Structural Determinants of RhoA Binding and Nucleotide Exchange in Leukemia-associated Rho Guanine-Nucleotide Exchange Factor

Rho guanine-nucleotide exchange factors (RhoGEFs) activate Rho GTPases, and thereby regulate cytoskeletal structure, gene transcription, and cell migration. Leukemia-associated RhoGEF (LARG) belongs to a small subfamily of RhoGEFs that are RhoA-selective and directly activated by the Gα12/13 family of heterotrimeric G proteins. Herein we describe the atomic structures of the catalytic Dbl homology (DH) and pleckstrin homology (PH) domains of LARG alone and in complex with RhoA. These structures demonstrate that the DH/PH domains of LARG can undergo a dramatic conformational change upon binding RhoA, wherein both the DH and PH domains directly engage RhoA. Through mutational analysis we show that full nucleotide exchange activity requires a novel N-terminal extension on the DH domain that is predicted to exist in a broader family of RhoGEFs that includes p115-RhoGEF, Lbc, Lfc, Net1, and Xpln, and identify regions within the LARG PH domain that contribute to its ability to facilitate nucleotide exchange in vitro. In crystals of the DH/PH-RhoA complex, the active site of RhoA adopts two distinct GDP-excluding conformations among the four unique complexes in the asymmetric unit. Similar changes were previously observed in structures of nucleotide-free Ras and Ef-Tu. A potential protein-docking site on the LARG PH domain is also evident and appears to be conserved throughout the Lbc subfamily of RhoGEFs. Rho guanine-nucleotide exchange factors (RhoGEFs) activate Rho GTPases, and thereby regulate cytoskeletal structure, gene transcription, and cell migration. Leukemia-associated RhoGEF (LARG) belongs to a small subfamily of RhoGEFs that are RhoA-selective and directly activated by the Gα12/13 family of heterotrimeric G proteins. Herein we describe the atomic structures of the catalytic Dbl homology (DH) and pleckstrin homology (PH) domains of LARG alone and in complex with RhoA. These structures demonstrate that the DH/PH domains of LARG can undergo a dramatic conformational change upon binding RhoA, wherein both the DH and PH domains directly engage RhoA. Through mutational analysis we show that full nucleotide exchange activity requires a novel N-terminal extension on the DH domain that is predicted to exist in a broader family of RhoGEFs that includes p115-RhoGEF, Lbc, Lfc, Net1, and Xpln, and identify regions within the LARG PH domain that contribute to its ability to facilitate nucleotide exchange in vitro. In crystals of the DH/PH-RhoA complex, the active site of RhoA adopts two distinct GDP-excluding conformations among the four unique complexes in the asymmetric unit. Similar changes were previously observed in structures of nucleotide-free Ras and Ef-Tu. A potential protein-docking site on the LARG PH domain is also evident and appears to be conserved throughout the Lbc subfamily of RhoGEFs. Rho GTPases are molecular switches that cycle between an active GTP-bound and an inactive GDP-bound state. In their activated form, Rho GTPases bind to effector proteins that regulate the actin cytoskeleton, gene expression, and cell cycle progression (1Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3927) Google Scholar). Members of the three principal Rho GTPase subfamilies (Rho, Cdc42, and Rac) exert distinct morphological effects on cells and promote transcriptional activation through unique pathways. All are thought to play important roles in cellular transformation and metastasis (2Jaffe A.B. Hall A. Adv. Cancer Res. 2002; 84: 57-80Crossref PubMed Scopus (255) Google Scholar, 3Sahai E. Marshall C.J. Nat. Rev. Cancer. 2002; 2: 133-142Crossref PubMed Scopus (1241) Google Scholar). Rho GTPases are converted into their active, GTP-bound form by a large family of ∼50 guanine-nucleotide exchange factors (RhoGEFs) 1The abbreviations used are: RhoGEF, Rho guanine-nucleotide exchange factor; DH, Dbl homology; PH, pleckstrin homology; LARG, leukemia-associated RhoGEF; PEG, polyethylene glycol; mant-GDP, N-methylanthraniloyl-GDP; r.m.s.d., root mean square deviation; RH, regulator of G protein signaling (RGS) homology; MIRAS, multiple isomorphous replacement with anomalous scattering. that have a catalytic domain homologous to that of the Dbl oncoprotein (the DH domain) (4Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (996) Google Scholar). In most Dbl family RhoGEFs, the DH domain is positioned immediately N-terminal to a pleckstrin homology (PH) domain. Structures of DH/PH tandem domains from several RhoGEFs have been determined, either alone (Sos, Trio-N, and Dbs) (5Soisson S.M. Nimnual A.S. Uy M. Bar-Sagi D. Kuriyan J. Cell. 1998; 95: 259-268Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 6Skowronek K.R. Guo F. Zheng Y. Nassar N. J. Biol. Chem. 2004; 279: 37895-37907Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 7Worthylake D.K. Rossman K.L. Sondek J. Structure. 2004; 12: 1079-1086Abstract Full Text Full Text PDF Scopus (30) Google Scholar) or in complex with their substrate GTPases (Tiam1-Rac1, Dbs-Cdc42, Dbs-RhoA, and intersectin-Cdc42) (8Worthylake D.K. Rossman K.L. Sondek J. Nature. 2000; 408: 682-688Crossref PubMed Scopus (308) Google Scholar, 9Rossman K.L. Worthylake D.K. Snyder J.T. Siderovski D.P. Campbell S.L. Sondek J. EMBO J. 2002; 21: 1315-1326Crossref PubMed Scopus (190) Google Scholar, 10Snyder J.T. Worthylake D.K. Rossman K.L. Betts L. Pruitt W.M. Siderovski D.P. Der C.J. Sondek J. Nat. Struct. Biol. 2002; 9: 468-475Crossref PubMed Scopus (193) Google Scholar). The DH domain is an oblong helical bundle that facilitates nucleotide exchange by forming a stable complex with a nucleotide-free conformation of the Rho GTPase. The bulk of the DH domain-GTPase interface is formed between residues in the α1, α5, and α6 segments of the DH domain and the switch 1 and 2 elements of the GTPase. These contacts are highly conserved and define the basis for disruption of the nucleotide and magnesium binding sites of the GTPase. Based on the various DH/PH-GTPase crystal structures and biochemical studies (10Snyder J.T. Worthylake D.K. Rossman K.L. Betts L. Pruitt W.M. Siderovski D.P. Der C.J. Sondek J. Nat. Struct. Biol. 2002; 9: 468-475Crossref PubMed Scopus (193) Google Scholar), the α4-α5 loop region of the DH domain was shown to be important for determining specificity, because it interacts with subfamily-specific residues near the N terminus of the GTPase. Tiam1 is selective for Rac1, intersectin for Cdc42, and Dbs for both Cdc42 and RhoA. However, no structures have yet been reported for a RhoA-selective DH domain. The core of the RhoGEF PH domain is a flattened, seven-stranded β-barrel capped with a characteristic C-terminal helix (αC). The role of the PH domain is complex and varies among RhoGEF subfamilies. The PH domain can help localize Rho-GEFs to membranes (6Skowronek K.R. Guo F. Zheng Y. Nassar N. J. Biol. Chem. 2004; 279: 37895-37907Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 11Zheng Y. Zangrilli D. Cerione R.A. Eva A. J. Biol. Chem. 1996; 271: 19017-19020Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 12Whitehead I.P. Lambert Q.T. Glaven J.A. Abe K. Rossman K.L. Mahon G.M. Trzaskos J.M. Kay R. Campbell S.L. Der C.J. Mol. Cell. Biol. 1999; 19: 7759-7770Crossref PubMed Google Scholar, 13Fuentes E.J. Karnoub A.E. Booden M.A. Der C.J. Campbell S.L. J. Biol. Chem. 2003; 278: 21188-21196Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), which is an important step for many cytosolic RhoGEFs, because Rho GTPases are geranylgeranylated at their C termini and therefore typically membrane-associated. The PH domain may also target the RhoGEF directly to the cytoskeleton where many downstream effectors of RhoA are found (14Glaven J.A. Whitehead I. Bagrodia S. Kay R. Cerione R.A. J. Biol. Chem. 1999; 274: 2279-2285Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 15Olson M.F. Sterpetti P. Nagata K. Toksoz D. Hall A. Oncogene. 1997; 15: 2827-2831Crossref PubMed Scopus (62) Google Scholar, 16Bellanger J.M. Estrach S. Schmidt S. Briancon-Marjollet A. Zugasti O. Fromont S. Debant A. Biol. Cell. 2003; 95: 625-634Crossref PubMed Scopus (32) Google Scholar). In some cases, the PH domain also seems to play a role in regulating catalytic activity (15Olson M.F. Sterpetti P. Nagata K. Toksoz D. Hall A. Oncogene. 1997; 15: 2827-2831Crossref PubMed Scopus (62) Google Scholar, 17Baumeister M.A. Martinu L. Rossman K.L. Sondek J. Lemmon M.A. Chou M.M. J. Biol. Chem. 2003; 278: 11457-11464Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 18Rossman K.L. Cheng L. Mahon G.M. Rojas R.J. Snyder J.T. Whitehead I.P. Sondek J. J. Biol. Chem. 2003; 278: 18393-18400Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 19Palmby T.R. Abe K. Der C.J. J. Biol. Chem. 2002; 277: 39350-39359Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). For example, residues within the PH domain of Dbs interact directly with the bound Rho GTPase (9Rossman K.L. Worthylake D.K. Snyder J.T. Siderovski D.P. Campbell S.L. Sondek J. EMBO J. 2002; 21: 1315-1326Crossref PubMed Scopus (190) Google Scholar, 10Snyder J.T. Worthylake D.K. Rossman K.L. Betts L. Pruitt W.M. Siderovski D.P. Der C.J. Sondek J. Nat. Struct. Biol. 2002; 9: 468-475Crossref PubMed Scopus (193) Google Scholar) and enhance in vitro nucleotide exchange on Cdc42 and RhoA 11- and 24-fold, respectively (9Rossman K.L. Worthylake D.K. Snyder J.T. Siderovski D.P. Campbell S.L. Sondek J. EMBO J. 2002; 21: 1315-1326Crossref PubMed Scopus (190) Google Scholar, 20Rossman K.L. Campbell S.L. Methods Enzymol. 2000; 325: 25-38Crossref PubMed Google Scholar). Conversely, the PH domains of Sos and the C-terminal DH/PH domains of Trio appear to inhibit nucleotide exchange (16Bellanger J.M. Estrach S. Schmidt S. Briancon-Marjollet A. Zugasti O. Fromont S. Debant A. Biol. Cell. 2003; 95: 625-634Crossref PubMed Scopus (32) Google Scholar, 21Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Crossref PubMed Scopus (390) Google Scholar). The relative orientation of the PH domain with respect to the DH domain is similar in the structures of intersectin, Dbs, and Trio-N, suggesting a common functional role. However, only in the Dbs-RhoA and -Cdc42 complexes have direct contacts between the PH domain and the GTPase been observed. These contacts are important for PH domain-assisted nucleotide exchange in vitro and Dbs function in vivo (18Rossman K.L. Cheng L. Mahon G.M. Rojas R.J. Snyder J.T. Whitehead I.P. Sondek J. J. Biol. Chem. 2003; 278: 18393-18400Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Intersectin does not form analogous contacts between its PH domain and Cdc42 and does not exhibit PH domain-assisted nucleotide exchange (22Pruitt W.M. Karnoub A.E. Rakauskas A.C. Guipponi M. Antonarakis S.E. Kurakin A. Kay B.K. Sondek J. Siderovski D.P. Der C.J. Biochim. Biophys. Acta. 2003; 1640: 61-68Crossref PubMed Scopus (17) Google Scholar). The PH domain of Trio-N assists in nucleotide exchange (16Bellanger J.M. Estrach S. Schmidt S. Briancon-Marjollet A. Zugasti O. Fromont S. Debant A. Biol. Cell. 2003; 95: 625-634Crossref PubMed Scopus (32) Google Scholar), however a structure of the Trio-N DH/PH domains in complex with their substrate GTPase is not available. Leukemia-associated RhoGEF (LARG) and its close homologs, p115-RhoGEF and PDZ-RhoGEF, are RhoA-selective RhoGEFs that are directly regulated by activated Ga12/13 proteins and thereby play a key role in oncogenic transformation induced by G protein-coupled receptors (23Whitehead I.P. Zohn I.E. Der C.J. Oncogene. 2001; 20: 1547-1555Crossref PubMed Scopus (82) Google Scholar, 24Fukuhara S. Chikumi H. Gutkind J.S. Oncogene. 2001; 20: 1661-1668Crossref PubMed Scopus (200) Google Scholar, 25Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (950) Google Scholar). All three Rho-GEFs contain a regulator of G protein signaling (RGS) homology (RH) domain positioned ∼200 residues N-terminal to their DH/PH domains and are therefore referred to as the RH-Rho-GEFs (24Fukuhara S. Chikumi H. Gutkind J.S. Oncogene. 2001; 20: 1661-1668Crossref PubMed Scopus (200) Google Scholar). The RH domain binds to and serves as a GTPase-activating protein for activated Gα12/13 (26Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (743) Google Scholar, 27Suzuki N. Nakamura S. Mano H. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 733-738Crossref PubMed Scopus (175) Google Scholar). At the same time, the binding of Gα12/13 (27Suzuki N. Nakamura S. Mano H. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 733-738Crossref PubMed Scopus (175) Google Scholar, 28Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (679) Google Scholar) and possibly Gαq (29Booden M.A. Siderovski D.P. Der C.J. Mol. Cell. Biol. 2002; 22: 4053-4061Crossref PubMed Scopus (149) Google Scholar) stimulates nucleotide exchange on RhoA. The PH domains of LARG (30Reuther G.W. Lambert Q.T. Booden M.A. Wennerberg K. Becknell B. Marcucci G. Sondek J. Caligiuri M.A. Der C.J. J. Biol. Chem. 2001; 276: 27145-27151Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and p115-RhoGEF (31Wells C.D. Gutowski S. Bollag G. Sternweis P.C. J. Biol. Chem. 2001; 276: 28897-28905Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) are required for full catalytic activity in vitro and enhance nucleotide exchange ∼2- and 14- to 24-fold, respectively. Whereas the contributions of the LARG and other RhoGEF PH domains toward nucleotide exchange in vitro are relatively small (2- to 24-fold), it is anticipated that they have much more profound effects in vivo, as was shown for Dbs (18Rossman K.L. Cheng L. Mahon G.M. Rojas R.J. Snyder J.T. Whitehead I.P. Sondek J. J. Biol. Chem. 2003; 278: 18393-18400Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). To better understand the structure and regulation of human LARG, we initiated crystallographic studies of its DH/PH domains (32Kristelly R. Earnest B.T. Krishnamoorthy L. Tesmer J.J. Acta Crystallogr. D. Biol. Crystallogr. 2003; 59: 1859-1862Crossref PubMed Scopus (19) Google Scholar). Herein we report atomic structures of the DH/PH domains of LARG and their complex with a soluble (unprenylated) form of human RhoA. These structures reveal novel interactions between the LARG DH and PH domains and RhoA. Using site-directed mutagenesis and fluorescence-based nucleotide-exchange assays, we show that these interactions are important for LARG-mediated nucleotide exchange in vitro. Cloning, Expression, and Protein Purification—The cloning, expression, and purification of the LARG DH/PH fragment and TEV protein were as previously described (32Kristelly R. Earnest B.T. Krishnamoorthy L. Tesmer J.J. Acta Crystallogr. D. Biol. Crystallogr. 2003; 59: 1859-1862Crossref PubMed Scopus (19) Google Scholar). DNA encoding the LARG DH domain (residues 765–986) was cloned into a modified pMAL expression vector (pMALc2H10T) using BamHI and SalI restriction sites. The pMALc2H10T expression vector was generated by inserting oligonucleotides encoding a decahistidine (H10) tag followed by a TEV protease recognition site between AvaI and EcoRI of the pMALc2X vector (New England Biolabs). Expression and purification of the LARG DH domain was as described previously for the DH/PH domains except that protein was expressed at 20 °C, the MBP-DH fusion protein was dialyzed against buffer containing 100 mm NaCl, and finally digested with 2% (w/w) TEV protease. Fractions containing the DH domain were pooled, concentrated to ∼5 mg/ml, and stored at -80 °C. The DNA sequence encoding 1–193 of human RhoA was cloned from the pGEXKG-RhoA vector (T. Kozasa, University of Illinois at Chicago, Medical Center) into pMALc2H10T using the EcoRI and SalI restriction sites. Protein expression from Rosetta (DE3) pLysS cells transformed with the pMALc2H10T-RhoA vector was induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside at 30 °C and harvested after 4–6 h. Lysis and purification was as described above for the LARG DH domain, except that cells were lysed in the presence of 50 μm GDP. Buffers for nickel-nitrilotriacetic acid columns also contained 10% glycerol, 10 mm MgCl2, and 5 μm GDP, and the gel filtration buffer contained 1 mm MgCl2 and 40 μm GDP. RhoA was concentrated to ∼5 mg/ml and stored at -80 °C. The coding region for residues 1–192 of human Rac1 and residues 1–191 of human Cdc42 were amplified from pCDNA-rac1 and pCDNA-cdc42 (gifts from S. Dharmawardhane, University of Texas at Austin), and then cloned into the pMALC2H10T vector using the EcoRI/SalI sites. The proteins were then purified as described above for RhoA. Purification of the RhoA-DH/PH Complex—The DH/PH domains of LARG were mixed with a 2-fold molar excess of RhoA and diluted 10-fold with complex buffer (20 mm HEPES, pH 8.0, 150 mm NaCl, 10 mm EDTA, 2 mm dithiothreitol). After incubation for 10 min on ice, the complex was loaded onto an S200 16/60 size-exclusion column pre-equilibrated in complex buffer supplemented with 1 mm EDTA. Fractions containing the 1:1 RhoA-DH/PH complex were pooled, concentrated to about 8 mg/ml, and stored at -80 °C until crystallization. Crystallization and Data Collection—Crystallization of and data collection from the LARG DH/PH domains were described previously (32Kristelly R. Earnest B.T. Krishnamoorthy L. Tesmer J.J. Acta Crystallogr. D. Biol. Crystallogr. 2003; 59: 1859-1862Crossref PubMed Scopus (19) Google Scholar) (Table I). DH/PH-RhoA complex crystals were formed by vapor diffusion using wells containing 50 mm sodium phosphate, pH 7.4, 11% PEG 8K, 0.6 m NaCl, and 5 mm EDTA. The crystals grew as long rods that can approach 1 mm in length and have 72% solvent content. Native data from the DH/PH-RhoA complex were collected from a single crystal at 90 K harvested in cryoprotectant solution (15% PEG 400, 50 mm sodium phosphate, pH 7.4, 20 mm HEPES, pH 8.0, 15% PEG 8K, 0.6 m NaCl, 5 mm EDTA, and 2 mm dithiothreitol) on beam line 8.2.1 at the Advanced Light Source, Lawrence Berkeley National Lab (Table I). Data were reduced by HKL2000 (33Otwinoski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38782) Google Scholar).Table IData collection and refinement statisticsDH/PHDH/PH-RhoAWavelength (Å)1.0001.069Space groupC2C2Unit cell (Å, °)a = 193.6a = 294.9b = 45.9b = 95.0c = 74.7c = 157.0β = 107.5β = 94.0Resolution limit (Å)2.073.2Unique reflections (total)35,784 (814,907)64,268 (1,360,421)Completeness (%)aValues in parentheses refer to the highest resolution shell (DH/PH: 2.12–2.07 Å; DH/PH-RhoA: 3.31–3.2 Å)97.5 (93.7)aValues in parentheses refer to the highest resolution shell (DH/PH: 2.12–2.07 Å; DH/PH-RhoA: 3.31–3.2 Å)91.2 (80.4)Rsym (%)bRsym = Σ|I – Iavg|/ΣI, where the summation is over all symmetry-equivalent reflections, excluding reflections observed only once4.6 (36.5)8.3 (57.4)Average I/σ(I)21.0 (3.3)11.4 (1.4)Resolution range for refinement (Å)24 to 2.0715 to 3.22Total reflections used35,78460,392Number of protein atoms2,98017,001Number of water molecules910r.m.s.d. bond lengths (Å)0.020.02r.m.s.d. bond angles (°)1.71.8Rwork (%)cRwork = Σh ‖Fobs(h) – |Fcalc(h)‖/Σh|Fobs(h)|; no I/σ cutoff was used during refinement23.324.8Rfree (%)d5% of reflections were reserved from refinement for the calculation of Rfree27.629.1Average B-factor (Å2)36.530.4a Values in parentheses refer to the highest resolution shell (DH/PH: 2.12–2.07 Å; DH/PH-RhoA: 3.31–3.2 Å)b Rsym = Σ|I – Iavg|/ΣI, where the summation is over all symmetry-equivalent reflections, excluding reflections observed only oncec Rwork = Σh ‖Fobs(h) – |Fcalc(h)‖/Σh|Fobs(h)|; no I/σ cutoff was used during refinementd 5% of reflections were reserved from refinement for the calculation of Rfree Open table in a new tab Structure Determinations—The structure of the LARG DH/PH domains was determined using a combination of MIRAS and molecular replacement. Xenon and NaBr derivatives were generated as described previously (32Kristelly R. Earnest B.T. Krishnamoorthy L. Tesmer J.J. Acta Crystallogr. D. Biol. Crystallogr. 2003; 59: 1859-1862Crossref PubMed Scopus (19) Google Scholar) and a homology model of the LARG DH domain, based on that of intersectin (10Snyder J.T. Worthylake D.K. Rossman K.L. Betts L. Pruitt W.M. Siderovski D.P. Der C.J. Sondek J. Nat. Struct. Biol. 2002; 9: 468-475Crossref PubMed Scopus (193) Google Scholar), was manually placed into the resulting MIRAS-phased electron density map. Phases from molecular replacement and MIRAS were then combined, and the DH domain was refined using CNS (34Brünger 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. Acta Crystallogr. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (17025) Google Scholar). Subsequently, a solvent-flattened electron density map allowed placement of a homology model of the LARG PH domain. The structure was refined using rounds of maximum-likelihood refinement by either CNS or REFMAC (35Bailey S. Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (42) Google Scholar) alternating with model building in the program O (36Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13055) Google Scholar). In the final rounds of refinement, individual isotropic B-factors were used in conjunction with TLS refinement (37Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1660) Google Scholar). Modeling of the DH/PH domains was ultimately assisted by the structure of the DH/PH-RhoA complex, which facilitated interpretation of poorly ordered regions of the structure, particularly the N-terminal extension of the DH domain, and the β1-β2 and βN-αN loops of the PH domain. The 3.2-Å crystal structure of the LARG DH/PH-RhoA complex was determined by molecular replacement using as a search model the LARG DH domain modeled in complex with nucleotide-free RhoA (10Snyder J.T. Worthylake D.K. Rossman K.L. Betts L. Pruitt W.M. Siderovski D.P. Der C.J. Sondek J. Nat. Struct. Biol. 2002; 9: 468-475Crossref PubMed Scopus (193) Google Scholar). The PH domains of the four complexes in the asymmetric unit were later fit by hand. The structure was refined and built as described for the LARG DH/PH domains, except that 4-fold NCS restraints were imposed on structurally equivalent regions of each DH/PH-RhoA complex throughout refinement using REFMAC. As structural differences between subunits became apparent, these restraints were gradually loosened and/or eliminated. For all DH domains and RhoA subunits, main-chain and side-chain densities are well defined. The A and C chain PH domains are better ordered than the E and G PH domains and have even more ordered loops than the PH domain of the 2.1-Å uncomplexed structure. To verify the resulting model, σA-weighted phases (38Read R.J. Acta Crystallogr. Sect. A. 1986; 42: 140-149Crossref Scopus (2064) Google Scholar) were generated from the coordinates and refined with twenty cycles of solvent flattening and averaging in the program DM (39Cowtan K. Joint CCP4 ESF-EACBM Newsletter Prot. Crystallogr. 1994; 31: 34-38Google Scholar). Multidomain 4-fold averaging was used for all parts of the structure except for the RhoA subunits, which were subjected to 3-fold averaging owing to the observed conformational change in the B chain, whose density was omitted from averaging. Correlation between the averaged DM map and the 2|Fo| - |Fc| Fourier map generated by REFMAC was 95% for main-chain atoms and 92% for side-chain atoms. The omit map shown in Fig. 5a was generated in a similar fashion, except that the B chain of RhoA was left out of the initial model used to generate phases. Two residues in each subunit of LARG, Ser-833 and Asp-1054, fall within the disallowed region of the Ramachandran plot. In other atomic structures of DH domains, residues equivalent to Ser833 have the same strained backbone stereochemistry. Asp-1054 exists in the i+1 position of a type I β-turn in the PH domain, a position usually occupied by glycine. In the DH/PH structure, the residue Asn-765 of the DH domain and residues 999–1007 in the βN-αN loop and 1062–1074 in the β4 strand of the PH domain could not be modeled. In the "A" and "C" DH/PH chains of the LARG DH/PH-RhoA complex, Asp-765 at the N terminus and residues 1064–1074 in the β4 insertion of the PH domain do not have interpretable electron density and were not modeled. In the "E" and "G" DH/PH chains, the PH domains were substantially more disordered, and various additional loops between secondary structural elements could not be modeled. Refinement statistics for the LARG DH/PH and DH/PH-RhoA structure determinations are shown in Table I. Atomic coordinates and structure factor files have been deposited for the LARG DH/PH domain and the LARG DH/PH-RhoA complex in the Protein Data Bank with accession codes 1TXD and 1X86, respectively. Visual representations of the models were created using PyMOL (40.DeLano, W. (2002), 0.93 Ed., DeLano Scientific, San Carlos, CAGoogle Scholar). DH/PH and RhoA Mutagenesis and Purification of Mutants—DH/PH-W769A, DH/PH-W769D, DH/PH-ΔN, DH/PH-E1023A, DH/PH-E1023R, and DH/PH-S1118D mutants were generated in the pMALc2TH6-DH/PH bacterial expression vector by site-directed mutagenesis using the QuikChange mutagenesis kit (Stratagene). RhoA mutants RhoA-V33E, RhoA-K27T, RhoA-R68A, and RhoA-E97A were produced from pMALc2H10T-RhoA. The complete coding sequence for each of the mutant proteins was verified by DNA sequencing. The DH/PH and RhoA mutant constructs were expressed and purified as described above for the wild-type proteins. Nucleotide Exchange Assay—GTPases were loaded with N-methylanthraniloyl-GDP (mant-GDP; Jena Bioscience) by incubating 180 μm RhoA with a 10-fold molar excess of mant-GDP in loading buffer (20 mm HEPES, pH 8.0, 100 mm NaCl, 4 mm EDTA, 1 mm dithiothreitol) for 1.5 h on ice. Subsequently, the mant-GDP-loaded GTPase was stabilized by addition of MgCl2 to a final concentration of 10 mm and incubated for an additional 30 min on ice. mant-GDP-loaded GTPase was then exchanged into reaction buffer (20 mm HEPES, pH 8.0, 150 mm NaCl, 10 mm MgCl2, 1 mm dithiothreitol) via a G10 gel filtration column pre-equilibrated with reaction buffer to remove excess nucleotides. Fluorescence assays were performed on a Fluoromax-3 spectrophotometer at 25 °C (λex = 280 nm, λem = 430 nm, slits = 2/2 nm), in which 1 μm of each mant-GDP-loaded GTPase was incubated with 100 μm GTP in reaction buffer in a 200-μl cuvette. The exchange reaction was then started by the addition of 100 nm LARG fragment, and kobs was determined by modeling each trace as a one-phase exponential decay using the program Prism version 4.0. All proteins were purified to greater than 95% homogeneity as judged by SDS-PAGE, and their concentrations were determined using the BCA protein assay (Pierce). The Structure of the LARG DH/PH Domains—As in previously determined DH domain structures, the core of the LARG DH domain is comprised of six major helical segments (9Rossman K.L. Worthylake D.K. Snyder J.T. Siderovski D.P. Campbell S.L. Sondek J. EMBO J. 2002; 21: 1315-1326Crossref PubMed Scopus (190) Google Scholar), wherein segments 2, 3, and 5 are broken into several distinct α-helices (Fig. 1a). The LARG DH domain, however, has a novel N-terminal extension (residues 766–781) composed of two short helices, αN1 and αN2, that bury the side chain of Trp-769 against the α1 helix of the DH domain (Figs. 1a and 2a). This extension was included in the DH/PH fragment used for crystallization because of its high sequence homology among the Lbc subfamily of RhoGEFs (4Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (996) Google Scholar), which includes the RH-RhoGEFs, Lbc, Lfc, Net1, Xpln, and intersectin (Fig. 2b). Otherwise, the LARG DH domain is quite similar to that of its closest homolog of known structure, intersectin (r.m.s.d. of 1.3 Å for 187 equivalent Cα atoms), whose structure was determined without the corresponding N-terminal extension (10Snyder J.T. Worthylake D.K. Rossman K.L. Betts L. Pruitt W.M. Siderovski D.P. Der C.J. Sondek J. Nat. Struct. Biol. 2002; 9: 468-475Crossref PubMed Scopus (193) Google Scholar). The most pronounced difference between the LARG and intersectin DH domains occurs within the region spanning the α2-α3 loop and the first helix of the third helical segment, which is on the opposite side of the DH domain from the GTPase binding site.Fig. 2The αN1/αN2 extension of LARG. a, structure of the αN1/αN2 extension and its contacts with the switch 1 region of RhoA. Side chains that contribute to the small hydrophobic core of the extension are shown except for Gln789 from α1, whose side-chain packs against Trp-769. Several hydrogen bonds (dashed yellow lines) also likely stabilize the extension: the side chain of Glu-790 forms two backbone hydrogen bonds with the N terminus of αN1, and a backbone carbonyl in the α2-α3 loop forms a hydrogen bond with the side chain of Trp-769. The side chain of Gln-789 (not shown) also forms two backbone hydrogen bonds with the α2-α3 loop. b, sequence alignment of the αN1/αN2 extensions from Lbc subfamily RhoGEFs, and comparison with the N-terminal extension of Vav. Although they form distinct structures, the αN1/αN2 extension of LARG and an analogous N-terminal extension of Vav both ap