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• W2070791372 abstract "Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5′-(β,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr39 and Asp45 substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins. Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5′-(β,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr39 and Asp45 substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins. Small GTP-binding proteins (small G proteins or small GTPase) act as molecular switches and regulate a wide variety of important physiological functions in cells. Plants possess a specific subfamily of small GTPases called Rac/Rop (Rho-related GTPases from plants) (1.Yang Z. Small GTPases: versatile signaling switches in plants.Plant Cell. 2002; 14 (suppl.): S375-S388Crossref PubMed Scopus (336) Google Scholar, 2.Zheng Z.L. Yang Z. The Rop GTPase: an emerging signaling switch in plants.Plant Mol. Biol. 2000; 44: 1-9Crossref PubMed Scopus (155) Google Scholar), which have attracted recent interest due to their function as molecular switches in the regulation of various cellular responses (3.Nibau C. Wu H.M. Cheung A.Y. RAC/ROP GTPases: ‘hubs’ for signal integration and diversification in plants.Trends Plant Sci. 2006; 11: 309-315Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 4.Yang Z. Fu Y. ROP/RAC GTPase signaling.Curr. Opin. Plant Biol. 2007; 10: 490-494Crossref PubMed Scopus (108) Google Scholar, 5.Xu T. Wen M. Nagawa S. Fu Y. Chen J.G. Wu M.J. Perrot-Rechenmann C. Friml J. Jones A.M. Yang Z. Cell surface- and Rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis.Cell. 2010; 143: 99-110Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 6.Oda Y. Fukuda H. Initiation of cell wall pattern by a Rho- and microtubule-driven symmetry breaking.Science. 2012; 337: 1333-1336Crossref PubMed Scopus (178) Google Scholar). For example, OsRac1, a Rac/Rop from Oryza sativa, plays an important role in the regulation of rice immunity (7.Kawasaki T. Henmi K. Ono E. Hatakeyama S. Iwano M. Satoh H. Shimamoto K. The small GTP-binding protein Rac is a regulator of cell death in plants.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 10922-10926Crossref PubMed Scopus (304) Google Scholar, 8.Ono E. Wong H.L. Kawasaki T. Hasegawa M. Kodama O. Shimamoto K. Essential role of the small GTPase Rac in disease resistance of rice.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 759-764Crossref PubMed Scopus (320) Google Scholar, 9.Suharsono U. Fujisawa Y. Kawasaki T. Iwasaki Y. Satoh H. Shimamoto K. The heterotrimeric G protein α acts upstream of the small GTPase Rac in disease resistance of rice.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 13307-13312Crossref PubMed Scopus (215) Google Scholar, 10.Fujiwara M. Umemura K. Kawasaki T. Shimamoto K. Proteomics of Rac GTPase signaling reveals its predominant role in elicitor-induced defense response of cultured rice cells.Plant Physiol. 2006; 140: 734-745Crossref PubMed Scopus (63) Google Scholar, 11.Wong H.L. Pinontoan R. Hayashi K. Tabata R. Yaeno T. Hasegawa K. Kojima C. Yoshioka H. Iba K. Kawasaki T. Shimamoto K. Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension.Plant Cell. 2007; 19: 4022-4034Crossref PubMed Scopus (336) Google Scholar, 12.Kawano Y. Akamatsu A. Hayashi K. Housen Y. Okuda J. Yao A. Nakashima A. Takahashi H. Yoshida H. Wong H.L. Kawasaki T. Shimamoto K. Activation of a Rac GTPase by the NLR family disease resistance protein Pit plays a critical role in rice innate immunity.Cell Host Microbe. 2010; 7: 362-375Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 13.Akamatsu A. Wong H.L. Fujiwara M. Okuda J. Nishide K. Uno K. Imai K. Umemura K. Kawasaki T. Kawano Y. Shimamoto K. An OsCEBiP/OsCERK1-OsRacGEF1-OsRac1 module is an essential early component of chitin-induced rice immunity.Cell Host Microbe. 2013; 13: 465-476Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 14.Kawano Y. Chen L. Shimamoto K. The function of Rac small GTPase and associated proteins in rice innate immunity.Rice. 2010; 3: 112-121Crossref Scopus (28) Google Scholar, 15.Kawano Y. Shimamoto K. Early signaling network in rice PRR-mediated and R-mediated immunity.Curr. Opin. Plant Biol. 2013; 16: 496-504Crossref PubMed Scopus (50) Google Scholar), and NtRac5, from Nicotiana tabacum, regulates reactive oxygen species (ROS) 4The abbreviations used are: ROSreactive oxygen speciesGEFguanine nucleotide exchange factorCAconstitutively activatedDNdominant-negativeGMPPNPguanosine 5′-(β,γ-imido)triphosphateBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPDBProtein Data BankHSQCheteronuclear single quantum coherenceGTPγSguanosine 5′-O-(thiotriphosphate). production in pollen tubes (16.Potocký M. Pejchar P. Gutkowska M. Jiménez-Quesada M.J. Potocká A. Alché Jde D. Kost B. Žárský V. NADPH oxidase activity in pollen tubes is affected by calcium ions, signaling phospholipids and Rac/Rop GTPases.J. Plant Physiol. 2012; 169: 1654-1663Crossref PubMed Scopus (82) Google Scholar). The AtRop family members AtRop1, AtRop3, and AtRop5 redundantly regulate pollen tube growth in Arabidopsis thaliana (17.Craddock C. Lavagi I. Yang Z. New insights into Rho signaling from plant ROP/Rac GTPases.Trends Cell Biol. 2012; 22: 492-501Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), whereas AtRop2, AtRop4, and AtRop6 regulate root hair development (17.Craddock C. Lavagi I. Yang Z. New insights into Rho signaling from plant ROP/Rac GTPases.Trends Cell Biol. 2012; 22: 492-501Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Recently, the activation of AtRop2 and AtRop6 by auxin has been reported to regulate the subcellular distribution of auxin transporters PIN1 and PIN2, which control PIN-mediated pattern formation and morphogenesis in leaves and roots (18.Chen X. Naramoto S. Robert S. Tejos R. Löfke C. Lin D. Yang Z. Friml J. ABP1 and ROP6 GTPase signaling regulate clathrin-mediated endocytosis in Arabidopsis roots.Curr. Biol. 2012; 22: 1326-1332Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 19.Lin D. Nagawa S. Chen J. Cao L. Chen X. Xu T. Li H. Dhonukshe P. Yamamuro C. Friml J. Scheres B. Fu Y. Yang Z. A ROP GTPase-dependent auxin signaling pathway regulates the subcellular distribution of PIN2 in Arabidopsis roots.Curr. Biol. 2012; 22: 1319-1325Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 20.Nagawa S. Xu T. Lin D. Dhonukshe P. Zhang X. Friml J. Scheres B. Fu Y. Yang Z. ROP GTPase-dependent actin microfilaments promote PIN1 polarization by localized inhibition of clathrin-dependent endocytosis.PLoS Biol. 2012; 10: e1001299Crossref PubMed Scopus (161) Google Scholar). AtRop10 and AtRop11 are specific negative regulators of abscisic acid responses (21.Zheng Z.L. Nafisi M. Tam A. Li H. Crowell D.N. Chary S.N. Schroeder J.I. Shen J. Yang Z. Plasma membrane-associated ROP10 small GTPase is a specific negative regulator of abscisic acid responses in Arabidopsis.Plant Cell. 2002; 14: 2787-2797Crossref PubMed Scopus (127) Google Scholar, 22.Li Z. Li Z. Gao X. Chinnusamy V. Bressan R. Wang Z.X. Zhu J.K. Wu J.W. Liu D. ROP11 GTPase negatively regulates ABA signaling by protecting ABI1 phosphatase activity from inhibition by the ABA receptor RCAR1/PYL9 in Arabidopsis.J. Integr. Plant. Biol. 2012; 54: 180-188Crossref PubMed Scopus (43) Google Scholar). In addition, AtRop9 functions as a signal integrator of auxin and abscisic acid signaling and plays an important role in embryo development and lateral root formation in A. thaliana (23.Nibau C. Tao L. Levasseur K. Wu H.M. Cheung A.Y. The Arabidopsis small GTPase AtRAC7/ROP9 is a modulator of auxin and abscisic acid signaling.J. Exp. Bot. 2013; 64: 3425-3437Crossref PubMed Scopus (19) Google Scholar). reactive oxygen species guanine nucleotide exchange factor constitutively activated dominant-negative guanosine 5′-(β,γ-imido)triphosphate 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol Protein Data Bank heteronuclear single quantum coherence guanosine 5′-O-(thiotriphosphate). Rac/Rop family proteins are composed of ∼200 amino acids and have masses of 20–24 kDa, similar to the animal small GTPases. They are inactive in the GDP-bound form and are activated by the binding of GTP. Several Rac/Rop structures have been reported, including AtRop5 (GDP-bound form), AtRop9 (GDP-bound form) (24.Sørmo C.G. Leiros I. Brembu T. Winge P. Os V. Bones A.M. The crystal structure of Arabidopsis thaliana RAC7/ROP9: the first RAS superfamily GTPase from the plant kingdom.Phytochemistry. 2006; 67: 2332-2340Crossref PubMed Scopus (18) Google Scholar), the AtRop4 (GDP-bound form)-guanine nucleotide exchange factor (GEF) complex (25.Thomas C. Fricke I. Scrima A. Berken A. Wittinghofer A. Structural evidence for a common intermediate in small G protein-GEF reactions.Mol. Cell. 2007; 25: 141-149Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and the AtRop7(apo)-GEF complex (26.Thomas C. Fricke I. Weyand M. Berken A. 3D structure of a binary ROP-PRONE complex: the final intermediate for a complete set of molecular snapshots of the RopGEF reaction.Biol. Chem. 2009; 390: 427-435Crossref PubMed Scopus (18) Google Scholar), but all of these structures are of inactive forms. Structural analysis of active-form animal small GTPases has revealed the biological processes associated with carcinogenic mutations and the biochemical mechanisms of carcinogenesis (27.Al-Mulla F. Milner-White E.J. Going J.J. Birnie G.D. Structural differences between valine-12 and aspartate-12 Ras proteins may modify carcinoma aggression.J. Pathol. 1999; 187: 433-438Crossref PubMed Scopus (90) Google Scholar). Hence, the structural determination of plant Rac/Rop proteins in their active form should be an important step in clarifying the mechanism of activation of target effectors. A constitutively activated mutant of OsRac1 (OsRac1 G19V, denoted as CA-OsRac1) has been reported to increase resistance to rice bacterial blight disease and subsequent cell death (7.Kawasaki T. Henmi K. Ono E. Hatakeyama S. Iwano M. Satoh H. Shimamoto K. The small GTP-binding protein Rac is a regulator of cell death in plants.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 10922-10926Crossref PubMed Scopus (304) Google Scholar, 8.Ono E. Wong H.L. Kawasaki T. Hasegawa M. Kodama O. Shimamoto K. Essential role of the small GTPase Rac in disease resistance of rice.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 759-764Crossref PubMed Scopus (320) Google Scholar). Conversely, a dominant-negative mutant (OsRac1 T24N, denoted as DN-OsRac1) was found to decrease the resistance reaction. Transgenic rice lines expressing CA-OsRac1, but not DN-OsRac1, displayed increased production of a phytoalexin and altered expression of defense-related genes (8.Ono E. Wong H.L. Kawasaki T. Hasegawa M. Kodama O. Shimamoto K. Essential role of the small GTPase Rac in disease resistance of rice.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 759-764Crossref PubMed Scopus (320) Google Scholar). Furthermore, overexpression of CA-OsRac1 induced ROS production in cultured rice cells (7.Kawasaki T. Henmi K. Ono E. Hatakeyama S. Iwano M. Satoh H. Shimamoto K. The small GTP-binding protein Rac is a regulator of cell death in plants.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 10922-10926Crossref PubMed Scopus (304) Google Scholar). These data clearly show that OsRac1 acts as a molecular switch during plant innate immunity. CA-OsRac1, but not DN-OsRac1, was also shown to interact directly with an NADPH oxidase, OsRbohB (O. sativa respiratory burst oxidase homolog B) (11.Wong H.L. Pinontoan R. Hayashi K. Tabata R. Yaeno T. Hasegawa K. Kojima C. Yoshioka H. Iba K. Kawasaki T. Shimamoto K. Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension.Plant Cell. 2007; 19: 4022-4034Crossref PubMed Scopus (336) Google Scholar). Transient coexpression of OsRac1 and OsRbohB in Nicotiana benthamiana leaves enhanced ROS production, supporting the notion that direct OsRac1-OsRbohB interactions activate NADPH oxidase in plants (11.Wong H.L. Pinontoan R. Hayashi K. Tabata R. Yaeno T. Hasegawa K. Kojima C. Yoshioka H. Iba K. Kawasaki T. Shimamoto K. Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension.Plant Cell. 2007; 19: 4022-4034Crossref PubMed Scopus (336) Google Scholar). Although the crystal structure of the N-terminal domain of OsRbohB has been reported (28.Oda T. Hashimoto H. Kuwabara N. Hayashi K. Kojima C. Kawasaki T. Shimamoto K. Sato M. Shimizu T. Crystallographic characterization of the N-terminal domain of a plant NADPH oxidase.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2008; 64: 867-869Crossref PubMed Scopus (7) Google Scholar, 29.Oda T. Hashimoto H. Kuwabara N. Akashi S. Hayashi K. Kojima C. Wong H.L. Kawasaki T. Shimamoto K. Sato M. Shimizu T. Structure of the N-terminal regulatory domain of a plant NADPH oxidase and its functional implications.J. Biol. Chem. 2010; 285: 1435-1445Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), the molecular mechanism by which OsRac1 activates OsRbohB for ROS production remains largely unknown. In this report, the crystal structure of OsRac1 in the active form (GMPPNP-bound) was determined in an effort to elucidate the molecular mechanism of ROS production in rice. Based on the structural information obtained, the OsRbohB-binding site on OsRac1 was predicted, and OsRbohB binding-deficient OsRac1 mutants were designed. The OsRbohB-binding activity of these mutants was evaluated by in vitro pulldown assays and NMR measurements, and the mutants were also analyzed by ROS production assays using rice cells. This study, together with our previous reports (11.Wong H.L. Pinontoan R. Hayashi K. Tabata R. Yaeno T. Hasegawa K. Kojima C. Yoshioka H. Iba K. Kawasaki T. Shimamoto K. Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension.Plant Cell. 2007; 19: 4022-4034Crossref PubMed Scopus (336) Google Scholar, 29.Oda T. Hashimoto H. Kuwabara N. Akashi S. Hayashi K. Kojima C. Wong H.L. Kawasaki T. Shimamoto K. Sato M. Shimizu T. Structure of the N-terminal regulatory domain of a plant NADPH oxidase and its functional implications.J. Biol. Chem. 2010; 285: 1435-1445Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), demonstrates that OsRac1 regulates ROS production through direct interactions with OsRbohB. cDNA encoding OsRac1(8–183) C32S/Q68L (denoted as OsRac1; see “Results and Discussion”) was cloned into the multiple cloning site of the pGEX-6P3 vector (GE Healthcare), and several mutations were introduced using the QuikChange site-directed mutagenesis kit (Stratagene). The resulting plasmids were used to transform Escherichia coli Rosetta (DE3) cells (Novagen), which were then grown in M9 medium until the cell suspension reached the appropriate turbidity. Chimeric proteins comprising GST fused to the N terminus of OsRac1 or its mutants were then overexpressed by the addition of 1 mm isopropyl 1-thio-β-d-galactopyranoside for 12 h at 15 °C, after which the cells were harvested by centrifugation. To obtain target proteins for NMR measurements, 0.5 g/liter [15N]ammonium chloride (99 atom % of 15N) was used as the sole nitrogen source in M9 medium. The overexpressed GST-fused OsRac1 proteins were initially purified by affinity chromatography using glutathione-Sepharose 4B resin (GE Healthcare). After enzymatic cleavage of the GST tag from target proteins using GST-human rhinovirus 3C protease, digestion products were passed through glutathione-Sepharose 4B resin, and the OsRac1 and mutant proteins were further purified by size exclusion column chromatography using Superdex 75 (GE Healthcare). To exchange nucleotide, OsRac1 proteins were incubated with a 25-fold molar excess of GMPPNP (Sigma) in 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 10 mm EDTA, and 2 mm DTT for 12 h at 4 °C. After the addition of 10 mm MgCl2, excess unbound nucleotides were removed using a Superdex 75 column. For crystallization and NMR measurements, purified proteins were concentrated by ultrafiltration using Amicon Ultra-10 filters to 4 mg/ml with 10 mm Tris-HCl (pH 7.5), 50 mm NaCl, 5 mm MgCl2, and 2 mm DTT and to 0.1 mg/ml with buffer A (50 mm BisTris (pH 6.8), 50 mm NaCl, 5 mm MgCl2, 2 mm CaCl2, and 2 mm DTT), respectively. For the prey protein in a GST pulldown assay, cDNA encoding OsRbohB(138–313) was cloned into pET32c (Novagen). Following overexpression of chimeric thioredoxin-His6-OsRbohB(138–313) in E. coli Rosetta (DE3) cells, protein was purified by affinity chromatography using nickel-nitrilotriacetic acid-agarose resin (Qiagen). After enzymatic cleavage of the thioredoxin-His6 tag from the target protein using recombinant enterokinase (Novagen), OsRbohB(138–313) was further purified by anion exchange and size exclusion chromatography using Superdex 75 in buffer A. For GST pulldown assays, purified OsRbohB(138–313) was concentrated to 0.6 mm by ultrafiltration using Ultra-10 filters with buffer A. OsRac1 complexed with the GTP analog GMPPNP was crystallized as described (30.Kosami K. Ohki I. Hayashi K. Tabata R. Usugi S. Kawasaki T. Fujiwara T. Nakagawa A. Shimamoto K. Kojima C. Purification, crystallization and preliminary X-ray crystallographic analysis of a rice Rac/Rop GTPase, OsRac1.Acta Crystallogr. F Struct. Biol. Commun. 2014; 70: 113-115Crossref PubMed Scopus (2) Google Scholar). In brief, OsRac1 crystals were obtained at 20 °C using the sitting-drop vapor-diffusion method by mixing 0.7 µl of 4 mg/ml purified protein with 0.7 µl of reservoir solution consisting of 100 mm MES (pH 6.0) and 10–30% PEG 6000. For the x-ray diffraction experiments, crystals in a reservoir solution supplemented with 25% (v/v) glycerol as a cryoprotectant were mounted in nylon loops (Hampton Research), flash-cooled using a 100 K dry nitrogen stream, and then kept under the nitrogen stream during data collection. X-ray diffraction data were collected from GMPPNP-bound OsRac1 crystals using a Rayonix MX225HE CCD detector installed on beamline BL44XU at SPring-8 (Harima, Japan). The camera was fixed at a distance of 260 mm from the crystal sample using an x-ray wavelength of 0.9000 Å. Data collection for native crystals was performed using an angular range of 240°, an oscillation step of 1.5°, and an exposure time of 1.0 s for each image. All data were integrated and scaled using HKL-2000 (31.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). Diffraction and intensity data collection statistics are summarized in Table 1.TABLE 1Data collection and refinement statistics of the OsRac1-GMPPNP-Mg2+ complex (OsRac1(GMPPNP))OsRac1(GMPPNP)Data collectionSpace groupP212121Cell constants (a, b, c) (Å)36.8, 59.1, 64.4Resolution (Å)50–1.9 (1.97–1.9)Redundancy8.2 (8.3)Completeness (%)99.8 (100)I/σ(I)19.7 (7.7)Rmerge (%)aRmerge = ΣhΣi|I(h)i − 〈I(h)〉|/ΣhΣiI(h)i, where I(h) is the intensity of reflection h, Σh is the sum of all measured reflections, and Σi is the sum of i measurements of reflection.9.5 (28.4)RefinementResolution range (Å)50–1.9No. of reflections10,901Rwork (%)bRwork and Rfree = Σhkl‖Fo|−|Fc‖)/Σhkl|Fo|, where the free reflections (5% of the total used) were held aside for Rfree throughout refinement.15.7Rfree (%)bRwork and Rfree = Σhkl‖Fo|−|Fc‖)/Σhkl|Fo|, where the free reflections (5% of the total used) were held aside for Rfree throughout refinement.20.3No. of atomsProtein1384Ligand and ion51Solvent90Average B-factor (Å2)16.1Root mean square deviationsBond length (Å)0.019Bond angles°2.2°Ramachandran analysisFavored (%)98.9Allowed (%)1.1Disallowed (%)0a Rmerge = ΣhΣi|I(h)i − 〈I(h)〉|/ΣhΣiI(h)i, where I(h) is the intensity of reflection h, Σh is the sum of all measured reflections, and Σi is the sum of i measurements of reflection.b Rwork and Rfree = Σhkl‖Fo|−|Fc‖)/Σhkl|Fo|, where the free reflections (5% of the total used) were held aside for Rfree throughout refinement. Open table in a new tab The crystal structure was solved by the molecular replacement method using Phaser (32.McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14440) Google Scholar). The tertiary structure coordinates of A. thaliana Rac7/Rop9 (Protein Data Bank (PDB) code 2J0V) (24.Sørmo C.G. Leiros I. Brembu T. Winge P. Os V. Bones A.M. The crystal structure of Arabidopsis thaliana RAC7/ROP9: the first RAS superfamily GTPase from the plant kingdom.Phytochemistry. 2006; 67: 2332-2340Crossref PubMed Scopus (18) Google Scholar) were used as a search model for OsRac1. Crystal structures of OsRac1 were rebuilt using Coot (33.Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17079) Google Scholar) and refined using REFMAC5 (34.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: 240-255Crossref PubMed Scopus (13854) Google Scholar) and CNS (35.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. Read R.J. Rice L.M. Simonson T. Warren G.L. Crystallography & NMR system: a new software suite for macromolecular structure determination.Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). Ramachandran plot analysis was performed using Rampage (36.Lovell S.C. Davis I.W. Arendall 3rd, W.B. de Bakker P.I.W. Word J.M. Prisant M.G. Richardson J.S. Richardson D.C. Structure validation by Cα geometry: Φ,Ψ and Cβ deviation.Proteins Struct. Funct. Genet. 2003; 50: 437-450Crossref PubMed Scopus (3847) Google Scholar). Final refinement statistics are summarized in Table 1. The solvent accessibility of each amino acid was analyzed using Naccess. Coordinates of the final model and structure factors of OsRac1 have been deposited in the PDB (code 4U5X). All structures in the figures were generated using PyMOL. Following immobilization of each GST-tagged protein onto a 50-µl gel volume of glutathione-Sepharose 4B resin, 20 µl of solution containing 0.6 mm purified OsRbohB(138–313) was added, and the mixture was incubated at 4 °C for 12 h. After washing the resin several times with fresh buffer solution (50 mm BisTris (pH 6.8), 50 mm KCl, 5 mm MgCl2, 2 mm CaCl2, 1 mm phenylmethylsulfonyl fluoride, and 3% (v/v) dimethyl sulfoxide), bound proteins were eluted and analyzed by SDS-PAGE. The SDS-polyacrylamide gels were stained with Coomassie Brilliant Blue. All NMR spectra were measured using a Bruker AVANCE 800 spectrometer equipped with a TXI cryogenic probe at 303 K, and the collected data were processed and analyzed using NMRPipe (37.Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes.J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11533) Google Scholar) and Sparky 3 NMR Assignment and Integration Software (University of California, San Francisco), respectively. All of the two-dimensional 1H-15N heteronuclear single quantum coherence (HSQC) experiments were performed using 0.1 mm uniformly 15N-labeled OsRac1 in 50 mm BisTris (pH 6.8), 50 mm KCl, 5 mm MgCl2, and 1 mm DTT in 90% H2O and 10% D2O. To generate rice suspension cells (Kinmaze) expressing CA-OsRac1, CA-OsRac1 Y39A, or CA-OsRac1 D45A, the coding regions of mutated OsRac1 were introduced into the p2K-GW binary vector (for transgenic plants expressing genes under the control of the maize ubiquitin promoter) using the Gateway system (Invitrogen). Agrobacterium tumefaciens-mediated transformation of rice calli was performed according to Hiei et al. (38.Hiei Y. Ohta S. Komari T. Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant J. 1994; 6: 271-282Crossref PubMed Scopus (2848) Google Scholar). Transformants selected by hygromycin resistance were subcultured in 22 ml of R2S medium every week and incubated on a rotary shaker (90 rpm) at 30 °C. Total RNA was extracted from rice suspension cultures using the RNeasy plant mini kit (Qiagen), and 1 µg was used as a template for reverse transcription using an oligo(dT) primer and SuperScript II (Invitrogen). PCR analyses were performed using specific primers for OsRac1 (5′-AGATAGGGCCTATCTTGCTGATCATC-3′ and 5′-CTAGAAGTTTCCTCCTAGCTGCAAGC-3′), hygromycin phosphotransferase (5′-GAGCCTGACCTATTGCATCTCC-3′ and 5′-GGCCTCCAGAAGAAGATGTTGG-3′), and ACT1 (5′-CAATCGTGAGAAGATGACCC-3′ and 5′-GTCCATCAGGAAGCTCGTAGC-3′). Rice suspension cells were subcultured for 4 days in fresh medium, and cells (∼20 mg) were placed into each well of 96-well white plates (Greiner Bio-One). Two-hundred microliters of 500 µm L-012 (Wako Chemicals) dissolved in medium was added to each well, and chemiluminescence was detected using an LAS-4000 mini luminescent image analyzer (Fujifilm) at 180 min. Emission intensity from each well was measured using ImageJ(http://rsbweb.nih.gov/ij/), and the following formula: (intensity of each well − intensity of background)/weight of suspension cells in each well. For the crystallization of active-form OsRac1, an OsRac1 mutant and the GTP analog GMPPNP were used to prevent GTP hydrolysis. The GTPase activity of the human small GTPase HsRhoA is significantly attenuated by substitution of glutamine with leucine at position 63 (39.Longenecker K. Read P. Lin S.K. Somlyo A.P. Nakamoto R.K. Derewenda Z.S. Structure of a constitutively activated RhoA mutant (Q63L) at 1.55 Å resolution.Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 876-880Crossref PubMed Scopus (27) Google Scholar). In this study, the corresponding OsRac1 mutant comprising the substitution Q68L was used. To increase the stability of the OsRac1 protein, Cys32 was substituted with serine, and the N and C termini were truncated. Hereafter, OsRac1 refers to this truncated mutant, OsRac1(8–183) C32S/Q68L. The OsRac1-GMPPNP-Mg2+ complex, designated as OsRac1(GMPPNP), was crystallized as described previously (30.Kosami K. Ohki I. Hayashi K. Tabata R. Usugi S. Kawasaki T. Fujiwara T. Nakagawa A. Shimamoto K. Kojima C. Purification, crystallization and preliminary X-ray crystallographic analysis of a rice Rac/Rop GTPase, OsRac1.Acta Crystallogr. F Struct. Biol. Commun. 2014; 70: 113-115Crossref PubMed Scopus (2) Google Scholar). The crystals gave strong and high-resolution x-ray diffraction. The diffraction data were collected to 1.9 Å resolution from one crystal (Table 1) and displayed orthorhombic P212121 symmetry with an estimated mosaicity of 0.41–0.62° and a Wilson B-factor of 12.7 Å2. The unit cell (a = 36.8, b = 59.1, and c = 64.4 Å) contains one molecule of OsRac1(GMPPNP) per asymmetric unit. The crystal structure of OsRac1(GMPPNP) was determined at 1.9 Å resolution with clear electron density for all atoms with Rwork = 15.7% and Rfree = 20.3% (Fig. 1A). OsRac1(GMPPNP) comprised a half-β-barrel-s" @default.
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• W2070791372 title "The Crystal Structure of the Plant Small GTPase OsRac1 Reveals Its Mode of Binding to NADPH Oxidase" @default.
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