crystallization communications
Purification, crystallization and preliminary X-ray
of the Kelch-like motif region of mouse Keap1aRIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan, bGraduate School of Comprehensive Human Sciences, Center for Tsukuba Advanced Research Alliance and ERATO Environmental Research Project, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan, cCellular Signaling Laboratory and Structurome Research Group, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan, and dDepartment of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
*Correspondence e-mail: yokoyama@biochem.s.u-tokyo.ac.jp
Keap1 (Kelch-like ECH-associating protein 1) is a negative regulator of the Nrf2 transcription factor in the cytoplasm. The Kelch/DGR (double-glycine repeat) domain of Keap1 associates with Nrf2 as well as with actin filaments. A recombinant protein containing both the Kelch/DGR domain and the C-terminal region of mouse Keap1 was expressed in Escherichia coli, purified to near-homogeneity and crystallized by the sitting-drop vapour-diffusion method. The crystal belongs to P61 or P65, with unit-cell parameters a = b = 102.95, c = 55.21 Å, and contains one molecule in the A complete diffraction data was collected to 2.25 Å resolution using an R-AXIS IV++ imaging plate mounted on an RA-Micro7 Cu Kα rotating-anode X-ray generator.
Keywords: Nrf2; transcription factor; Keap1; actin binding; antioxidants.
1. Introduction
Keap1 (Kelch-like ECH-associating protein 1) is a member of a large family of proteins that contain an N-terminal broad complex, tramtrack and bric-a-brac (BTB) domain (Bardwell & Treisman, 1994; Zollman et al., 1994; Chen et al., 1995) and a C-terminal Kelch/DGR (double-glycine repeat) domain (Xue & Cooley, 1993; Adams et al., 2000). Keap1 negatively regulates the Nrf2 transcription factor, a critical regulator of the antioxidant-response element (ARE) dependent expression of cellular defence enzymes against oxidative and xenobiotic stresses (Itoh, Ishii et al., 1999; Hayes et al., 2000; Nguyen et al., 2003). The induction of an array of ARE-dependent genes enhances cellular defences by restoring the cellular redox homeostasis, by minimizing oxidative DNA damage and by detoxifying reactive chemicals as well as carcinogens. Current models of the repressive regulation of the Nrf2 cellular defence system include nuclear deprivation of Nrf2 by physical sequestration through the Keap1–Nrf2 association in the cytoplasm (Itoh, Wakabayashi et al., 1999) as well as the Keap1-dependent polyubiquitination and proteasome-mediated degradation of Nrf2 (McMahon et al., 2003; Zhang & Hannink, 2003; Itoh et al., 2003).
Recent studies have shown that Keap1 acts as an Nrf2-specific adaptor for Cul3-based E3 ligases (Kobayashi et al., 2004; Cullinan et al., 2004). Keap1 has been suggested to bridge Nrf2 physically to a Cul3-based E3 ligase for ubiquitin transfer. By functioning in cytosolic Nrf2 sequestration or by working as an adaptor for transferring ubiquitin to the Nrf2 molecule, Keap1 essentially requires a physical interaction with the N-terminal Neh2 domain of Nrf2 through its Kelch/DGR (Gln315–Thr598) domain (Itoh, Wakabayashi et al., 1999). On the other hand, the Kelch/DGR domain is also important for co-localization with the actin cytoskeleton in the perinuclear region of the cytoplasm (Itoh, Wakabayashi et al., 1999; Kang et al., 2004). The Keap1–actin co-localization provides a scaffold for sequestering Nrf2 in the cytosol as well as for targeting Nrf2 to proteasome-mediated degradation, as the proteasome is also known to be co-localized with actin filaments and intermediate filaments. In addition, the extreme end of the C-terminal region (CTR; Ser599–Cys624) of mouse Keap1 has also been shown to be important for Keap1 repression of Nrf2 activity (Kang et al., 2004). Structural analyses of these two C-terminal domains, encompassing residues 309–624 (DGR and CTR domains; referred to hereafter as mouse Keap1-DC), will provide insights into the basis of the Keap1 interaction with either Nrf2 or actin filaments, which both have a great impact on the regulation of cellular responses against toxic environmental stresses. Here, we report the purification, crystallization and preliminary crystallographic analysis of Keap1-DC from mouse.
2. Materials and methods
2.1. Chemicals
The pTXB3 expression vector and the chitin-binding beads were purchased from New England Biolabs. Buffer reagents used for purification were purchased from Sigma unless stated otherwise.
2.2. Cloning
The DNA fragment encoding mouse Keap1-DC (Thr309–Cys624) as well as the Mxe intein/chitin-binding domain fusion was constructed by the PCR overlap extension method using mouse Keap1 cDNA and pTXB3 as templates. NcoI and BamHI restriction sites were added to the 5′ and 3′ ends, respectively, using the PCR primers. The resulting fragment was inserted into pET15b (Novagen) using the NcoI and BamHI sites.
2.3. Expression and purification
The expression vector containing mouse Keap1-DC with a C-terminally fused Mxe intein/chitin-binding domain was expressed in Escherichia coli BL21 CodonPlus (DE3)-RIL (Stratagene) cultured in Luria Broth (LB) medium at 310 K. Overexpression of the recombinant fusion protein was induced by adding isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM and by shifting the culture temperature to 298 K. After 18 h further incubation, the cells were harvested and resuspended in 20 mM Tris–HCl pH 8.3, 1%(v/v) Triton X-100, 1 mM EDTA, 10 µg ml−1 DNase I, 5 mM MgCl2, 2 mM dithiothreitol (DTT) and Complete protease inhibitor (Roche). Cells were mechanically lysed by sonication (Branson Sonifier 450) on ice. The soluble protein fraction was then recovered by centrifugation at 27 000g for 30 min at 277 K. The recombinant mouse Keap1-DC fusion protein was captured by chitin-binding beads. The protein-bound affinity beads were washed with 40 column volumes of buffer A (20 mM Tris–HCl pH 8.3, 1 mM EDTA and 1 mM benzamidine–HCl) containing 0.3 M NaCl and 1% Triton X-100, followed by 40 column volumes of buffer A. On-column protein cleavage for the retrieval of native mouse Keap1-DC was initiated by incubating the beads for 24 h with one column volume of buffer A in the presence of 50 mM DTT and 50 mM mercaptoethane sulfonate (Mesna) at 277 K. The recovered mouse Keap1-DC was further purified on anion-exchange (Q2, BioRad) and Superdex S75 26/60PG columns (Pharmacia). The purified mouse Keap1-DC protein has an extra methionine at the N-terminus for translation initiation and an extra tyrosine at the C-terminus of Cys624, which was left over from the fusion protein after cleavage. The protein solution was exchanged into a buffer containing 20 mM Tris–HCl pH 8.3, 20 mM DTT and 10 mM benzamidine–HCl and then concentrated to 4 mg ml−1. Finally, the protein was flash-frozen and stored in aliquots at 193 K.
2.4. Crystallization
Initial crystallization screening was performed with the sparse-matrix crystallization screening kits (Jancarik & Kim, 1991) Crystal Screens I and II from Hampton Research by the hanging-drop vapour-diffusion method in a 96-well plate (Corning, NY, USA). In each drop, 1.0 µl protein solution (4 mg ml−1 in 20 mM Tris–HCl pH 8.3, 20 mM DTT and 10 mM benzamidine–HCl) and 1.0 µl reservoir solution were mixed and equilibrated against 100 µl reservoir solution. Small hexagonal rounded crystals of approximately 0.05 mm in diameter were grown within a week in the drop corresponding to condition No. 15 of Crystal Screen II (1.0 M lithium sulfate, 0.5 M ammonium sulfate and 0.1 M sodium citrate pH 5.6). Further optimization was carried out by varying the precipitant concentration against pH using 24-well plates (Q Plate II, Hampton Research) with 1.5 µl drops of protein solution mixed with 1.5 µl reservoir solution and equilibrated against 500 µl reservoir solution.
2.5. Data collection
Diffraction data were collected under cryogenic conditions using a Rigaku RA-Micro7 Cu Kα rotating-anode X-ray generator operated at 40 kV and 20 mA and equipped with a Rigaku R-AXIS IV++ imaging-plate area detector and an X-stream low-temperature system. Prior to data collection, the crystals were transferred into a cryoprotectant containing 1.5 M lithium sulfate, 0.5 M ammonium sulfate and 0.1 M sodium citrate pH 5.2 for a few seconds and were then flash-cooled directly in a stream of cold gaseous N2 at 90 K. The crystal-to-detector distance was set to 180 mm and the oscillation range was 1° with an exposure time of 5 min. A complete diffraction data set was obtained to 2.25 Å resolution. The data were processed and scaled using the HKL2000 suite (Otwinowski & Minor, 1997).
3. Results
The mouse Keap1-DC protein was expressed and purified to near-homogeneity as confirmed by SDS–PAGE (Fig. 1). After optimizing the crystallization conditions, good hexagonal rounded crystals of approximately 0.2 mm in diameter (Fig. 2) were obtained in the presence of 0.8 M lithium sulfate, 0.5 M ammonium sulfate and 0.1 M sodium citrate pH 5.2. The crystal diffracted beyond 2.2 Å resolution using our in-house facility (Fig. 3) and belongs to the hexagonal P61 (or its P65). The statistics of the processed data are shown in Table 1. A total of 117 495 measured reflections were merged into 15 961 unique reflections with an Rmerge of 5.6% and 99.7% completeness to 2.25 Å resolution. Assuming that the crystal contains one molecule in the the molecular weight of the protein (34 674 Da) yields a solvent content of 49% and a Matthews coefficient of 2.4 Å3 Da−1 (Matthews, 1968).
|
The et al., 1998). The RCC1 consists of a seven-bladed β-propeller formed from internal repeats of 51–68 residues per blade. However, it showed poor sequence homology with the mouse Keap1-DC protein and also failed to produce the correct structure solution by using the RCC1 structure as a model. Hence, a search for suitable heavy-atom derivatives is in progress to solve the structure by the MIR or MAD method.
of RCC1 (regulator of chromosome condensation), which belongs to the Kelch-repeat superfamily, has recently been determined (RenaultAcknowledgements
Special thanks to the project secretarial staff for all their assistance, particularly that of Ms Tomoko Nakayama. This work was supported in part by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan and ERATO-JST.
References
Adams, J., Kelso, R. & Cooley, L. (2000). Trends Cell Biol. 10, 17–24. Web of Science CrossRef PubMed CAS Google Scholar
Bardwell, V. J. & Treisman, R. (1994). Genes Dev. 8, 1664–1677. CrossRef CAS PubMed Web of Science Google Scholar
Chen, W., Zollman, S., Couderc, J. L. & Laski, F. A. (1995). Mol. Cell. Biol. 15, 3424–3429. CAS PubMed Web of Science Google Scholar
Cullinan, S. B., Gordan, J. D., Jin, J., Harper, J. W. & Diehl, J. A. (2004). Mol. Cell. Biol. 24, 8477–8486. Web of Science CrossRef PubMed CAS Google Scholar
Hayes, J. D., Chanas, S. A., Henderson, C. J., McMahon, M., Sun, C., Moffat, G. J., Wolf, C. R. & Yamamoto, M. (2000). Biochem. Soc. Trans. 28, 33–41. Web of Science PubMed CAS Google Scholar
Itoh, K., Ishii, T., Wakabayashi, N. & Yamamoto, M. (1999). Free Radic. Res. 31, 319–324. Web of Science CrossRef PubMed CAS Google Scholar
Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, K., Igarashi, K., Engel, J. D. & Yamamoto, M. (1999). Genes Dev. 13, 76–86. Web of Science CrossRef CAS PubMed Google Scholar
Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, T., O'Connor, T. & Yamamoto, M. (2003). Genes Cells, 8, 379–391. Web of Science CrossRef PubMed CAS Google Scholar
Jancarik, J. & Kim, S.-H. (1991). J. Appl. Cryst. 24, 409–411. CrossRef CAS Web of Science IUCr Journals Google Scholar
Kang, M.-I., Kobayashi, A., Wakabayashi, N., King, S. G. & Yamamoto, M. (2004). Proc. Natl Acad. Sci. USA, 101, 2046–2051. Web of Science CrossRef PubMed CAS Google Scholar
Kobayashi, A., Kang, M. I., Okawa, H., Ohtsuji, M., Zenke, Y., Chiba, T., Igarashi, K. & Yamamoto, M. (2004). Mol. Cell. Biol. 24, 7130–7139. Web of Science CrossRef PubMed CAS Google Scholar
McMahon, M., Itoh, K., Yamamoto, M. & Hayes, J. D. (2003). J. Biol. Chem. 278, 21592–21600. Web of Science CrossRef PubMed CAS Google Scholar
Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497. CrossRef CAS PubMed Web of Science Google Scholar
Nguyen, T., Sherratt, P. J. & Pickett, C. B. (2003). Annu. Rev. Pharmacol. Toxicol. 43, 233–260. Web of Science CrossRef PubMed CAS Google Scholar
Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307–326. CrossRef CAS Web of Science Google Scholar
Renault, L., Nassar, N., Vetter, I., Becker, J., Kelbe, C., Roth, M. & Wittinghofer, A. (1998). Nature (London), 392, 97–101. Web of Science CAS PubMed Google Scholar
Xue, F. & Cooley, L. (1993). Cell, 72, 681–693. CrossRef CAS PubMed Web of Science Google Scholar
Zhang, D. D. & Hannink, M. (2003). Mol. Cell. Biol. 23, 8137–8151. Web of Science CrossRef PubMed CAS Google Scholar
Zollman, S., Godt, D., Prive, G. G., Couderc, J. L. & Laski, F. A. (1994). Proc. Natl Acad. Sci. USA, 91, 10717–10721. CrossRef CAS PubMed Web of Science Google Scholar
© International Union of Crystallography. Prior permission is not required to reproduce short quotations, tables and figures from this article, provided the original authors and source are cited. For more information, click here.