Solution Structure of the Transactivation Domain of ATF-2 Comprising a Zinc Finger-like Subdomain and a Flexible Subdomain Aritaka Nagadoi 1 , Ken-ichi Nakazawa 1 , Hiroko Uda 1 , Koichi Okuno 1 Toshio Maekawa 2 , Shunsuke Ishii 2 and Yoshifumi Nishimura 1,3 * 1 Graduate School of Integrated Science, Yokohama City University, 22-2 Seto Kanazawa-ku, Yokohama 236-0027, Japan 2 Laboratory of Molecular Genetics, Tsukuba Life Science Center, RIKEN, Koyadai Tsukuba, Ibaraki 305-0074, Japan 3 Cellular Signaling Laboratory The Institute of Physical and Chemical Research (RIKEN) 2-1 Hirosawa, Wako-shi Saitama 351-0198, Japan Activating transcription factor-2 (ATF-2) is a transcription factor that binds to cAMP response element (CRE). ATF-2 contains two functional domains, an N-terminal transactivation domain and a C-terminal DNA- binding domain. The DNA-binding domain contains the basic leucine zipper (bZip) motif. Here, the three-dimensional structure of the transact- ivation domain of ATF-2 has been determined by NMR. The transactiva- tion domain consists of two subdomains: the structure of an N-terminal half (N-subdomain) is well determined, while a C-terminal half (C-subdo- main) takes a highly ¯exible and disordered structure. The architecture of the N-subdomain is very similar to that of the well-known zinc ®nger motif found in DNA-binding domains, consisting of an antiparallel b-sheet and an a-helix. The zinc atom is tetrahedrally coordinated to two cysteine residues and two histidine residues. Amino acids that form the hydrophobic core in all of the DNA-binding zinc ®ngers are well con- served in the N-subdomain of the transactivation domain, whereas some amino acids that are responsible for binding to the phosphate backbone of DNA in the DNA-binding zinc ®ngers are substituted with other amino acids. The ¯exible C-subdomain, which contains two threonine residues that the stress-activated protein kinases phosphorylate, is likely to undergo a conformational change by speci®c binding to a target pro- tein. # 1999 Academic Press Keywords: ATF-2; three-dimensional structure; NMR; transactivation domain; zinc ®nger *Corresponding author Introduction Activating transcription factor 2 (ATF-2; Hai et al., 1989), also called cyclic AMP response element (CRE) binding protein-1 (CRE-BP1; Maekawa et al., 1989), is a member of the ATF/ CREB family of transcription factors that bind to CRE, which is an inducible enhancer in response to increased cAMP levels. ATF-2 binds to CRE as a homodimer or a heterodimer with c-Jun (Ivashkiv et al., 1990; Macgregor et al., 1990). The stress-acti- vated protein kinases (SAPK), such as Jun amino- terminal kinase (JNK) and p38, phosphorylate ATF-2 and stimulate its transactivating capacity (Gupta et al., 1995; Livingstone et al., 1995; van Dam et al., 1995). Since CREB and its related fac- tors such as CREM are activated through direct phosphorylation by cAMP-dependent protein kinase (PKA; Gonzalez & Montminy; 1989), the factors belonging to the ATF-2 and CREB sub- groups in the ATF/CREB family are linked to the distinct signaling cascades involving the SAPK and PKA pathways. The expression of ATF-2 is ubiqui- E-mail address of the corresponding author: nisimura@yokohama-cu.ac.jp Abbreviations used: ATF-2, activating transcription factor 2; bZip, basic leucine zipper; CRE, cAMP response element; CRE-BP1, CRE binding protein 1; DQF-COSY, double quantum ®ltered correlated spectroscopy; DTT, dithiothreitol; HSQC, heteronuclear single quantum coherence; IPTG, isopropyl-1-thio-b-D- galactopyranoside; JNK, Jun amino-terminal kinase; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; PDB, Protein Data Bank; 4D-SA, four-dimensional simulated annealing; SAPK, stress-activated protein kinase; TOCSY, total correlation spectroscopy; TTK, Tramtrack; YY1, Ying-Yang 1 protein. Article No. jmbi.1999.2620 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 287, 593±607 0022-2836/99/130593±15 $30.00/0 # 1999 Academic Press