The Crystal Structure of C3stau2 from Staphylococcus aureus and Its Complex with NAD
2003; Elsevier BV; Volume: 278; Issue: 46 Linguagem: Inglês
10.1074/jbc.m307719200
ISSN1083-351X
AutoresHazel R. Evans, J. Mark Sutton, Daniel E. Holloway, Joanne Ayriss, Clifford C. Shone, K. Ravi Acharya,
Tópico(s)Antimicrobial Resistance in Staphylococcus
ResumoThe C3stau2 exoenzyme from Staphylococcus aureus is a C3-like ADP-ribosyltransferase that ADP-ribosylates not only RhoA-C but also RhoE/Rnd3. In this study we have crystallized and determined the structure of C3stau2 in both its native form and in complex with NAD at 1.68- and 2.02-Å resolutions, respectively. The topology of C3stau2 is similar to that of C3bot1 from Clostridium botulinum (with which it shares 35% amino acid sequence identity) with the addition of two extra helices after strand β1. The native structure also features a novel orientation of the catalytic ARTT loop, which approximates the conformation seen for the "NAD bound" form of C3bot1. C3stau2 orients NAD similarly to C3bot1, and on binding NAD, C3stau2 undergoes a clasping motion and a rearrangement of the phosphate-nicotinamide binding loop, enclosing the NAD in the binding site. Comparison of these structures with those of C3bot1 and related toxins reveals a degree of divergence in the interactions with the adenine moiety among the ADP-ribosylating toxins that contrasts with the more conserved interactions with the nicotinamide. Comparison with C3bot1 gives some insight into the different protein substrate specificities of these enzymes. The C3stau2 exoenzyme from Staphylococcus aureus is a C3-like ADP-ribosyltransferase that ADP-ribosylates not only RhoA-C but also RhoE/Rnd3. In this study we have crystallized and determined the structure of C3stau2 in both its native form and in complex with NAD at 1.68- and 2.02-Å resolutions, respectively. The topology of C3stau2 is similar to that of C3bot1 from Clostridium botulinum (with which it shares 35% amino acid sequence identity) with the addition of two extra helices after strand β1. The native structure also features a novel orientation of the catalytic ARTT loop, which approximates the conformation seen for the "NAD bound" form of C3bot1. C3stau2 orients NAD similarly to C3bot1, and on binding NAD, C3stau2 undergoes a clasping motion and a rearrangement of the phosphate-nicotinamide binding loop, enclosing the NAD in the binding site. Comparison of these structures with those of C3bot1 and related toxins reveals a degree of divergence in the interactions with the adenine moiety among the ADP-ribosylating toxins that contrasts with the more conserved interactions with the nicotinamide. Comparison with C3bot1 gives some insight into the different protein substrate specificities of these enzymes. The family of C3 ADP-ribosyltransferases is a subgroup of the ADP-ribosyltransferase toxins that also include the A-B toxins such as diphtheria toxin and cholera toxin and the binary toxins, which include C2 from Clostridium botulinum, the vegetative insecticidal protein (VIP) 1The abbreviations used are: VIP, vegetative insecticidal protein; EDIN, epidermal differentiation inhibitor; MES, 4-morpholineethanesulfonic acid; r.m.s., root mean square; PN, phosphate-nicotinamide. from Bacillus cereus, and the Iota toxin from Clostridium perfringens. The targets for the C3 ADP-ribosyltransferases are mammalian Rho GT-Pases, but they are novel among the ADP-ribosylating toxins in that they lack a cell binding or translocation domain to allow entry into cells, and hence, their role in disease is not yet clear. However, the best-characterized member of this family, the C3 exoenzyme from C. botulinum, C3bot1, has long been used to research the function of the small mammalian GTPases. This is due to its ability to specifically ADP-ribosylate and, therefore, inactivate RhoA, -B, and- C (1Sekine A. Fujiwara M. Narumiya S. J. Biol. Chem. 1989; 264: 8602-8605Abstract Full Text PDF PubMed Google Scholar) but not the related proteins Rac and Cdc42 (2Aktories K. Braun U. Rosener S. Just I. Hall A. Biochem. Biophys. Res. Commun. 1989; 158: 209-213Crossref PubMed Scopus (220) Google Scholar, 3Chardin P. Boquet P. Madaule P. Popoff M.R. Rubin E.J. Gill D.M. EMBO J. 1989; 8: 1087-1092Crossref PubMed Scopus (448) Google Scholar, 4Just I. Mohr C. Schallehn G. Menard L. Didsbury J.R. Vandekerckhove J. van Damme J. Aktories K. J. Biol. Chem. 1992; 267: 10274-10280Abstract Full Text PDF PubMed Google Scholar). C3bot1 has been described as the prototype for this family of ADP-ribosyltransferases, which also includes C3 from Clostridium limosium (C3lim) (4Just I. Mohr C. Schallehn G. Menard L. Didsbury J.R. Vandekerckhove J. van Damme J. Aktories K. J. Biol. Chem. 1992; 267: 10274-10280Abstract Full Text PDF PubMed Google Scholar), B. cereus (C3cer) (5Just I. Selzer J. Jung M. van Damme J. Vandekerckhove J. Aktories K. Biochemistry. 1995; 34: 334-340Crossref PubMed Scopus (57) Google Scholar), and the epidermal differentiation inhibitor (EDIN) (6Inoue S. Sugai M. Murooka Y. Paik S.Y. Hong Y.M. Ohgai H. Suginaka H. Biochem. Biophys. Res. Commun. 1991; 174: 459-464Crossref PubMed Scopus (58) Google Scholar) from Staphylococcus aureus. The two isoforms of C3 from C. botulinum, known as C3bot1 (7Popoff M.R. Hauser D. Boquet P. Eklund M.W. Gill D.M. Infect. Immun. 1991; 59: 3673-3679Crossref PubMed Google Scholar, 8Aktories K. Rosener S. Blaschke U. Gursharan S. Chhatwal G.S. Eur. J. Biochem. 1988; 172: 445-450Crossref PubMed Scopus (128) Google Scholar) and C3bot2 (9Nemoto Y. Namba T. Kozaki S. Narumiya S. J. Biol. Chem. 1991; 266: 19312-19319Abstract Full Text PDF PubMed Google Scholar), have so far been assumed to represent the whole family and have attracted the most research. Recently, however, the existence of a subgroup of the family has emerged with the discovery of two proteins from S. aureus named C3stau2 (or EDIN B) (10Wilde C. Chhatwal G.S. Schmalzing G. Aktories K. Just I. J. Biol. Chem. 2001; 276: 9537-9542Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 11Wilde C. Just I. Aktories K. Biochemistry. 2002; 41: 1539-1544Crossref PubMed Scopus (33) Google Scholar, 12Yamaguchi T. Nishifuji K. Sasaki M. Fudaba Y. Aepfelbacher M. Takata T. Ohara M. Komatsuzawa H. Amagai M. Sugai M. Infect. Immun. 2002; 70: 5835-5845Crossref PubMed Scopus (200) Google Scholar) and C3stau3 (or EDIN C) (13Yamaguchi T. Hayashi T. Takami H. Ohnishi M. Murata T. Nakayama K. Asakawa K. Ohara M. Komatsuzawa H. Sugai M. Infect. Immun. 2001; 69: 7760-7771Crossref PubMed Scopus (120) Google Scholar). Whereas the C3s from C. botulinum and C. limosium have 63% sequence identity (4Just I. Mohr C. Schallehn G. Menard L. Didsbury J.R. Vandekerckhove J. van Damme J. Aktories K. J. Biol. Chem. 1992; 267: 10274-10280Abstract Full Text PDF PubMed Google Scholar), the C3stau exoenzymes have only 35% sequence identity with the clostridial C3s, although they are 65% identical to each other (Fig. 1). Interestingly, C3stau2, and very recently, EDIN (C3stau1) have been shown to have substrate specificities different from that of C3bot1, ribosylating the related GTPases RhoE and Rnd3 as well as RhoA-C (10Wilde C. Chhatwal G.S. Schmalzing G. Aktories K. Just I. J. Biol. Chem. 2001; 276: 9537-9542Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 14Wilde C. Vogelsgesang M. Aktories K. Biochemistry. 2003; 42: 9693-9702Crossref Scopus (39) Google Scholar). Inactivation of RhoA-C after C3-mediated ADP-ribosylation at Asn-41 impacts on cell functions in various ways, reflecting the many roles of Rho in mammalian cells. Through the use of C3bot as a research tool it has been shown that Rho is involved in many cell activities through its regulation of the cytoskeleton and transcription. These include cell cycle progression, chemotaxis, cell transformation, and apoptosis (15Ridley A.J. Hall A. GTPases. Oxford University Press, Oxford2001: 89-136Google Scholar). RhoE and Rnd3 are isoforms, identical except for a 15-residue N-terminal extension on Rnd3, that are antagonistic to RhoA (16Guash R.M. Scambler P. Jones G.E. Ridley A.J. Mol. Cell. Biol. 1998; 18: 4761-4771Crossref PubMed Scopus (194) Google Scholar, 17Nobes C.D. Lauritzen I. Mattei M.-G. Paris S. Hall A. Chardin P. J. Cell Biol. 1998; 141: 187-197Crossref PubMed Scopus (311) Google Scholar). Unusually, they bind GTP but lack GTPase activity. This renders them constitutively active (18Foster R. Hu K.-Q. Lu Y. Nolan K.M. Thissen J. Settleman J. Mol. Cell. Biol. 1996; 16: 2689-2699Crossref PubMed Scopus (246) Google Scholar), and they are, therefore, speculated to reduce RhoA activity by sequestering the exchange factors required to activate it (10Wilde C. Chhatwal G.S. Schmalzing G. Aktories K. Just I. J. Biol. Chem. 2001; 276: 9537-9542Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Surprisingly, although parallel C3stau2-mediated ribosylation of RhoA and RhoA antagonists might be presumed to produce a different cell morphology to that produced by C3bot1 exposure, both proteins produce the same cell rounding effect in fibroblasts (10Wilde C. Chhatwal G.S. Schmalzing G. Aktories K. Just I. J. Biol. Chem. 2001; 276: 9537-9542Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The role of the clostridial C3 exoenzymes in pathogenesis is uncertain due to the lack of an obvious cell entry mechanism. The C3stau exoenzymes, however, may have a more obvious role due to the ability of S. aureus to invade host cells, circumventing the need for a binding or translocation domain. It has been shown that EDIN and C3bot1 can prevent wound healing in vivo, indicating a possible role for the C3stau exoenzymes in S. aureus infection (19Aepfelbacher M. Essler M. Huber E. Sugai M. Weber P.C. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1623-1629Crossref PubMed Scopus (97) Google Scholar). Additionally, preliminary findings show that some clinical isolates produce C3stau exoenzymes, C3stau2 in particular (20Czech A. Yamaguchi T. Bader L. Linder S. Kaminski K. Sugai M. Aepfelbacher M. J. Infect. Dis. 2001; 184: 785-788Crossref PubMed Scopus (30) Google Scholar), and another study reports two infections of EDIN-expressing methicillin-resistant S. aureus in neonates suffering acquired subglottic stenosis due to impaired wound healing (21Yamada Y. Sugai M. Woo M. Nishida N. Sugimoto T. Arch. Dis. Child Fetal Neonatal Ed. 2001; 84: 38-39Crossref PubMed Google Scholar). To visualize the structural features of this novel ADP-ribosyltransferase, we have used x-ray crystallography to determine the three-dimensional structure of C3stau2 at 1.68-Å resolution and in complex with NAD at 2.02-Å resolution. Comparison of C3stau2 with the structures of C3bot1 (22Han S. Arvai A.S. Clancy S.B. Tainer J.A. J. Mol. Biol. 2001; 305: 95-107Crossref PubMed Scopus (133) Google Scholar, 23Menetrey J. Flatau G. Stura E.A. Charbonnier J.B. Gas F. Teulon J.M. Le Du M.H. Boquet P. Menez A. J. Biol. Chem. 2002; 277: 30950-30957Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), the VIP from B. cereus (24Han S. Craig J.A. Putnam C.D. Carozzi N.B. Tainer J.A. Nat. Struct. Biol. 1999; 6: 932-936Crossref PubMed Scopus (213) Google Scholar) (Fig. 1), and the Iota toxin from C. perfringens (25Tsuge H. Nagahama M. Nishimura H. Hisatsune J. Sakaguchi Y. Itogawa Y. Katunuma N. Sakurai J. J. Mol. Biol. 2003; 325: 471-483Crossref PubMed Scopus (89) Google Scholar), with each of which it has ∼30% sequence identity, confirms a similar topology and active site features. On binding NAD, C3stau2 encloses the NAD via a clasping motion that brings into range residues important for the interaction. C3stau2 also possesses novel features in its variable domain and position of helix α3 that may govern target protein specificity. Cloning and Expression—A synthetic gene encoding C3stau2 was synthesized using codon bias optimized for expression in Escherichia coli. A series of overlapping oligonucleotides was synthesized (Sigma-Genosys) (Table I). In addition to the structural oligonucleotides, flanking primers were designed to introduce a 5′ and a 3′ BamHI site and sequence encoding a Factor Xa cleavage site immediately before the initial alanine of the mature C3stau2 peptide sequence. The oligonucleotides, at a concentration of 25 nmol/μl for the structural primers and 500 nmol/μl for the flanking primers, were assembled in a single PCR reaction using VENT polymerase (New England Biolabs) in a buffer containing 10 mm KCl, 20 mm Tris-HCl, pH 8.8, at 25 °C, 10 mm (NH4)2SO4, 4.5 mm MgSO4, 0.1% Triton X-100, 1 mm dNTPs. A PCR amplification of 30 cycles was carried out using the parameters 94 °C for 1 min, 56 °C for 1 min, 72 °C for 1 min followed by 72 °C for 10 min. The PCR product was analyzed on a 1.2% agarose gel in Tris-acetate-EDTA electrophoresis buffer, and a principal band of ∼750 base pairs was excised from the gel. The band was extracted from the gel using QIAEX II gel extraction resin (Qiagen) and subcloned into the sequencing vector PCR4.0 TOPO (Invitrogen) according to the manufacturer's instructions. The clone was sequenced and showed two point mutations in the regions of primers C3sBamXa.for and C3s10.rev. These were corrected by amplification with these primers followed by extension to give the full-length clone and re-amplification with the flanking primers. Cloning and sequence verification were carried out as described above.Table IOligonucleotides synthesized for cloning The numbering indicates the order of the oligonucleotides with the forward primers numbered 5′-3′ along the sense strand and the reverse primers numbered 5′-3′ along the anti-sense strand.Oligo nameSequenceC3s1.forGCTGAAACCAAAAACTTCACCGACCTGGTTGAAGCTACCAAATGGGGTAACTCTCTGATCAAATCTGCTAAATACTC TTCC3s2.forTAAAGACAAAATGGCTATCTACAACTACACCAAAAACTCTTCTCCGATCAACACCCCGCTGCGTTCTGCTAACGGTG ACGTTAACAAACTGC3s3.forTCTGAAAACATCCAGGAACAGGTTCGTCAGCTGGACTCTACCATCTCTAAATCTGTTACCCCGGACTCTGTTTACGT TTACCGC3s4.forTCTGCTGAACCTGGACTACCTGTCTTCTATCACCGGTTTCACCCGTGAAGACCTGCACATGCTGCAGCAGACCAACA ACGGC3s5.forTCAGTACAACGAAGCTCTGGTTTCTAAACTGAACAACCTGATGAACTCTCGTATCTACCGTGAAAACGGTTACTCTT CTACCCC3s6.forAGCTGGTTTCTGGTGCTGCTCTGGCTGGTCGTCCGATCGAACTGAAACTGGAACTGCCGAAAGGTACCAAAGCTGCC3s7.forTTACATCGACTCTAAAGAACTGACCGCTTACCCCGGTCAGCAGGAAGTTCTGCTGCCGCGTGGTACCGAATACGCTGC3s8.forTTGGTTCTGTTAAACTGTCTGACAACAAACGTAAAATCATCATCACCGCTGTTGTTTTCAAGAAGC3s9.revGACAGTTTAACAGAACCAACAGCGTATTCG GTACCACGCGGCC3s10.revGTTCTTTAGAGTCGATGTAAGCAGCTTTGGT ACCTTTCGGC3s11.revGAGCAGCACCAGAAACCAGCTGGGTAGAAGAG TAACCGTTTTCACC3s12.revCCAGAGCTTCGTTGTACTGACCGTTGTTGG TCTGCTGCAGCC3s13.revCAGGTAGTCCAGGTTCAGCAGACGGTAAACGT AAACAGAGTCCC3s14.revGTTCCTGGATGTTTTCAGACAGTTTGTTAA CGT CACCGTTAGCC3s15.revGTAGATAGCCATTTTGTCTTTAGAAGAGTATTTA GCAGATTTGATCAGC3sBamXa.forGGGATCCATCGAAGGTCGTGCTGAAACCAAAAACTTCACCGC3sBam.revCGGATCCTTATCACTTCTTGAAAACAACAGCGG Open table in a new tab Protein Expression and Purification—For expression of the C3stau2 fragment, a modified malE fusion vector was generated to ensure that the gene could not be disseminated to other bacteria. The ApaI-HindIII fragment from the expression vector pMALc2x (New England Biolabs) was isolated and subcloned into the vector pBC SK+ (Stratagene) to generate the vector pBCmalE. The construction of the vector was confirmed by restriction digest and sequencing. A BamHI fragment containing the C3stau2-coding sequence was subcloned into BamHI-digested pBCmalE, and the clone was verified. For expression, the clone E. coli TB1 pBCmalE C3stau2 was grown overnight at 30 °C in Terrific Broth supplemented with 35 μg/ml chloramphenicol and 0.5% (w/v) glucose. The overnight culture was diluted 1:10 in fresh media and grown for 4 h (A 600 ∼ 3.9). The culture was induced with isopropyl-1-thio-β-d-galactopyranoside at a final concentration of 500 μm and grown for a further2h30minat25 °C before harvest. The cells were collected by centrifugation and resuspended in 20 mm MES/NaOH, pH 5.8. The cells were lysed by sonication, and the cell lysate was collected by centrifugation. The C3stau2-MBP fusion protein was initially purified by cation exchange chromatography. The protein bound to a SP-Sepharose column equilibrated with 20 mm MES/NaOH, pH 5.8, and was eluted on an ascending NaCl gradient as a single peak. The fractions corresponding to the peak were pooled, and 150 units of Factor Xa were added per half-liter of cells to cleave the fusion protein. During cleavage, the protein was dialyzed for 24 h at room temperature against a buffer containing 20 mm Na-HEPES, pH 7.3, 50 mm NaCl, and 5 mm CaCl2. The cleaved C3stau2 bound to the SP-Sepharose column equilibrated in 20 mm Na-HEPES, pH 7.3, 50 mm NaCl and was eluted again on an ascending salt gradient. Final purification and de-salting was achieved by gel filtration. The protein was concentrated in a 10-kDa cut-off centrifugal concentrator (Amicon) and applied to a Superdex 200 gel filtration column (Amersham Biosciences) equilibrated in 20 mm Na-HEPES, pH 7.3, 50 mm NaCl. The purified protein was finally concentrated to 35 mg/ml and stored in 20 mm Na-HEPES, pH 7.3, 50 mm NaCl at -70 °C. Purity was greater that 95% as judged by SDS-PAGE. The specific ADP-ribosylating activity of the preparation was 0.19 ± 0.01 mol/mol enzyme/min as measured by the labeling of 19 μm RhoA by 11 μm C3stau2 in the presence of 20 μm [adenylate-32P]NAD at 37 °C. Crystallization—Single crystals were grown by the hanging drop vapor diffusion method at 22 °C. 1 μl of C3stau2 (8.75–10.25 mg/ml) was mixed with 1 μl of well solution (100 mm sodium cacodylate, pH 6.4–6.6, 29–30% PEG (polyethylene glycol) 8000). Orthorhombic crystals grew within 1–3 weeks. Seeding produced larger numbers of crystals, which grew within 3 days and were suitable for cryo-diffraction. Structure Determination—Native crystals were soaked in cryoprotectant (0.1 m sodium cacodylate, pH 6.5, 30% PEG 8000, 20% glycerol) and cryo-cooled to 100 K. The crystals diffracted to 1.68 Å on station PX 9.6 at the Synchrotron Radiation Source at Daresbury, UK, and data were collected using an ADSC-CCD detector. The data were processed and scaled with HKL2000 (26Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38777) Google Scholar). The symmetry and systematic absences were consistent with the space group P212121. A Matthews coefficient (27Matthews B.W. J. Mol. Biol. 1968; 33: 479-491Google Scholar) of 2.0 indicated that there was one C3stau2 monomer per asymmetric unit and that the crystals contained 38.8% solvent. Data reduction was carried out using the program TRUNCATE (28CCP4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar). Detailed crystallographic statistics are presented in Table II.Table IICrystallographic statisticsForm of C3stau2NativeNAD complexData collectionSpace groupP212121P212121Maximum resolution (Å)1.682.02Unit cell dimensions (Å)a = 39.62 b = 64.53 c = 74.98a = 41.90 b = 65.08 c = 75.07Measured reflections409,845327,780Unique reflections22,61914,088Completeness (%) (last shell)aLast shell is 1.74—1.68 and 2.09—2.02 Å for native and NAD complex, respectively99.4 (97.5)96.9 (96.6)Mean I/σ(I) (last shell)aLast shell is 1.74—1.68 and 2.09—2.02 Å for native and NAD complex, respectively20.0 (5.3)15.8 (4.1)R sym (%)bR sym = ΣhΣi|I(h) — I i(h)|/ΣhΣi I(h), where I i(h) and I(h) are the ith and the mean measurements of the intensity of reflection h, respectively (last shell)aLast shell is 1.74—1.68 and 2.09—2.02 Å for native and NAD complex, respectively8.5 (29.5)8.8 (34.1)RefinementR cryst (%)cR cryst = Σh|F o — F c|/Σh F o where F o and F c are the observed and calculated structure factor amplitudes of reflection h, respectively17.021.4R free (%)dR free is equal to R sym for a randomly selected 850—950 reflections not used in the refinement (30)23.726.9Number of protein atoms1,6601,660Number of solvent atoms284154Number of ligand atoms41Deviation from ideality (r.m.s.) Bond lengths (Å)0.0040.006 Bond angles (°)1.31.3 Average B-factors (Å2) Protein atoms15.324.5 Solvent molecules19.027.3 Ligand atoms31.1a Last shell is 1.74—1.68 and 2.09—2.02 Å for native and NAD complex, respectivelyb R sym = ΣhΣi|I(h) — I i(h)|/ΣhΣi I(h), where I i(h) and I(h) are the ith and the mean measurements of the intensity of reflection h, respectivelyc R cryst = Σh|F o — F c|/Σh F o where F o and F c are the observed and calculated structure factor amplitudes of reflection h, respectivelyd R free is equal to R sym for a randomly selected 850—950 reflections not used in the refinement (30Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3894) Google Scholar) Open table in a new tab Initial phases were determined by molecular replacement with MOLREP (28CCP4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar) using residues 47–246 of the C3bot1 structure (22Han S. Arvai A.S. Clancy S.B. Tainer J.A. J. Mol. Biol. 2001; 305: 95-107Crossref PubMed Scopus (133) Google Scholar) as a search model. The best solution had an R-factor of 56% and a correlation coefficient of 31.4%. Initial refinement was carried out using CNS version 1.0 (29Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. GrosseKunstleve 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. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (17024) Google Scholar) at 2.6-Å resolution, increasing to 1.68 Å. The behavior of the R-free value (30Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3894) Google Scholar) was monitored throughout. Insertion of water molecules and final refinement were performed in SHELX (31Sheldrick G.M. Gould R.O. Acta Crystallogr. Sect. B. 1995; 51: 423-431Crossref PubMed Scopus (149) Google Scholar). All the water molecules had peak heights above 3σ in the F o - F c map and temperature factors less than 40 Å2. The program O (32Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13039) Google Scholar) was used to rebuild the model and visualize the maps. The final model contains 1660 protein atoms and 284 water molecules and has a crystallographic R-factor of 17.0% and an R-free value of 23.7%. The N and C termini were clearly visible in the structure, but residues 197–199 were slightly disordered and have been modeled on the basis of weak density. The extremities of side chains of 85, 94, 116, 170, and 200 have been modeled with an occupancy of 0 due to insufficient density. Detailed model statistics are given in Table II. To study the C3stau2·NAD complex, native crystals were soaked in crystallization reservoir solution supplemented with 25 mm NAD for 1 h and then briefly in the above cryoprotectant before cryo-cooling to 100 K. The crystals diffracted to 2.02 Å on station PX 14.2 at the Synchrotron Radiation Source, Daresbury, UK, and the data were collected using an ADSC-CCD detector. The data were processed and scaled as above. The native C3stau2 structure was used as an initial model that was improved by rigid body refinement of these data. Refinement and water-picking were performed using CNS. The water positions were checked to have heights above 3σ in the F o - F c map and to have temperature factors less than 50 Å2. NAD was added to the model at a position where continuous difference density was observed in the F o - F c map at 2σ level. Most of the model is well ordered except for the extremities of residues 30, 56, and 116, which have been modeled at 0 occupancy. Met-105 and Asn-124 were modeled in two conformations with both conformations at 0.5 occupancy. The final model had an R-factor of 21.4% and an R-free of 26.9%. The details of data processing and refinement are given in Table II. Both structures were checked with PROCHECK (33Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). All figures were created with MOLSCRIPT (34Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) rendered with POVRAY (www.povray.org). Overall Topology—The overall structure of C3stau2 is similar to that of C3bot1, i.e. a mixed α/β fold with a β-sandwich core (Fig. 2A) (22Han S. Arvai A.S. Clancy S.B. Tainer J.A. J. Mol. Biol. 2001; 305: 95-107Crossref PubMed Scopus (133) Google Scholar, 23Menetrey J. Flatau G. Stura E.A. Charbonnier J.B. Gas F. Teulon J.M. Le Du M.H. Boquet P. Menez A. J. Biol. Chem. 2002; 277: 30950-30957Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Structural alignment of C3stau2 with C3bot1 (Fig. 2B) shows that the main differences are restricted to the region of helices α5 and α6 (residues 92–126) in C3stau2, which corresponds to the loop between β1 and α5 in C3bot1 (residues 135–162) (Fig. 1). This region in C3stau2 includes seven extra residues that contribute to two extra helices comprising residues 99–108 (α5) and 115–125 (α6). This extended variable region is linked by main chain hydrogen bonding to the loop between strands β7 and β8 (residues 197–202), which also deviates from its counterpart in C3bot1 (r.m.s. deviation of Cα atoms is 1.6 Å). Here, the main chain oxygens of residues Asn-199 and Lys-200 are bonded to the main chain nitrogen of Tyr-114 and the side-chain nitrogen of Gln-133, respectively. NAD was added to the second model after initial refinement against the native C3stau structure showed clear difference density at 2σ level. The complexed form has the same topology as the native form of C3stau2 with a Cα r.m.s. deviation of 0.83 Å. Analysis of the Ramachandran (φ-Ψ) plot showed that all residues lie in the allowed regions for both structures. The ARTT Loop—The bacterial ADP-ribosylating toxins all share a conserved glutamate that is essential for ADP-ribosylation. In C3stau2, this residue (Glu-180) is located between strands β5 and β6 at the end of the second turn of what has been termed for C3bot1, the "ADP-ribosylating turn turn" (or ARTT) motif. The ARTT loop been shown to be essential for binding the NAD and positioning the C3·NAD·Rho complex for ADP-ribosyl transfer by mutational analysis (11Wilde C. Just I. Aktories K. Biochemistry. 2002; 41: 1539-1544Crossref PubMed Scopus (33) Google Scholar, 23Menetrey J. Flatau G. Stura E.A. Charbonnier J.B. Gas F. Teulon J.M. Le Du M.H. Boquet P. Menez A. J. Biol. Chem. 2002; 277: 30950-30957Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 25Tsuge H. Nagahama M. Nishimura H. Hisatsune J. Sakaguchi Y. Itogawa Y. Katunuma N. Sakurai J. J. Mol. Biol. 2003; 325: 471-483Crossref PubMed Scopus (89) Google Scholar) and was shown to change orientation in C3bot1 on NAD binding (23Menetrey J. Flatau G. Stura E.A. Charbonnier J.B. Gas F. Teulon J.M. Le Du M.H. Boquet P. Menez A. J. Biol. Chem. 2002; 277: 30950-30957Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In particular, three residues of the ARTT loop have been shown to be essential for C3stau2 ADP-ribosylation. As well as the catalytic glutamate on turn 2, there is a conserved glutamine (or glutamate) thought to be essential for positioning the ternary C3·NAD·Rho complex on turn 2 and a conserved tyrosine (or phenylalanine) on turn 1 necessary for Rho binding (11Wilde C. Just I. Aktories K. Biochemistry. 2002; 41: 1539-1544Crossref PubMed Scopus (33) Google Scholar). Despite the conserved role of the ARTT loop in ADP-ribosylation, the loop is orientated differently in C3stau2 and C3bot1. In the native structure of C3stau2, the backbone of the first turn of the ARTT loop is shifted by up to 5.1 Å compared with the backbone of the same turn in C3bot1 (Fig. 3), although this does not affect the conserved position of Glu-180. The three C3-like exoenzymes from S. aureus are unusual among the C3-like and binary toxins discovered so far in that the loops immediately before their ARTT loops contain an extra two residues. These two residues appear to be responsible for positioning the ARTT loop in a conformation identical not to that of the native C3bot1 structure but to that of the C3bot1·NAD complex, the NAD-bound conformation. The loop is well ordered with B-factors (mean = 10.2 Å2 over 8 atoms) below average (15.3 Å2) for the structure and appears optimally placed for ribosylation. It is not surprising then that upon binding of NAD, the ARTT loop undergoes just a slight conformational change (Fig. 3). The r.m.s. deviation for the ARTT loop between the native and the NAD-complexed structures over 8 Cα atoms is 0.1 Å. As expected, Glu-180 can be seen hydrogen bonding to the nicotinamide ribose nitrogen, NO2′ (Fig. 4), an interaction for which it does not need to move. In C3stau2, the position of Glu-180 is not stabilized by hydrogen bonding to the phosphate-nicotinamide (PN) loop as seen in C3bot1. However, Glu-180 does interact with Ser-138, the first serine in the STX motif (also known as the STS motif), as seen in C3bot1. The YX STX motif has been identified as a conserved motif across the ADP-ribosyltransferase toxins, and in the case of the C3 exoenzymes from S. aureus, this motif has the sequence YS STQ. This motif has generally been considered to help stabilize the NAD binding pocket, and mutational analysis of this motif in diphtheria toxin and cholera toxin has helped distinguish differences in the NAD hydrolysis mechanism between the two types of A-B toxins (36Dolan K.M. Lindenmayer G. Olson J.C. Biochemistry. 2000; 39: 8266-8275Crossref PubMed Scopus (15) Google Scholar). Also, mutation of the first serine of the STX motif has shown it to be essential for NAD hydrolysis and ADP-ribosylation in the Clostridium difficile Iota (binary) toxin (37Nagahama M. Sakaguchi Y. Kobayashi S. Ochi S. Sakurai J. J. Bacteriol. 2000; 182: 2096-2103Crossref PubMed Scopus (41) Google Scholar) and to be likely to help stabilize the transition state with the catalytic glutamate (38Sakurai J. Nagahama M. Hisatsune J. Katunuma N. Tsuge H. Adv. Enzyme Regul. 2003; 43: 361-377Crossref PubMed
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