Crystal structure of unphosphorylated STAT3 core fragment Zhiyong Ren a , Xiang Mao a , Claudia Mertens b , Ravi Krishnaraj b , Jie Qin a , Pijus K. Mandal c , Michael J. Romanowski d , John S. McMurray c , Xiaomin Chen a, * a Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Unit 1000, Houston, TX 77030, USA b Laboratory of Molecular Cell Biology, The Rockefeller University, New York, NY, USA c Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, TX, USA d Sunesis Pharmaceuticals, Inc., South San Francisco, CA, USA article info Article history: Received 18 March 2008 Available online 21 April 2008 Keywords: STAT Signal transduction Dimerization abstract Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcriptional factors that play an important role in cytokine and growth factor signaling. Here we report a 3.05 Å-resolution crystal structure of an unphosphorylated STAT3 core fragment. The overall monomeric structure is very similar to that of the phosphorylated STAT3 core fragment. However, the dimer interface observed in the unphosphorylated STAT1 core fragment structure is absent in the STAT3 structure. Solution studies fur- ther demonstrate that the core fragment of STAT3 is primarily monomeric. Mutations corresponding to those in STAT1, which lead to disruption of the core fragment interface and prolonged tyrosine phosphor- ylation, show little or no effect on the tyrosine phosphorylation kinetics of STAT3. These results highlight the structural and biochemical differences between STAT3 and STAT1, and suggest different regulation mechanisms of these two proteins. Ó 2008 Elsevier Inc. All rights reserved. STAT proteins are latent cytoplasmic transcriptional factors that play an essential role in cytokine and growth factor signaling. Mammalian STATs share six structural regions: N-domain (ND), coiled-coil domain (CCD), DNA-binding domain (DBD), linker do- main, SH2 domain, and transcriptional activation domain (Fig. 1A). Upon receptor activation, a single tyrosine residue (Y705 in STAT3) is phosphorylated. Once phosphorylated, STATs form homo- or hetero-dimers via reciprocal SH2-phosphotyrosine interactions, translocate into the nucleus, and activate gene tran- scription [1–3]. Crystal structures of unphosphorylated STAT1 and STAT5 showed that the core fragment (residues 130 to 680) forms a re- ciprocal dimer involving CCD and DBD [4,5]. This so-called ‘‘anti- parallel” dimer has been shown to play an important role in STAT1 dephosphorylation [6,7]. To explore whether this interface is con- served in other STATs, we set out to determine the crystal structure of unphosphorylated STAT3. Materials and methods Molecular cloning, protein and peptidomimetic preparations. DNA sequence of corresponding mouse STAT3 residues 127–688 was cloned into pET20b(+) (Novagen). This protein was purified as pub- lished [8]. The peptidomimetic compound, PM50D (Fig. 1B), was synthesized as published [9]. QuikChange (Stratagene) was used to generate STAT3 mutations in the pRc/CMV vector. All of the STAT constructs and mutants were confirmed by sequencing. Crystallization, data collection, structure determination, and refinement. The complex of STAT3 (residues 127–688) and PM50D was prepared by mixing the protein with the peptide in a 1:1.5 molar ratio. Crystals in hanging drops were obtained at 4 °C by mixing 0.8 ll of 20 mg/ml protein-PM50D mixture, 0.8 ll of res- ervoir solution (100 mM Bis–Tris, pH 5.5–5.9, 500 mM (NH 4 ) 2 SO 4 , 10–12% PEG3350), 0.4 ll 500 mM KCl, and 0.5 ll 250 mM NaI. The best crystals (600  80  80 lm 3 ) were obtained in 3–4 days. Crystals were transferred through a series of cryoprotection solutions with increasing concentrations of glycerol (100 mM Bis–Tris, pH 5.5, 500 mM (NH 4 ) 2 SO 4 , 12% PEG3350, 100 mM KCl, 50 mM NaI, and 5%–25% glycerol) and flash frozen in liquid N 2 . Dif- fraction data were collected at Advanced Light Source (ALS) Beam- line 8.3.1 and processed using HKL2000 [10]. Crystals grew in the trigonal space group P3 1 21 with unit cell dimensions a = b = 254.8 and c = 123.8 Å. There are two molecules of STAT3 per asymmetric unit. The structure was determined by molecular replacement using Phaser [11]. The published STAT3 structure [12] was used as the search model. Molecular model was then modified in O [13] and refined with CNS [14]. Partial twinning was detected using CNS and the refinement was carried out using the twinning operator h, k, l and twinning fraction of 0.373. Multiangle light scattering (MALS) analysis. Purified proteins [STAT3a (residues 1–770), STAT3 (residues 127–688), and STAT3b 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.049 * Corresponding author. Fax: +1 713 834 6273. E-mail address: xichen@mdacc.tmc.edu (X. Chen). Biochemical and Biophysical Research Communications 374 (2008) 1–5 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc