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• W2150652405 abstract "Cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells, which are mediated by the DNA-damaging CdtB subunit. Here we report the first x-ray structure of an isolated CdtB subunit (Escherichia coli-II CdtB, EcCdtB). In conjunction with previous structural and biochemical observations, active site structural comparisons between free and holotoxin-assembled CdtBs suggested that CDT intoxication is contingent upon holotoxin disassembly. Solution NMR structural and 15N relaxation studies of free EcCdtB revealed disorder in the interface with the CdtA and CdtC subunits (residues Gly233–Asp242). Residues Leu186–Thr209 of EcCdtB, which encompasses tandem arginine residues essential for nuclear translocation and intoxication, were also disordered in solution. In stark contrast, nearly identical well defined α-helix and β-strand secondary structures were observed in this region of the free and holotoxin CdtB crystallographic models, suggesting that distinct changes in structural ordering characterize subunit disassembly and nuclear localization factor binding functions. Cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells, which are mediated by the DNA-damaging CdtB subunit. Here we report the first x-ray structure of an isolated CdtB subunit (Escherichia coli-II CdtB, EcCdtB). In conjunction with previous structural and biochemical observations, active site structural comparisons between free and holotoxin-assembled CdtBs suggested that CDT intoxication is contingent upon holotoxin disassembly. Solution NMR structural and 15N relaxation studies of free EcCdtB revealed disorder in the interface with the CdtA and CdtC subunits (residues Gly233–Asp242). Residues Leu186–Thr209 of EcCdtB, which encompasses tandem arginine residues essential for nuclear translocation and intoxication, were also disordered in solution. In stark contrast, nearly identical well defined α-helix and β-strand secondary structures were observed in this region of the free and holotoxin CdtB crystallographic models, suggesting that distinct changes in structural ordering characterize subunit disassembly and nuclear localization factor binding functions. Cytolethal distending toxin (CDT) 4The abbreviations used are: CDT, cytolethal distending toxin; EcCdtB, B subunit of CDT from E. coli; HdCDT, H. ducreyi holotoxin structure; AaCDT, A. actinomycetemcomitans holotoxin structure; NLS, nuclear localization sequence; hHdCdtB, B subunit within holotoxin crystal structure of CDT from H. ducreyi; hAaCdtB, B subunit within holotoxin crystal structure of CDT from A. actinomycetemcomitans; r.m.s.d., root mean square deviation; NOE, heteronuclear nuclear Overhauser effect; R1, longitudinal relaxation time constant; R2, transverse relaxation time constant. is a DNA-damaging bacterial toxin produced by a number of important bacterial pathogens (1Pickett C.L. Whitehouse C.A. Trends Microbiol. 1999; 7: 292-297Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 2Dreyfus L.A. Burns D. Bacterial Protein Toxins. ASM Press, Washington, D. C.2003: 257-270Google Scholar). The cytopathic effects associated with CDT intoxication are a direct result of chromosomal DNA damage inflicted by the CdtB subunit, a homolog of eukaryotic type I DNase (3Lara-Tejero M. Galan J.E. Science. 2000; 290: 354-357Crossref PubMed Scopus (422) Google Scholar, 4Elwell C.A. Dreyfus L.A. Mol. Microbiol. 2000; 37: 952-963Crossref PubMed Scopus (235) Google Scholar). CdtB-mediated chromosome strand breakage signals the induction of an ATM (ataxia telangiectasia-mutated)- or ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent mitotic checkpoint resulting in the sequestration of Cdc25, a block in the cell cycle at the G2/M boundary, cellular distension, and ultimately cell death by endoreduplication or apoptosis (2Dreyfus L.A. Burns D. Bacterial Protein Toxins. ASM Press, Washington, D. C.2003: 257-270Google Scholar, 5de Rycke J. Oswald E. FEMS Microbiol. Lett. 2001; 203: 141-148Crossref PubMed Google Scholar). The CDT holotoxin is a heterotrimer composed of CdtA, CdtB, and CdtC subunits. Although the catalyst for CDT-mediated apoptosis is ultimately attributed to the DNase activity of CdtB, all three subunits are required for cellular entry of CDT (6Lara-Tejero M. Galan J.E. Infect. Immun. 2001; 69: 4358-4365Crossref PubMed Scopus (262) Google Scholar, 7Elwell C. Chao K.L. Patel K. Dreyfus L. Infect. Immun. 2001; 69: 3418-3422Crossref PubMed Scopus (114) Google Scholar). The CdtA and CdtC subunits are lectin-like domains that bind carbohydrate residues on the target cell surface, whereby CDT is subsequently internalized by receptor-mediated endocytosis (8Cortes-Bratti X. Chaves-Olarte E. Lagergard T. Thelestam M. Infect. Immun. 2000; 68: 6903-6911Crossref PubMed Scopus (82) Google Scholar, 9Guerra L. Teter K. Lilley B.N. Stenerlow B. Holmes R.K. Ploegh H.L. Sandvig K. Thelestam M. Frisan T. Cell. Microbiol. 2005; 7: 921-934Crossref PubMed Scopus (90) Google Scholar). The Haemophilus ducreyi holotoxin structure (HdCDT) has demonstrated that CDT is an AB type toxin, with the catalytically active A subunit of the AB toxin corresponding to the CdtB subunit (10Nesic D. Hsu Y. Stebbins C.E. Nature. 2004; 429: 429-433Crossref PubMed Scopus (239) Google Scholar). Inspection of this structure and of the structurally similar CDT holotoxin from Actinobacillus actinomycetemcomitans (AaCDT) (11Yamada T. Komoto J. Saiki K. Konishi K. Takusagawa F. Protein Sci. 2006; 15: 362-372Crossref PubMed Scopus (40) Google Scholar) revealed that one face of the CdtA-CdtC dimer binds the CdtB subunit. Mutagenesis studies of HdCDT suggest that another face of the dimer with a grooved ricin B-chain fold mediates cell surface binding (10Nesic D. Hsu Y. Stebbins C.E. Nature. 2004; 429: 429-433Crossref PubMed Scopus (239) Google Scholar). Trafficking of CDT to the nucleus is thought to involve retrograde transport through the Golgi and the ultimate translocation of CdtB from the endoplasmic reticulum to the nuclear envelope by an apparent ERAD (endoplasmic reticulum-associated degradation)-independent step (9Guerra L. Teter K. Lilley B.N. Stenerlow B. Holmes R.K. Ploegh H.L. Sandvig K. Thelestam M. Frisan T. Cell. Microbiol. 2005; 7: 921-934Crossref PubMed Scopus (90) Google Scholar). CdtB traverses the nuclear envelope by virtue of at least one nuclear localization sequence (NLS), which is recognized by unknown cognate nuclear localization factors (12McSweeney L.A. Dreyfus L.A. Cell. Microbiol. 2004; 6: 447-458Crossref PubMed Scopus (79) Google Scholar). Although CdtB disassembly from the CDT holotoxin has not been established, direct deposition of the isolated EcCdtB subunit into the cytoplasm of HeLa cells by electroporation promotes cell cycle arrest at G2/M, demonstrating that free EcCdtB locates to the nucleus and retains toxicity (7Elwell C. Chao K.L. Patel K. Dreyfus L. Infect. Immun. 2001; 69: 3418-3422Crossref PubMed Scopus (114) Google Scholar). Significantly, toxicity determined by this avenue is dependent on the integrity of two EcCdtB arginine residues (Arg191-Arg192) within the characterized NLS (12McSweeney L.A. Dreyfus L.A. Cell. Microbiol. 2004; 6: 447-458Crossref PubMed Scopus (79) Google Scholar). Here, we report the first high-resolution structure of a free CdtB subunit. The nearly identical active site conformations in the free and holotoxin states of CdtB suggest that the much lower enzymatic activity of this subunit within assembled CDT is mediated by interactions with CdtC rather than intramolecular structural rearrangement within CdtB upon holotoxin disassembly (10Nesic D. Hsu Y. Stebbins C.E. Nature. 2004; 429: 429-433Crossref PubMed Scopus (239) Google Scholar). In addition, NMR 15N relaxation studies identify two regions of dynamic disorder within the ensemble of solution conformers of free EcCdtB, suggesting that distinct structural transitions involving ordered and disordered states underlie CdtB functional activation (holotoxin disassembly) and binding to cognate nuclear localization factors. Crystallization and X-ray Data Collection—The cdtB gene used for these studies was derived from Escherichia coli strain 9142–88 (O128:H–) (13Pickett C.L. Cottle D.L. Pesci E.C. Bikah G. Infect. Immun. 1994; 62: 1046-1051Crossref PubMed Google Scholar) and cloned as described previously (14Aragon V. Chao K. Dreyfus L.A. Infect. Immun. 1997; 65: 3774-3780Crossref PubMed Google Scholar). Three distinct CDT operons have been characterized from various E. coli strains and are designated CDT-I, CDT-II, and CDT-III (2Dreyfus L.A. Burns D. Bacterial Protein Toxins. ASM Press, Washington, D. C.2003: 257-270Google Scholar). E. coli 9142-88 produces CDT-II, and thus the CdtB used in this work is designated EcCdtB-II, referred to herein as EcCdtB. Recombinant EcCdtB protein corresponding to the mature subunit (residues 1–251) plus an 11-residue N-terminal linker including six histidine residues was crystallized by microbatch techniques as described elsewhere (15Hontz J.H. Villar-Lecumberri M.T. Dreyfus L.A. Yoder M.D. Acta Crystallogr. 2006; F62: 192-195Google Scholar). The EcCdtB toxin preparation possessed identical toxicity and enzymatic activity as reported previously (7Elwell C. Chao K.L. Patel K. Dreyfus L. Infect. Immun. 2001; 69: 3418-3422Crossref PubMed Scopus (114) Google Scholar). X-ray data collected at Advanced Photon Source 22BM on a Mar300 charge-coupled device detector to 1.73 Å were processed by HKL2000 (16Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar), and phases were determined by molecular replacement as implemented in AMoRe (17Navazza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar) using the H. ducreyi CdtB subunit from the holotoxin structure (hHdCdtB) as the search model (Protein Data Bank code 1SR4) (10Nesic D. Hsu Y. Stebbins C.E. Nature. 2004; 429: 429-433Crossref PubMed Scopus (239) Google Scholar). The hHdCdtB phasing model included residues 28–81, 93–134, 144–163, 194–211, 225–233, 237–241, and 267–281. The remaining residues were added by iterative cycles of manual model building in O (18Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and model refinement by simulated annealing and conjugate gradient minimization as implemented in CNS (Crystallography and NMR System) (19Brunger 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. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Solvent molecules were added using a combination of default parameters in CNS and ARP/wARP (20Lamzin V.S. Perrakis A. Wilson K.S. Rossman M.G. Arnold E. Crystallography of Biological Macromolecules. Kluwer Academic Publishers, Dordrecht, The Netherlands2001: 720-722Google Scholar). Final refinement was performed using maximum likelihood with restrained refinement by Refmac5 (21Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) in the CCP4 suite (22Collaborative Computer Project Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). Secondary structure assignments were determined by the DSSP program (23Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12421) Google Scholar). Root mean square deviations (r.m.s.d.) between CdtB structures were calculated using SwissPdbViewer, version 3.7 (24Guex N. Peitsch M.C. Protein Data Bank Quarterly Newsletter. 2006; 77: 7Google Scholar), and using Iterative Magic Fit to align the structures. After alignment, regions were manually defined for the r.m.s.d. calculation. NMR Spectroscopy—All NMR data were recorded on a 14.1 T Varian Inova spectrometer (599.7 MHz for 1H). The recombinant EcCdtB used for all NMR studies was identical to that for crystallographic studies. The details of EcCdtB NMR sample preparation, resonance assignment determination, extent of assignments (25Villar-Lecumberri M.T. Potter B.M. Wang Z. Dreyfus L. Laity J.H. J. Biomol. NMR 2006. 2006; 34Google Scholar), and random coil chemical shifts (26Wishart D.S. Sykes B.D. Richards F.M. J. Mol. Biol. 1991; 222: 311-333Crossref PubMed Scopus (1791) Google Scholar, 27Braun D. Wider G. Wuthrich K. J. Am. Chem. Soc. 1994; 116: 8466-8469Crossref Scopus (147) Google Scholar) have been reported. The general approach used for 15N relaxation data collection is that of Potter et al. (28Potter B.M. Feng L.S. Parasuram P. Matskevich V.A. Wilson J.A. Andrews G.K. Laity J.H. J. Biol. Chem. 2005; 280: 28529-28540Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). EcCdtB steady-state heteronuclear NOEs (15N-{1H}NOE), 15N longitudinal (R1) and transverse (R2) relaxation time constants were measured by the steady-state, inversion-recovery, and Carr-Purcell-Meiboom-Gill methods, respectively (29Palmer III, A.G. Cavanagh J. Wright P.E. Rance M. J. Magn. Reson. 1991; 93: 151-170Google Scholar, 30Stone M.J. Fairbrother W.J. Palmer III, A.G. Reizer J. Saier Jr., M.H. Wright P.E. Biochemistry. 1992; 31: 4394-4406Crossref PubMed Scopus (234) Google Scholar, 31Farrow N.A. Zhang O. Forman-Kay J.D. Kay L.E. Biochemistry. 1995; 34: 868-878Crossref PubMed Scopus (288) Google Scholar) using standard water flip-back methods (32Grzesiek S. Bax A. J. Am. Chem. Soc. 1993; 115: 12594Google Scholar). R1 and R2 spectra relaxation delays were as follows: 0.03, 0.05, 0.07, 0.09, 0.12, 0.15, 0.20, 0.35, 0.50, 0.65, 0.70, 0.90, 1.20, 1.60, 2.00, 2.35, 2.75 and 3.50 s for R1; 0.01, 0.03, 0.05, 0.07, 0.09, 0.11, 0.13, 0.15, 0.17, 0.21 and 0.25 s for R2. Multiple R1 and R2 spectra were used to estimate the precision of the peak intensities. Five pairs of 15N-{1H}NOE spectra were collected with and without 1H saturation to allow for estimation of experimental uncertainty. A 7-s recovery delay was used for all 15N relaxation experiments. R1 and R2 time constants were calculated with CurveFit1.30. 5A. G. Palmer III, Columbia University, New York. Hydrodynamic and model-free analysis (33Mandel A.M. Akke M. Palmer III, A.G. J. Mol. Biol. 1995; 246: 144-163Crossref PubMed Scopus (906) Google Scholar, 34Lipari G. Szabo A. J. Am. Chem. Soc. 1984; 104: 4546-4549Crossref Scopus (3409) Google Scholar, 35Lipari G. Szabo A. J. Am. Chem. Soc. 1984; 104: 4559-4570Crossref Scopus (1877) Google Scholar) was carried out with TENSOR2 (36Dosset P. Hus J.C. Blackledge M. Marion D. J. Biomol. NMR. 2000; 16: 23-28Crossref PubMed Scopus (446) Google Scholar) as described previously (28Potter B.M. Feng L.S. Parasuram P. Matskevich V.A. Wilson J.A. Andrews G.K. Laity J.H. J. Biol. Chem. 2005; 280: 28529-28540Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Crystal Structure of Free EcCdtB—The structure of EcCdtB was determined to a resolution of 1.73 Å using x-ray diffraction analysis. Phasing information was obtained by molecular replacement using core regions of the CdtB subunit from the H. ducreyi holotoxin (10Nesic D. Hsu Y. Stebbins C.E. Nature. 2004; 429: 429-433Crossref PubMed Scopus (239) Google Scholar). A summary of x-ray data and refinement statistics for the EcCdtB structure is presented in Table 1. The final EcCdtB model consists of the entire mature subunit, residues 1–251 plus two residues of the 11-residue His tag linker, and 160 water molecules. A sample electron density map is shown in Fig. 1. Coordinates and structure factors have been deposited in the Protein Data Bank with accession code 2F1N.TABLE 1Data collection and refinement statistics for E. coli cdtBData collectionSpace groupP212121Unit cell (Å)a = 39.88, b = 47.54, c = 114.89Molecules per asymmetric unit1Estimated solvent content (%)34.1Total reflections collected102,947Unique reflections23,348Completeness (%)aValues indicated in parentheses are for the highest resolution bin.99.1 (98.8)I/σI16.8 (4.7)Average redundancy4.5 (3.5)Mosaicity (°)0.42Rsym (%)bRsym = ∑|I(hkl) — 〈I(hkl) 〉|/∑Ihkl where 〈I(hkl) 〉 is average intensity over symmetry equivalents.6.6 (21.2)Refinement statisticsResolution (Å)57.7-1.73 (1.78-1.73)Rfactor (%)cRfactor = ∑|Fobs(hkl) — Fcalc(hkl)|/∑hklFobs(hkl).18.4 (22.7)Rfree (%)dRfree was calculated using randomly selected 5.1% of reflections.21.7 (29.1)No. of protein atomseNonhydrogen protein atoms only.2098No. of water molecules160r.m.s.d. from ideal geometryBond lengths (Å)0.02Bond angles (°)1.75Average B-factor (Å2)20.2Main chain B-factor18.4Side chains B-factor22.3Water molecules B-factor29.4a Values indicated in parentheses are for the highest resolution bin.b Rsym = ∑|I(hkl) — 〈I(hkl) 〉|/∑Ihkl where 〈I(hkl) 〉 is average intensity over symmetry equivalents.c Rfactor = ∑|Fobs(hkl) — Fcalc(hkl)|/∑hklFobs(hkl).d Rfree was calculated using randomly selected 5.1% of reflections.e Nonhydrogen protein atoms only. Open table in a new tab The EcCdtB structure belongs to the α + β structural class and consists of a β-sandwich with α-helices on the exterior (Fig. 2A). Although EcCdtB has low sequence identity with the DNase-I family of enzymes including bovine DNase I (19.4% identity), human HapI DNA repair endonuclease (17.3% identity), and E. coli EXO III (18.6% identity), the protein adopts a very similar fold. Differences between the DNase I and CdtB family folds include the number of strands in the β-sheets and the number of connecting helices. As shown in Fig. 2B, the CdtB fold is composed of 8- and 6-stranded β-sheets (10Nesic D. Hsu Y. Stebbins C.E. Nature. 2004; 429: 429-433Crossref PubMed Scopus (239) Google Scholar, 11Yamada T. Komoto J. Saiki K. Konishi K. Takusagawa F. Protein Sci. 2006; 15: 362-372Crossref PubMed Scopus (40) Google Scholar), whereas the canonical DNase I fold has two 6-stranded sheets (37Suck D. Lahm A. Oefner C. Nature. 1988; 332: 464-468Crossref PubMed Scopus (242) Google Scholar). In the CdtB fold, two additional antiparallel β-strands (3 and 4) occur at the C-terminal end of β-sheet 1. The α-helix connecting β-strands 2 and 3 in the DNase I family fold is missing in the corresponding region connecting β-strands 2 and 3 in the CdtB family fold. Active Site of EcCdtB—The active site of EcCdtB contains two histidine residues (His136 and His243) that are essential for catalysis of DNA phosphodiester cleavage (Fig. 3) (3Lara-Tejero M. Galan J.E. Science. 2000; 290: 354-357Crossref PubMed Scopus (422) Google Scholar, 4Elwell C.A. Dreyfus L.A. Mol. Microbiol. 2000; 37: 952-963Crossref PubMed Scopus (235) Google Scholar). Moreover, based on structural similarities to DNase I, catalytic enhancement attributed to a raising of the His243 pKa in EcCdtB is likely to be contributed by Asp211 through side chain carboxylate-imidazole hydrogen bonding (37Suck D. Lahm A. Oefner C. Nature. 1988; 332: 464-468Crossref PubMed Scopus (242) Google Scholar, 38Weston S.A. Lahm A. Suck D. J. Mol. Biol. 1992; 226: 1237-1256Crossref PubMed Scopus (250) Google Scholar, 39Jones S.J. Worrall A.F. Connolly B.A. J. Mol. Biol. 1996; 264: 1154-1163Crossref PubMed Scopus (72) Google Scholar). The solvent structure surrounding these three conserved amino acids in EcCdtB and hHdCdtB shows striking structural conservation. The lower resolution of the A. actinomycetemcomitans CdtB subunit from the holotoxin structure (hAaCdtB) makes the comparison of solvent structure more difficult and is not included in Fig. 3. Three additional residues that make direct DNA substrate contacts in DNase I are located in structurally conserved positions in EcCdtB (Arg95, Arg120, and Asn176). All of these important catalytic and substrate binding residues, which are absolutely conserved in all known CDTs, have remarkably similar side chain positions in the assembled holotoxin (10Nesic D. Hsu Y. Stebbins C.E. Nature. 2004; 429: 429-433Crossref PubMed Scopus (239) Google Scholar, 11Yamada T. Komoto J. Saiki K. Konishi K. Takusagawa F. Protein Sci. 2006; 15: 362-372Crossref PubMed Scopus (40) Google Scholar) and free EcCdtB structures (Fig. 3). Comparison of Free EcCdtB with CdtB in Holotoxins—The 1.02-Å r.m.s.d. measured for 216 Cα atoms from aligned, non-gapped residues of free EcCdtB and the homologous hHdCdtB subunit (48% identity) crystal structures demonstrates that conformational rearrangements accompanying holotoxin assembly are not global (Fig. 4). If only residues within regular secondary structure elements are considered (111 residues), the r.m.s.d. between Cα atoms from the two CdtB proteins drops to 0.70 Å. Given the 97% CdtB sequence identity between H. ducreyi and A. actinomycetemcomitans CdtB subunits, it is not surprising that over the entire structure, the Cα r.m.s.d. from these holotoxin-associated proteins is somewhat lower (0.43 Å, see Fig. 4). Indeed, the backbone topology of all three subunits from these two CDT holotoxins is nearly identical (10Nesic D. Hsu Y. Stebbins C.E. Nature. 2004; 429: 429-433Crossref PubMed Scopus (239) Google Scholar, 11Yamada T. Komoto J. Saiki K. Konishi K. Takusagawa F. Protein Sci. 2006; 15: 362-372Crossref PubMed Scopus (40) Google Scholar). Although the structures of CdtB in the free and assembled forms are very similar, the CdtB crystal structures show significant protein backbone variation between free (EcCdtB) and holotoxin (hHdCdtB and hAaCdtB) subunits in the loop region joining the two C-terminal β-sheet 2 strands β13 and β14 (residues Ile230–Pro245 of EcCdtB, Fig. 4). Close inspection of the free EcCdtB structural alignments with hHdCdtB and hAaCdtB revealed that most of this region of CdtB is in the subunit interface with CdtA and CdtC (supplemental Fig. 1). This interface is occupied largely by residues Leu263–His274 in hHdCdtB and hAaCdtB (corresponding to EcCdtB residues Tyr232–His243) and contains a three-residue α-helix (residues Leu263–Gln265) in both proteins that is not formed by the corresponding residues of the free EcCdtB subunit (Tyr232–Ala234). Although the r.m.s.d. between Cα atoms of hHdCdtB and hAaCdtb in this region are only 0.30 Å, the corresponding Cα r.m.s.d. between EcCdtB and hHdCdtB is 5.64 Å in the same region. NLS Regions—A tandem arginine sequence located in helix E of EcCdtB (Arg191-Arg192, Fig. 5), which is part of a larger monopartite or bipartite NLS (40Dingwall C. Laskey R.A. Trends Biochem. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1713) Google Scholar), is essential for nuclear translocation and CDT-mediated intoxication (12McSweeney L.A. Dreyfus L.A. Cell. Microbiol. 2004; 6: 447-458Crossref PubMed Scopus (79) Google Scholar). Although a corresponding tandem arginine sequence within an NLS for HdC-dtB and AaCdtB has not been characterized experimentally, the expanded structural alignment in the vicinity of the EcCdtB NLS region with the holotoxin subunits shown in Fig. 5 reveals that the hHdCdtB and hAaCdtB Arg249-Arg250 side chain positions face out into solution in close proximity to the EcCdtB Arg191-Arg192 side chains, even though the locations of these arginine pairs in the backbone topology of the respective CdtB orthologs are quite different. Solution NMR Structural and 15N Relaxation Studies of Free EcCdtB—To identify potential ensemble variations in structure and backbone motions that could be related to CdtB-dependent CDT biological function, NMR solution studies of EcCdtB were carried out with an identical EcCdtB protein to that used for crystallographic studies (15Hontz J.H. Villar-Lecumberri M.T. Dreyfus L.A. Yoder M.D. Acta Crystallogr. 2006; F62: 192-195Google Scholar, 25Villar-Lecumberri M.T. Potter B.M. Wang Z. Dreyfus L. Laity J.H. J. Biomol. NMR 2006. 2006; 34Google Scholar). Although the quality of the NMR spectra were generally good for the uniformly 2H-, 15N-, and 13C-labeled EcCdtB protein, 29 residues concentrated primarily within the first 100 N-terminal residues of free EcCdtB were characterized by μs-ms time scale exchange broadening to the extent that the corresponding 1H-15N-HSQC resonances were unobservable (25Villar-Lecumberri M.T. Potter B.M. Wang Z. Dreyfus L. Laity J.H. J. Biomol. NMR 2006. 2006; 34Google Scholar). The comparison of backbone EcCdtB residue 1HN, 15N, 13Cα, and 13CO NMR chemical shift differences with those of the corresponding residues in small “random coil” peptides shown in Fig. 6A is a good indicator of protein secondary structure (26Wishart D.S. Sykes B.D. Richards F.M. J. Mol. Biol. 1991; 222: 311-333Crossref PubMed Scopus (1791) Google Scholar, 27Braun D. Wider G. Wuthrich K. J. Am. Chem. Soc. 1994; 116: 8466-8469Crossref Scopus (147) Google Scholar). For the majority of the EcCdtB protein, the secondary structure predictions match those observed in the crystallographic model of the protein (Fig. 2). However, the 13Cα and 13CO chemical shift differences from random coil in the regions of EcCdtB encompassing helix A (E20-I31), β-strand 2 (Ile39–Gln43), and β-strand 5 (Gln81–Ser87) are not completely consistent with the observed secondary structural elements in the crystal structure, although these regions have large gaps in chemical shift information because of conformational exchange (Fig. 6A) (25Villar-Lecumberri M.T. Potter B.M. Wang Z. Dreyfus L. Laity J.H. J. Biomol. NMR 2006. 2006; 34Google Scholar). More significantly, the region of EcCdtB encompassing residues Leu186–Thr209 (Fig. 6A, blue bars) has backbone 1HN, 15N, 13Cα, and 13CO NMR chemical shifts remarkably similar to those of random coil. This disorder (blue region in Fig. 6C) is somewhat unexpected given the elements of regular secondary structure (helix E and β-strand 11) observed in the same region of the EcCdtB crystal structure (Fig. 2A). Similar trends were observed for residues Glu233–Asp242 of EcCdtB (red regions in Fig. 6, A and C), although, in general, the chemical differences from random coil in this region are larger than those in the other disordered region (residues Leu186–Thr209). Complementary 15N relaxation NMR spectra were recorded and analyzed for the free EcCdtB protein in solution (Fig. 6B). The majority of EcCdtB residues are characterized by fast internal ns-ps (15N-{1H}NOE) and slower “tumbling” ns (R2/R1) solution dynamic properties consistent with a 29-kDa protein. This 15N relaxation data fit well to an isotropic hydrodynamic model for EcCdtB, which produced a rotational correlation time of 13.6 ns using those regions of defined solution secondary structure predicted from the NMR chemical shift data shown in Fig. 6A. However, the disordered regions of EcCdtB evident from chemical shift comparisons (Leu186–Thr209 and Gly233–Asp242) were likewise characterized by fast internal and tumbling dynamic properties characteristic of disordered polypeptides (41Palmer III, A.G. Chem. Rev. 2004; 104: 3623-3640Crossref PubMed Scopus (706) Google Scholar). Residues Ser74–Gln80 of EcCdtB, which connect β-strands 3 and 4 (green regions in Fig. 6, A–C) also had 15N relaxation properties consistent with a flexible loop. Model-free analysis (34Lipari G. Szabo A. J. Am. Chem. Soc. 1984; 104: 4546-4549Crossref Scopus (3409) Google Scholar, 35Lipari G. Szabo A. J. Am. Chem. Soc. 1984; 104: 4559-4570Crossref Scopus (1877) Google Scholar) of fast internal motions within well structured regions of CdtB reveal consistent small amplitude (mostly <100 ps) motions, in contrast to larger amplitude motions in the three regions described above characterized by higher ns-ps mobility (data not shown). We note that sufficient electron density for model determination was evident in all three regions with solution disorder (Fig. 1 and data not shown). Backbone ϕ and ψ dihedral angles from the solution structure were calculated for those residues of EcCdtB in ordered regions for which a reliable angle could be obtained using the TALOS method (42Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2740) Google Scholar). A comparison of 100 such TALOS-derived ϕ and ψ dihedral angles with those extracted from the crystal structure shown in supplemental Fig. 2 suggests an EcCdtB ensemble average solution conformation very similar to that in the crystal state for the ordered regions of the protein. Analysis of Crystal Contacts and Correlation to Regions of Solution Disorder—Given the NMR results indicating solution disorder in the three regions of EcCdtB, crystal contacts were analyzed as a possible explanation for the structural order in the same regions of the corresponding crystallographic model. The EcCdtB crystals used for x-ray diffraction analysis have a relatively low solvent content of 34% (Table 1). Of the 253 residues in the final model, 110 (43%) make crystal contacts. Regions showing symmetry contacts are spread throughout the protein (Fig. 6D). The disordered regions at Ser74–Gln80 and Gly233–Asp242 in the solution state of the protein contain significant crystal contacts. In the Gly233–Asp242 region, all residues make crystal contacts with symmetry related molecules, with five of the 10 residues forming hydrogen bonds with symmetry-related molecules. In contrast, in the longest disordered region (Leu186–Thr209, blue in Fig. 6, A–C), crystal contacts are localized around the five-residue helix E. Although it has been established that CdtB is the catalytically active subunit of the CDT toxin and that CdtA and CdtC are necessary for cell surface binding and cellular entry (1Pickett C.L. Whitehouse C.A. Trends Microbiol. 1999; 7: 292-297Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 2D" @default.
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