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• W1957224860 abstract "C3 exoenzyme is a mono-ADP-ribosyltransferase (ART) that catalyzes transfer of an ADP-ribose moiety from NAD+ to Rho GTPases. C3 has long been used to study the diverse regulatory functions of Rho GTPases. How C3 recognizes its substrate and how ADP-ribosylation proceeds are still poorly understood. Crystal structures of C3-RhoA complex reveal that C3 recognizes RhoA via the switch I, switch II, and interswitch regions. In C3-RhoA(GTP) and C3-RhoA(GDP), switch I and II adopt the GDP and GTP conformations, respectively, which explains why C3 can ADP-ribosylate both nucleotide forms. Based on structural information, we successfully changed Cdc42 to an active substrate with combined mutations in the C3-Rho GTPase interface. Moreover, the structure reflects the close relationship among Gln-183 in the QXE motif (C3), a modified Asn-41 residue (RhoA) and NC1 of NAD(H), which suggests that C3 is the prototype ART. These structures show directly for the first time that the ARTT loop is the key to target protein recognition, and they also serve to bridge the gaps among independent studies of Rho GTPases and C3. C3 exoenzyme is a mono-ADP-ribosyltransferase (ART) that catalyzes transfer of an ADP-ribose moiety from NAD+ to Rho GTPases. C3 has long been used to study the diverse regulatory functions of Rho GTPases. How C3 recognizes its substrate and how ADP-ribosylation proceeds are still poorly understood. Crystal structures of C3-RhoA complex reveal that C3 recognizes RhoA via the switch I, switch II, and interswitch regions. In C3-RhoA(GTP) and C3-RhoA(GDP), switch I and II adopt the GDP and GTP conformations, respectively, which explains why C3 can ADP-ribosylate both nucleotide forms. Based on structural information, we successfully changed Cdc42 to an active substrate with combined mutations in the C3-Rho GTPase interface. Moreover, the structure reflects the close relationship among Gln-183 in the QXE motif (C3), a modified Asn-41 residue (RhoA) and NC1 of NAD(H), which suggests that C3 is the prototype ART. These structures show directly for the first time that the ARTT loop is the key to target protein recognition, and they also serve to bridge the gaps among independent studies of Rho GTPases and C3. Rho GTPases (∼20 kDa) are key regulators of cytoskeletal dynamics, affecting such processes as morphogenesis, cell migration, neuronal development, and cell division and adhesion (1Heasman S.J. Ridley A.J. Mammalian Rho GTPases: new insights into their functions from in vivo studies.Nat. Rev. Mol. Cell Biol. 2008; 9: 690-701Crossref PubMed Scopus (1432) Google Scholar, 2Hall A. Rho GTPases and the actin cytoskeleton.Science. 1998; 279: 509-514Crossref PubMed Scopus (5216) Google Scholar) and functioning as molecular switches that control signal transduction pathways from plasma membrane receptors to the cytoskeleton. The switching mechanism is thought to be mediated by conformational changes in two switch regions (I and II). Switching between an inactive GDP-bound conformation and an active GTP-bound one (*Rho(GDP) or Rho(GTP) indicates the Rho-bound nucleotide) (3Ihara K. Muraguchi S. Kato M. Shimizu T. Shirakawa M. Kuroda S. Kaibuchi K. Hakoshima T. Crystal structure of human RhoA in a dominantly active form complexed with a GTP analogue.J. Biol. Chem. 1998; 273: 9656-9666Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 4Wei Y. Zhang Y. Derewenda U. Liu X. Minor W. Nakamoto R.K. Somlyo A.V. Somlyo A.P. Derewenda Z.S. Crystal structure of RhoA-GDP and its functional implications.Nat. Struct. Biol. 1997; 4: 699-703Crossref PubMed Scopus (153) Google Scholar5Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Structure at 1.65 A of RhoA and its GTPase-activating protein in complex with a transition-state analogue.Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar) is tightly controlled by three regulatory proteins: (i) the guanine nucleotide exchange factor (GEF) 2The abbreviations used are: GEFguanine nucleotide exchange factorARTADP-ribosyltransferaseGDIguanine nucleotide dissociation inhibitorARTTADP-ribosylating toxin turn-turnPNphosphate nicotinamidePDBProtein Data Bankr.m.s.d.root mean square deviation. activates Rho GTPase by exchanging GDP for GTP; (ii) the GTPase-activating protein (GAP) inactivates Rho GTPase by increasing its intrinsic rate of GTP-hydrolysis; and (iii) the guanine nucleotide dissociation inhibitor (GDI) sequesters isoprenylated Rho GTPase in the cytosol away from the membrane. guanine nucleotide exchange factor ADP-ribosyltransferase guanine nucleotide dissociation inhibitor ADP-ribosylating toxin turn-turn phosphate nicotinamide Protein Data Bank root mean square deviation. ADP-ribosylation is an important post-translational protein modification known to be catalyzed by bacterial toxins as well as eukaryotic endogenous ADP-ribosyltransferases (ARTs). Based on their target specificity, bacterial ARTs are traditionally classified into several types. Interestingly, however, there is a prominent structural similarity between the Rho GTPase-specific C3 exoenzyme and the binary toxin enzymatic units Ia (6Tsuge H. Nagahama M. Nishimura H. Hisatsune J. Sakaguchi Y. Itogawa Y. Katunuma N. Sakurai J. Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin.J. Mol. Biol. 2003; 325: 471-483Crossref PubMed Scopus (88) Google Scholar), C2I (7Schleberger C. Hochmann H. Barth H. Aktories K. Schulz G.E. Structure and action of the binary C2 toxin from Clostridium botulinum.J. Mol. Biol. 2006; 364: 705-715Crossref PubMed Scopus (95) Google Scholar), VIP2 (8Han S. Craig J.A. Putnam C.D. Carozzi N.B. Tainer J.A. Evolution and mechanism from structures of an ADP-ribosylating toxin and NAD complex.Nat. Struct. Biol. 1999; 6: 932-936Crossref PubMed Scopus (208) Google Scholar), and CDTa (9Sundriyal A. Roberts A.K. Shone C.C. Acharya K.R. Structural basis for substrate recognition in the enzymatic component of ADP-ribosyltransferase toxin CDTa from Clostridium difficile.J. Biol. Chem. 2009; 284: 28713-28719Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), even though they have different substrate specificities (10Han S. Arvai A.S. Clancy S.B. Tainer J.A. Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis.J. Mol. Biol. 2001; 305: 95-107Crossref PubMed Scopus (129) Google Scholar); C3-like ARTs (25 kDa) catalyze the ADP-ribosylation of Rho GTPases on Asn-41 (11Sekine A. Fujiwara M. Narumiya S. Asparagine residue in the Rho gene product is the modification site for botulinum ADP-ribosyltransferase.J. Biol. Chem. 1989; 264: 8602-8605Abstract Full Text PDF PubMed Google Scholar) and Ia-like ARTs modify actin on Arg-177 (6Tsuge H. Nagahama M. Nishimura H. Hisatsune J. Sakaguchi Y. Itogawa Y. Katunuma N. Sakurai J. Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin.J. Mol. Biol. 2003; 325: 471-483Crossref PubMed Scopus (88) Google Scholar). C3 exoenyzme has been used extensively to examine the function of Rho GTPases (12Aktories K. Hall A. Botulinum ADP-ribosyltransferase C3: a new tool to study low molecular weight GTP-binding proteins.Trends Pharmacol. Sci. 1989; 10: 415-418Abstract Full Text PDF PubMed Scopus (119) Google Scholar13Ridley A.J. Hall A. Snails, Swiss, and serum: the solution for Rac ‘n’ Rho.Cell. 2004; 116: S23-S25Abstract Full Text PDF PubMed Google Scholar, 14Ridley A.J. Hall A. The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3824) Google Scholar, 15Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. The small GTP-binding protein Rac regulates growth factor-induced membrane ruffling.Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3071) Google Scholar16Nobes C.D. Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3728) Google Scholar). C3-like ARTs are expressed by various Gram-positive bacteria and have been characterized on the basis of their substrate specificity. So far, seven C3-like isoforms have been described: two isoforms of C3 from Clostridium botulinum (C3bot1 and C3bot2) (17Aktories K. Weller U. Chhatwal G.S. Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin.FEBS Lett. 1987; 212: 109-113Crossref PubMed Scopus (172) Google Scholar18Ohashi Y. Narumiya S. ADP-ribosylation of a Mr 21,000 membrane protein by type D botulinum toxin.J. Biol. Chem. 1987; 262: 1430-1433Abstract Full Text PDF PubMed Google Scholar, 19Rubin E.J. Gill D.M. Boquet P. Popoff M.R. Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum.Mol. Cell. Biol. 1988; 8: 418-426Crossref PubMed Scopus (235) Google Scholar, 20Popoff M. Boquet P. Gill D.M. Eklund M.W. DNA sequence of exoenzyme C3, an ADP-ribosyltransferase encoded by Clostridium botulinum C and D phages.Nucleic Acids Res. 1990; 181291 Crossref PubMed Scopus (25) Google Scholar21Nemoto Y. Namba T. Kozaki S. Narumiya S. Clostridium botulinum C3 ADP-ribosyltransferase gene: cloning, sequencing, and expression of a functional protein in Escherichia coli.J. Biol. Chem. 1991; 266: 19312-19319Abstract Full Text PDF PubMed Google Scholar), C3lim from Clostridium limosum (22Just I. Mohr C. Schallehn G. Menard L. Didsbury J.R. Vandekerckhove J. van Damme J. Aktories K. Purification and characterization of an ADP-ribosyltransferase produced by Clostridium limosum.J. Biol. Chem. 1992; 267: 10274-10280Abstract Full Text PDF PubMed Google Scholar), C3 from Bacillus cereus (C3cer) (23Just I. Selzer J. Jung M. van Damme J. Vandekerckhove J. Aktories K. Rho-ADP-ribosylating exoenzyme from Bacillus cereus: purification, characterization, and identification of the NAD-binding site.Biochemistry. 1995; 34: 334-340Crossref PubMed Scopus (57) Google Scholar), and three isoforms of C3 from Staphylococcus aureus (C3stau1, C3stau2, and C3stau3) (24Inoue S. Sugai M. Murooka Y. Paik S.Y. Hong Y.M. Ohgai H. Suginaka H. Molecular cloning and sequencing of the epidermal cell differentiation inhibitor gene from Staphylococcus aureus.Biochem. Biophys. Res. Commun. 1991; 174: 459-464Crossref PubMed Scopus (56) Google Scholar25Wilde C. Just I. Aktories K. Structure-function analysis of the Rho-ADP-ribosylating exoenzyme C3stau2 from Staphylococcus aureus.Biochemistry. 2002; 41: 1539-1544Crossref PubMed Scopus (33) Google Scholar, 26Yamaguchi T. Hayashi T. Takami H. Ohnishi M. Murata T. Nakayama K. Asakawa K. Ohara M. Komatsuzawa H. Sugai M. Complete nucleotide sequence of a Staphylococcus aureus exfoliative toxin B plasmid and identification of a novel ADP-ribosyltransferase, EDIN-C.Infect. Immun. 2001; 69: 7760-7771Crossref PubMed Scopus (119) Google Scholar27Wilde C. Chhatwal G.S. Aktories K. C3stau, a new member of the family of C3-like ADP-ribosyltransferases.Trends Microbiol. 2002; 10: 5-7Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). C3bot, C3lim, and C3cer preferentially ADP-ribosylate RhoA, RhoB, and RhoC but not Rac1 or Cdc42 (28Wilde C. Vogelsgesang M. Aktories K. Rho-specific Bacillus cereus ADP-ribosyltransferase C3cer cloning and characterization.Biochemistry. 2003; 42: 9694-9702Crossref PubMed Scopus (39) Google Scholar). ADP-ribosylation of Rho GTPases by C3-like ARTs prevents GEF binding, thereby inhibiting subsequent binding of Rho GTPases to effector proteins (29Sehr P. Joseph G. Genth H. Just I. Pick E. Aktories K. Glucosylation and ADP ribosylation of Rho proteins: effects on nucleotide binding, GTPase activity, and effector coupling.Biochemistry. 1998; 37: 5296-5304Crossref PubMed Scopus (170) Google Scholar). Moreover, ADP-ribosylation increases the ability of RhoA to complex with GDI (30Genth H. Gerhard R. Maeda A. Amano M. Kaibuchi K. Aktories K. Just I. Entrapment of Rho ADP-ribosylated by Clostridium botulinum C3 exoenzyme in the Rho-guanine nucleotide dissociation inhibitor-1 complex.J. Biol. Chem. 2003; 278: 28523-28527Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), so that ADP-ribosylated RhoA is found exclusively in the cytosolic fraction of C3-treated cells. This C3-induced sequestration most likely blocks Rho-dependent signaling (31Aktories K. Wilde C. Vogelsgesang M. Rho-modifying C3-like ADP-ribosyltransferases.Rev. Physiol. Biochem. Pharmacol. 2004; 152: 1-22Crossref PubMed Scopus (97) Google Scholar). The first structural details of a C3-like ART were obtained from C3bot1 in a NAD+-free state using x-ray crystallography (10Han S. Arvai A.S. Clancy S.B. Tainer J.A. Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis.J. Mol. Biol. 2001; 305: 95-107Crossref PubMed Scopus (129) Google Scholar). Han and Tainer (32Han S. Tainer J.A. The ARTT motif and a unified structural understanding of substrate recognition in ADP-ribosylating bacterial toxins and eukaryotic ADP-ribosyltransferases.Int. J. Med. Microbiol. 2002; 291: 523-529Crossref PubMed Scopus (77) Google Scholar) proposed that the bipartite ADP-ribosylating toxin turn-turn (ARTT) loop, which consists of turns 1 and 2, is responsible for substrate recognition and is thus crucial for the ARTase activity of C3-like exoenzymes and binary toxins. This finding has prompted the examination of the conformation and significance of the ARTT loop within the NAD+-bound and -free structures of C3 (33Ménétrey J. Flatau G. Stura E.A. Charbonnier J.B. Gas F. Teulon J.M. Le Du M.H. Boquet P. Menez A. NAD binding induces conformational changes in Rho ADP-ribosylating Clostridium botulinum C3 exoenzyme.J. Biol. Chem. 2002; 277: 30950-30957Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 34Evans H.R. Sutton J.M. Holloway D.E. Ayriss J. Shone C.C. Acharya K.R. The crystal structure of C3stau2 from Staphylococcus aureus and its complex with NAD.J. Biol. Chem. 2003; 278: 45924-45930Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, recognition of RhoA by the ARTT loop has never been directly verified, because the structure of a C3-like ART in complex with a protein substrate has never been determined. On the other hand, several RhoA amino acid residues involved in recognition by C3bot have been identified through mutational analysis (35Wilde C. Genth H. Aktories K. Just I. Recognition of RhoA by Clostridium botulinum C3 exoenzyme.J. Biol. Chem. 2000; 275: 16478-16483Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The complex structure C3-RhoA is a long sought after piece of this C3 field study. How C3-like ART recognizes Rho GTPase and how the ADP-ribosylation of asparagine occurs are major issues to be addressed. Here we have reported the crystal structures of apo(NAD+-free)-C3-RhoA(GTP), NADH-C3-RhoA(GTP), and NADH-C3-RhoA(GDP). These structures provide the first direct evidence that the ARTT loop is essential for target protein recognition and not only explain the recognition between C3 and RhoA but also provide insight into the asparagine ART reaction. His-tagged C3cer (UniProt ID: Q8KNY0) was designed and subcloned into the pRham vector, after which the plasmid was transformed into DH10B cells using electroporation. Cells containing pRham-C3cer plasmid were selected on LB agar plates containing kanamycin. A single colony was inoculated into 1.5 liters of LB medium (containing 25 μg/ml kanamycin, 0.2% rhamnose, and 0.05% glucose) and cultured overnight at 37 °C with vigorous shaking. The cells were then harvested by centrifugation and stored at −80 °C. The frozen cells were later thawed, resuspended with 50 ml of buffer CA (50 mm Tris-HCl (pH 8.0), 300 mm NaCl, and 5 mm imidazole), disrupted using a French press, and centrifuged at 180,000 × g for 40 min. The resultant supernatant was loaded onto a Ni-NTA agarose column. After washing the column with buffer CA to remove the unbound residues, the His-tagged sample was eluted using buffer CB (50 mm Tris-HCl (pH 8.0), 300 mm NaCl, and 300 mm imidazole). The His tag was then cleaved using His-tagged TEV (tobacco etch virus) protease overnight at 10 °C. The resultant mixture was concentrated, and the buffer in the mixture was exchanged for buffer CA without imidazole using an Amicon Ultra filter (Millipore). To remove the His tag and His-tagged TEV protease, the sample was run on a nickel-nitrilotriacetic acid-agarose column, after which the concentrated sample was loaded onto a Superdex 75 size-exclusion column (GE Healthcare) and eluted with buffer CC (10 mm Tris-HCl (pH 7.4) and 100 mm NaCl). The C3cer fractions were collected and concentrated to ∼20 mg/ml and stored at −80 °C. The C-terminal 14 residues of RhoA (UniProt ID: P61586) were truncated, and Phe25 was substituted with Asn as described elsewhere (36Derewenda U. Oleksy A. Stevenson A.S. Korczynska J. Dauter Z. Somlyo A.P. Otlewski J. Somlyo A.V. Derewenda Z.S. The crystal structure of RhoA in complex with the DH/PH fragment of PDZRhoGEF, an activator of the Ca(2+) sensitization pathway in smooth muscle.Structure. 2004; 12: 1955-1965Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Truncated and mutated RhoA was subcloned into the pGEX 4T-1 vector, after which the plasmid was transformed into BL21(DE3) cells using heat shock. Cells containing pGEX 4T-1-RhoA plasmid were selected on LB agar plates containing ampicillin, and a single colony was inoculated into a small volume of LB medium and precultured overnight at 37 °C. The preculture was transferred into 1.5 liters of LB medium containing 100 μg/ml ampicillin, and the cells were cultured for 4 h at 37 °C with vigorous shaking. After inducing expression with isopropyl-1-thio-β-d-galactopyranoside at a final concentration of 0.5 mm, the culture was incubated for an additional 22 h at 20 °C. The cells were then harvested by centrifugation and stored at −80 °C. The frozen cells were later thawed, resuspended with 50 ml of buffer RA (50 mm Tris-HCl (pH 7.4), 5 mm MgCl2, and 5 mm DTT) containing complete EDTA-free (Roche Applied Science), passed through a French press, and centrifuged at 17,000 × g for 50 min at 4 °C. The supernatant was loaded onto a glutathione-Sepharose 4B column (GE Healthcare), which was then washed with buffer RA to remove the unbound residues. The GST tag was cleaved in buffer RA containing 2.5 mm CaCl2 and 350 units of thrombin for 18 h at room temperature on the Sepharose 4B column. The sample and the thrombin mixture were then eluted and concentrated using an Amicon Ultra filter, after which the concentrated mixture was loaded onto a Superdex 75 size-exclusion column and eluted with buffer RC (10 mm Tris-HCl (pH 8.0), 1 mm MgCl2, and 1 mm DTT). RhoA fractions were collected, concentrated to ∼20 mg/ml, and stored at −80 °C. Cdc42 (UniProt ID: P60953-1) in which the C-terminal 14 residues were truncated was subcloned into pGEX 4T-1 vector, after which the plasmid was transformed into BL21(DE3) cells using heat shock. Protein expression was the same as described in RhoA. RhoA and Cdc42 mutants were constructed by site-directed mutagenesis using a PrimeSTAR mutagenesis basal kit (TaKaRa Bio Inc.) according to the manufacturer's instructions. Protein expression was the same as described for RhoA. To obtain apo-C3cer-RhoA crystals, C3cer and RhoA samples were mixed at a 1:1 molar ratio before the addition of GTPγS or GDP to a final concentration of 1 mm. Crystallization was carried out using the hanging drop vapor diffusion method against a reservoir solution containing 100 mm MES (pH 6.4) and 20% PEG 1500 at 4 °C. The drop was composed of equal volumes of the protein and reservoir solutions. In 4 to 6 weeks, rod-like crystals appeared in a cluster for both C3cer-RhoA(GTP) and C3cer-RhoA(GDP). NADH cannot be an ADP-ribosylation substrate of C3. To obtain crystals of the NADH-bound C3cer-RhoA(GTP or GDP) complex, a small portion of the clustered crystals was soaked in buffer containing 10 mm NADH with 30% ethylene glycol as a cryoprotectant for 1 h at 4 °C. The crystals were plunged into a stream of nitrogen gas at 100 K. Data collection for the apo- and NADH-soaked C3cer-RhoA(GDP) was performed at 100 K using an x-ray wavelength of 1.0 Å with an ADSC Quantum 210r detector system on beamlines BL-5A and AR-NW12A, respectively, at the KEK Photon Factory. The data for NADH-soaked C3cer-RhoA(GTP) were collected in-house using an x-ray wavelength of 1.54 Å with a MicroMax-007 HF generator and RAXISVII (Rigaku). A total of 360 frames of each of the data sets were collected for each crystal with 0.5° oscillations. The best data set was collected at 1.8 Å for apo-C3cer-RhoA(GTP). The datasets of NADH-C3cer-RhoA(GTP) and NADH-C3cer-RhoA(GDP) were collected at 2.5 Å, respectively. The diffraction images for all crystals were indexed, integrated, and scaled using the programs DENZO and SCALEPACK from the HKL2000 suite (37Otwinowski Z. Minor W. Processing of x-ray diffraction data collected in oscillation mode.Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). The space group for all crystals was P32, and the asymmetric unit contained one C3cer and one RhoA(GTP or GDP). Data collection statistics and cell constants are summarized in Table 1.TABLE 1Data collection and structure refinement statistics of C3cer-RhoA complexesapo-C3cer-RhoA(GTP)NADH-C3cer-RhoA(GTP)NADH-C3cer-RhoA(GDP)Data collection groupP32P32P32 Cell dimensionsa, b, c (Å)50.75, 50.75, 135.8750.42, 50.42, 136.6750.48, 50.48, 136.67α, β, γ (°)90.0, 90.0, 120.090.0, 90.0, 120.090.0, 90.0, 120.0 X-ray sourcePF BL-5ARigaku MicroMax-007 HFPF-AR NW-12A Wavelength (Å)1.001.541.00 Resolution range (Å)50.00–1.80 (1.86–1.80)50.00–2.50 (2.59–2.50)50.00–2.50 (2.59–2.50) Observed reflections205,28073,14163,158 RmeasaRmeas = Σhkl{N(hkl)/[N(hkl) − 1]}1/2Σi|Ii(hkl) − 〈I(hkl)〉|/ΣhklΣiIi(hkl), where Ii(hkl) are the observed intensities.0.074 (0.859)0.090 (0.912)0.127 (1.336) RpimbRpim = Σhkl{1/[N(hkl) − 1]}1/2Σi|Ii(hkl) − 〈I(hkl)〉|/ΣhklΣiIi(hkl), where Ii(hkl) are the observed intensities, 〈I(hkl)〉 is the average intensity, and N(hkl) is the multiplicity of reflection hkl.0.031 (0.367)0.038 (0.428)0.059 (0.628) CC½cCC½ = percentage of correlation between intensities from random half-data sets. CC½ is calculated according to the formula found in Ref. 54.0.998 (0.823)0.998 (0.703)0.994 (0.449) I/σI (%)21.4 (2.2)21.0 (2.6)17.4 (2.0) Completeness (%)100.0 (100.0)99.9 (99.9)99.6 (100.0) Redundancy5.7 (5.4)5.5 (4.4)4.7 (4.4)Refinement Resolution (Å)45.29-1.8045.56-2.5045.56-2.50 Rwork/Rfree (%)dRwork = Σhkl‖Fobs | − |Fcalc‖/Σhkl|Fobs|. Rfree is the cross-validation R-factor for the test set (5%) of reflections omitted from model refinement.17.7 / 22.319.6 / 25.421.1 / 24.6 Overall B-factors (Å2)eObtained by TLS refinement in Refmac5 of CCP4i.32.849.745.9 r.m.s.d.Bond lengths (Å)0.0080.0100.020Bond angles (°)1.341.251.74 Ramachandran plotFavored regions (%)91.289.493.5Allowed regions (%)8.810.66.5Outliers (%)0.00.00.0 PDB ID4XSG4XSH5BWMa Rmeas = Σhkl{N(hkl)/[N(hkl) − 1]}1/2Σi|Ii(hkl) − 〈I(hkl)〉|/ΣhklΣiIi(hkl), where Ii(hkl) are the observed intensities.b Rpim = Σhkl{1/[N(hkl) − 1]}1/2Σi|Ii(hkl) − 〈I(hkl)〉|/ΣhklΣiIi(hkl), where Ii(hkl) are the observed intensities, 〈I(hkl)〉 is the average intensity, and N(hkl) is the multiplicity of reflection hkl.c CC½ = percentage of correlation between intensities from random half-data sets. CC½ is calculated according to the formula found in Ref. 54Karplus P.A. Diederichs K. Linking crystallographic model and data quality.Science. 2012; 336: 1030-1033Crossref PubMed Scopus (1294) Google Scholar.d Rwork = Σhkl‖Fobs | − |Fcalc‖/Σhkl|Fobs|. Rfree is the cross-validation R-factor for the test set (5%) of reflections omitted from model refinement.e Obtained by TLS refinement in Refmac5 of CCP4i. Open table in a new tab The structure of apo-C3cer-RhoA(GTP) was determined using the molecular replacement method with the Phaser-MR program in the Phenix suite (38Adams P.D. Afonine P.V. Bunkóczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.W. Kapral G.J. Grosse-Kunstleve R.W. McCoy A.J. Moriarty N.W. Oeffner R. Read R.J. Richardson D.C. Richardson J.S. Terwilliger T.C. Zwart P.H. PHENIX: a comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (16434) Google Scholar). The structures of C3lim (PDB code: 3BW8 (39Vogelsgesang M. Stieglitz B. Herrmann C. Pautsch A. Aktories K. Crystal structure of the Clostridium limosum C3 exoenzyme.FEBS Lett. 2008; 582: 1032-1036Crossref PubMed Scopus (15) Google Scholar)) and RhoA (PDB code: 1A2B (3Ihara K. Muraguchi S. Kato M. Shimizu T. Shirakawa M. Kuroda S. Kaibuchi K. Hakoshima T. Crystal structure of human RhoA in a dominantly active form complexed with a GTP analogue.J. Biol. Chem. 1998; 273: 9656-9666Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar)) served as templates. The structure was refined iteratively using Phenix.refine in the Phenix suite (40Afonine P.V. Grosse-Kunstleve R.W. Echols N. Headd J.J. Moriarty N.W. Mustyakimov M. Terwilliger T.C. Urzhumtsev A. Zwart P.H. Adams P.D. Towards automated crystallographic structure refinement with phenix.refine.Acta Crystallogr. D Biol. Crystallogr. 2012; 68: 352-367Crossref PubMed Scopus (3371) Google Scholar), REFMAC5 in the CCP4i suite (41Murshudov 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 (13853) Google Scholar), and Coot (42Emsley P. Cowtan K. Coot: model-building tools for molecular graphics.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23224) Google Scholar). A comparison of the structures and the preparation of the figures were done using PyMOL (43DeLano W.L. The PyMOL Molecular Graphics System. Schrodinger, LLC, New York2010Google Scholar). Finally, the structure was refined and validated using the PDB_REDO Web server (44Joosten R.P. Joosten K. Cohen S.X. Vriend G. Perrakis A. Automatic rebuilding and optimization of crystallographic structures in the Protein Data Bank.Bioinformatics. 2011; 27: 3392-3398Crossref PubMed Scopus (74) Google Scholar). For both NADH-bound C3cer-RhoA(GTP) and NADH-bound C3cer-RhoA(GDP), using the resolved apostructure served as the template, and the structures were determined and improved using the same methods used for the apostructure. The final model statistics for all structures are summarized in Table 1. The crystal assay was conducted as follows. Crystals of the C3-RhoA complex were washed with mother liquor twice. Biotin-NAD+ (50 μm) was then added to the mother liquor containing the C3-RhoA complex, and the mixture was kept at room temperature for 4 h. These samples were then subjected to SDS-PAGE. The gel was washed twice with PBS, stained with streptavidin-FITC (250 nm) overnight, washed twice with PBS again, and scanned using a Typhoon FLA9000 laser scanner (General Electric). The solution assay was conducted as follows. In the experiment shown in Fig. 2, C3cer (0.3 μm) and GST-RhoA (3 μm) or C3cer (0.3 μm) and non-tagged RhoA (3 μm) were mixed together in buffer RC. In the mutational experiment (Fig. 5), C3cer (0.03 μm) and GST-RhoA (3 μm) or C3cer (0.3 μm) and GST-Cdc42 (mutants) (3 μm) were mixed together in buffer RC. Biotin-NAD+ (3.5 μm) was then added to the mixture, which was kept at 37 °C for 10 min. Thereafter, the same protocol used for the crystal assay was applied.FIGURE 5.ADP-ribosylation of RhoA and Cdc42 mutants by C3cer A, ADP-ribosylation of RhoA single point mutations. B, ADP-ribosylation of Cdc42 combined mutations. Wild type and mutants of RhoA and Cdc42 were purified as GST-tagged protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT) we used C3cer for crystallization of the complexes. The structure of C3cer is unknown, but its sequence shows identity with C3bot1 (38%), C3bot2 (36%), C3lim (36%), C3stau1 (29%), and C3stau2 (29%) (Fig. 1A). To investigate how C3cer recognizes RhoA, we determined the crystal structure of apo(NAD+-free)-C3cer complexed with human RhoA(GTP) at 1.8 Å resolution (Fig. 2A and Table 1). Then, by soaking the apo-C3-RhoA(GTP) crystal with NADH, we obtained the structure of NADH-bound C3-RhoA(GTP) at 2.5 Å resolution (Fig. 2B). Using the same approach with C3-RhoA(GDP), we obtained the structure of NADH-C3-RhoA(GDP) at 2.5 Å resolution (Fig. 2C). The overall structure of C3cer is similar to those of other C3 exoenzymes (i.e. a mixed α/β-fold with a β-sandwich core). Structural alignment of C3cer with C3bot1 shows that the main differences are restricted to two 310-helices in C3cer (Fig. 1A). There was no large conformational change between apo-C3-RhoA(GTP) and NADH-C3-RhoA(GTP), except in the phosphate nicotinamide (PN) loop, which is one of the RhoA binding regions. Only in the PN loop, which includes Tyr-151, does a large conformational change from the apo to the NADH structure occur. On the other hand, a comparison between NADH-C3-RhoA(GTP) and NADH-C3-RhoA(GDP) showe" @default.
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• W1957224860 title "Rho GTPase Recognition by C3 Exoenzyme Based on C3-RhoA Complex Structure" @default.
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