MKK7/JNKK2. Nat Cell Biol 6, 146-153 14743220.
[28] Schrag, J.D., Jiralerspong, S., Banville, M., Jaramillo, M.L., and O’Connor-McCourt, M.D. (2008). The crystal structure and dimerization interface of GADD45gamma. Proc Natl Acad Sci U S A 105, 6566-6571 18445651.
[29] Schuck, P., Perugini, M.A., Gonzales, N.R., Howlett, G.J., and Schubert, D. (2002). Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems. Biophys J 82, 1096-1111 11806949.
[30] Shao, S., Wang, Y., Jin, S., Song, Y., Wang, X., Fan, W., Zhao, Z., Fu, M., Tong, T., Dong, L., (2006). Gadd45a interacts with aurora-A and inhibits its kinase activity. J Biol Chem 281, 28943-28950 16772293.
[31] Smith, G.B., and Mocarski, E.S. (2005). Contribution of GADD45 family members to cell death suppression by cellular Bcl-xL and cytomegalovirus vMIA. J Virol 79, 14923-14932 16282491.
[32] Smith, M.L., Chen, I.T., Zhan, Q., Bae, I., Chen, C.Y., Gilmer, T.M., Kastan, M.B., O’Connor, P.M., and Fornace, A.J. Jr. (1994). Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 266, 1376-1380 7973727.
[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., and Fornace, A.J. Jr. (2000). p53-mediated DNA repair responses to UV radiation: studies of mouse cells lacking p53, p21, and/or gadd45 genes. Mol Cell Biol 20, 3705-3714 10779360.
[34] Sun, L., Gong, R., Wan, B., Huang, X., Wu, C., Zhang, X., Zhao, S., and Yu, L. (2003). 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 30, 249-253 14672412.
[35] Takekawa, M., and Saito, H. (1998). A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell 95, 521-530 9827804.
[36] Thyss, R., Virolle, V., Imbert, V., Peyron, J.F., Aberdam, D., and Virolle, T. (2005). NF-kappaB/Egr-1/Gadd45 are sequentially activated upon UVB irradiation to mediate epidermal cell death. EMBO J 24, 128-137 15616591.
[37] Tornatore, L., Marasco, D., Dathan, N., Vitale, R.M., Benedetti, E., Papa, S., Franzoso, G., Ruvo, M., and Monti, S.M. (2008). Gadd45 beta forms a homodimeric complex that binds tightly to MKK7. J Mol Biol 378, 97-111 18343408.
[38] Vairapandi, M., Azam, N., Balliet, A.G., Hoffman, B., and Liebermann, D.A. (2000). Characterization of MyD118, Gadd45, and proliferating cell nuclear antigen (PCNA) interacting domains. PCNA impedes MyD118 AND Gadd45-mediated negative growth control. J Biol Chem 275, 16810-16819 10828065.
[39] Vairapandi, M., Balliet, A.G., Fornace, A.J. Jr, Hoffman, B., and Liebermann, D.A. (1996). The differentiation primary response gene MyD118, related to GADD45, encodes for a nuclear protein which interacts with PCNA and p21WAF1/CIP1. Oncogene 12, 2579-2594 8700517.
[40] Vairapandi, M., Balliet, A.G., Hoffman, B., and Liebermann, D.A. (2002). 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 192, 327-338 12124778.
[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, and Harris, C.C. (1999). GADD45 induction of a G2/M cell cycle checkpoint. Proc Natl Acad Sci U S A 96, 3706-3711 10097101.
[42] Zhan, Q., Antinore, M.J., Wang, X.W., Carrier, F., Smith, M.L., Harris, C.C., and Fornace, A.J. Jr. (1999). Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53-regulated protein Gadd45. Oncogene 18, 2892-2900 10362260.
[43] Zhao, H., Jin, S., Antinore, M.J., Lung, F.D., Fan, F., Blanck, P., Roller, P., Fornace, A.J. Jr, and Zhan, Q. (2000). The central region of Gadd45 is required for its interaction with p21/WAF1. Exp Cell Res 258, 92-100 10912791