Structural and mechanistic basis of immunity toward endonuclease colicins.
Colin KleanthousAndrew M. HemmingsUlrike C. KühlmannAnsgar J. PommerNeil FergusonSheena E. RadfordGeoffrey R. MooreRichard James
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Homing endonucleases have great potential as tools for targeted gene therapy and gene correction, but identifying variants of these enzymes capable of cleaving specific DNA targets of interest is necessary before the widespread use of such technologies is possible. We identified homologues of the LAGLIDADG homing endonuclease I-AniI and their putative target insertion sites by BLAST searches followed by examination of the sequences of the flanking genomic regions. Amino acid substitutions in these homologues that were located close to the target site DNA, and thus potentially conferring differences in target specificity, were grafted onto the I-AniI scaffold. Many of these grafts exhibited novel and unexpected specificities. These findings show that the information present in genomic data can be exploited for endonuclease specificity redesign. Homing endonucleases have great potential as tools for targeted gene therapy and gene correction, but identifying variants of these enzymes capable of cleaving specific DNA targets of interest is necessary before the widespread use of such technologies is possible. We identified homologues of the LAGLIDADG homing endonuclease I-AniI and their putative target insertion sites by BLAST searches followed by examination of the sequences of the flanking genomic regions. Amino acid substitutions in these homologues that were located close to the target site DNA, and thus potentially conferring differences in target specificity, were grafted onto the I-AniI scaffold. Many of these grafts exhibited novel and unexpected specificities. These findings show that the information present in genomic data can be exploited for endonuclease specificity redesign.
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Reconstitution of Colicin E 2 -Induced Deoxyribonucleic Acid Degradation in Spheroplast Preparations
Spheroplasts are insensitive to colicin E 2 and do not show deoxyribonucleic acid (DNA) degradation even in the presence of massive amounts of E 2 . However, when both endonuclease I and E 2 were present, spheroplast DNA was degraded by an endonucleolytic activity which gave rise primarily to double-strand DNA cleavages, producing fragments having an average molecular weight of 9 × 10 6 . Pancreatic ribonuclease could substitute for colicin E 2 in the reconstitution system, but pancreatic deoxyribonuclease could not replace endonuclease I. However, colicin E 2 could not activate transfer ribonucleic acid-inhibited endonuclease I in an in vitro system where pancreatic ribonuclease caused full stimulation.
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We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers (Mosbahi, K., Lemaître, C., Keeble, A. H., Mobasheri, H., Morel, B., James, R., Moore, G. R., Lea, E. J., and Kleanthous, C. (2002) Nat. Struct. Biol. 9, 476-484). It was proposed that the E9 DNase mediates its own translocation across the cytoplasmic membrane and that the formation of ion channels is essential to this process. Here we describe changes to the structure and stability of the E9 DNase that accompany interaction of the protein with phospholipid vesicles. Formation of the protein-lipid complex at pH 7.5 resulted in a red-shift of the intrinsic protein fluorescence emission maximum (λmax) from 333 to 346 nm. At pH 4.0, where the E9 DNase lacks tertiary structure but retains secondary structure, DOPG induced a blue-shift in λmax, from 354 to 342 nm. Changes in λmax were specific for anionic phospholipid vesicles at both pHs, suggesting electrostatics play a role in this association. The effects of phospholipid were negated by Im9 binding, the high affinity, acidic, exosite inhibitor protein, but not by zinc, which binds at the active site. Fluorescence-quenching experiments further demonstrated that similar protein-phospholipid complexes are formed regardless of whether the E9 DNase is initially in its native conformation. Consistent with these observations, chemical and thermal denaturation data as well as proteolytic susceptibility experiments showed that association with negatively charged phospholipids destabilize the E9 DNase. We suggest that formation of a destabilizing protein-lipid complex pre-empts channel formation by the E9 DNase and constitutes the initial step in its translocation across the Escherichia coli inner membrane. We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers (Mosbahi, K., Lemaître, C., Keeble, A. H., Mobasheri, H., Morel, B., James, R., Moore, G. R., Lea, E. J., and Kleanthous, C. (2002) Nat. Struct. Biol. 9, 476-484). It was proposed that the E9 DNase mediates its own translocation across the cytoplasmic membrane and that the formation of ion channels is essential to this process. Here we describe changes to the structure and stability of the E9 DNase that accompany interaction of the protein with phospholipid vesicles. Formation of the protein-lipid complex at pH 7.5 resulted in a red-shift of the intrinsic protein fluorescence emission maximum (λmax) from 333 to 346 nm. At pH 4.0, where the E9 DNase lacks tertiary structure but retains secondary structure, DOPG induced a blue-shift in λmax, from 354 to 342 nm. Changes in λmax were specific for anionic phospholipid vesicles at both pHs, suggesting electrostatics play a role in this association. The effects of phospholipid were negated by Im9 binding, the high affinity, acidic, exosite inhibitor protein, but not by zinc, which binds at the active site. Fluorescence-quenching experiments further demonstrated that similar protein-phospholipid complexes are formed regardless of whether the E9 DNase is initially in its native conformation. Consistent with these observations, chemical and thermal denaturation data as well as proteolytic susceptibility experiments showed that association with negatively charged phospholipids destabilize the E9 DNase. We suggest that formation of a destabilizing protein-lipid complex pre-empts channel formation by the E9 DNase and constitutes the initial step in its translocation across the Escherichia coli inner membrane. Unraveling the interactions and mechanisms that enable proteins to cross biological membranes is of considerable interest, as the ability to target specific exogenous enzymes to the cytosol is likely to facilitate the design and discovery of novel chemotherapeutic agents. Many protein toxins have evolved to deliver a cytotoxic domain or subunit to the cytoplasm of susceptible cells, and so they provide an invaluable tool for studying protein translocation from the extracellular environment to their cellular targets, often located in the cytoplasm (1Falnes P.Ø. Sandvig K. Curr. Opin. Cell Biol. 2000; 12: 407-413Crossref PubMed Scopus (247) Google Scholar). The transition from the water-soluble to membrane-bound state has perhaps been most intensely studied in the pore-forming colicins (2Stroud R.M. Reiling K. Wiener M. Freymann D. Curr. Opin. Struct. Biol. 1998; 8: 525-533Crossref PubMed Scopus (64) Google Scholar, 3Zakharov S.D. Cramer W.A. Biochim. Biophys. Acta. 2002; 1565: 333-346Crossref PubMed Scopus (81) Google Scholar). This family of bacterial toxins, like all colicins, share a common three-domain structure, with receptor-binding and translocation domains that facilitate binding to the cell surface and mediate delivery of the channel-forming cytotoxic domain to the inner membrane. Cell death occurs as a consequence of ion channel formation across the cytoplasmic membrane, inducing depolarization of the membrane. Association of the cytotoxic domain with the membrane is thought to lead to destabilization and unfolding of the protein, yielding a “molten globule-like” state of loosely interacting helices (4van der Goot F.G. González-Mañas J.M. Lakey J.H. Pattus F. Nature. 1991; 354: 408-410Crossref PubMed Scopus (424) Google Scholar, 5Muga A. González-Mañas J.M. Lakey J.H. Pattus F. Surewicz W.K. J. Biol. Chem. 1993; 268: 1553-1557Abstract Full Text PDF PubMed Google Scholar), from which a hydrophobic helical hairpin is able to spontaneously insert into the membrane (6Slatin S.L. Qiu X.-Q. Jakes K.S. Finkelstein A. Nature. 1994; 371: 158-161Crossref PubMed Scopus (151) Google Scholar, 7Jakes K.S. Kienker P.K. Finkelstein A. Q. Rev. Biophys. 1999; 32: 189-205Crossref PubMed Scopus (34) Google Scholar). Electrostatic interactions are known to play an important role in mediating this interaction, particularly in the initial formation of the colicin-lipid complex (5Muga A. González-Mañas J.M. Lakey J.H. Pattus F. Surewicz W.K. J. Biol. Chem. 1993; 268: 1553-1557Abstract Full Text PDF PubMed Google Scholar, 8Zakharov S.D. Heymann J.B. Zhang Y.L. Cramer W.A. Biophys. J. 1996; 70: 2774-2783Abstract Full Text PDF PubMed Scopus (38) Google Scholar). The major phospholipid constituents of the Escherichia coli cytoplasmic membrane are phosphatidylethanolamine, phosphatidylglycerol (PG), 1The abbreviations used are: PG, phosphatidylglycerol; DOPG, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; λmax, intrinsic protein fluorescence emission maximum; E9 DNase, the isolated 15-kDa endonuclease domain of colicin E9; Im9, the colicin E9 immunity protein; KPi, potassium phosphate; RL-P, lipid:protein molar ratio; Tm, melting temperature; ANS, 8-anilinonaphthalene-1-sulfonic acid; ECP, eosinophil cationic protein. and cardiolipin. The most abundant of these, phosphatidylethanolamine, is zwitterionic and usually accounts for around 70-80% of total cytoplasmic membrane phospholipid. Both PG and cardiolipin are anionic and account for the remaining 20-30% of phospholipid in an approximate 2:1 ratio (9Kadner R.J. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, DC1996: 58-87Google Scholar). Both PG and cardiolipin are widely distributed throughout Gram-negative and Gram-positive bacteria and are, to a large degree, responsible for the overall anionic character of bacterial membranes. Our current work focuses on the mechanism by which the cytotoxic DNase domain of the microbial toxin colicin E9 is able to translocate into the cytoplasm of susceptible cells to reach its cellular target, the bacterial chromosome. The E9 DNase domain shares no extensive sequence or structural similarities with the pore-forming colicins but does share with this group of toxins the ability to form ion channels in planar lipid bilayers (10Mosbahi K. Lemaître C. Keeble A.H. Mobasheri H. Morel B. James R. Moore G.R. Lea E.J. Kleanthous C. Nat. Struct. Biol. 2002; 9: 476-484Crossref PubMed Scopus (48) Google Scholar). However, in contrast to the pore-forming colicins, which kill cells through depolarization of the inner membrane, the channels formed by the E9 DNase domain do not in themselves cause cell death, because a mutant protein lacking DNase activity is still able to induce channel formation in planar lipid bilayers but is not cytotoxic (10Mosbahi K. Lemaître C. Keeble A.H. Mobasheri H. Morel B. James R. Moore G.R. Lea E.J. Kleanthous C. Nat. Struct. Biol. 2002; 9: 476-484Crossref PubMed Scopus (48) Google Scholar). Instead, it was proposed that the observed channels are related to the ability of the E9 DNase domain to translocate across the inner membrane and must somehow reseal on entry of the domain into the cytoplasm. This possibility was inferred from the observation that the introduction of a specific disulphide bond in the E9 DNase domain had little effect upon the endonuclease activity of the DNase domain but abolished both channel activity and colicin cytotoxicity. The 134-residue colicin E9 DNase domain is monomeric in solution (11Pommer A.J. Wallis R. Moore G.R. James R. Kleanthous C. Biochem. J. 1998; 334: 387-392Crossref PubMed Scopus (56) Google Scholar) and, like all the enzymatic colicins, forms a high affinity complex with its cognate immunity protein, Im9 (12Wallis R. Moore G.R. James R. Kleanthous C. Biochemistry. 1995; 34: 13743-13750Crossref PubMed Scopus (146) Google Scholar, 13Walker D. Moore G.R. James R. Kleanthous C. Biochemistry. 2003; 42: 4161-4171Crossref PubMed Scopus (47) Google Scholar). The immunity protein serves to protect the producing cell from the lethal effects of the toxin but must be jettisoned before translocation into the target cell. Unusually for an enzyme-inhibitor complex, the immunity protein does not bind directly to the active site of the E9 DNase, but rather to an adjacent exosite (14Kleanthous C. Kühlmann U.C. Pommer A. Ferguson N. Radford S.E. Moore G.R. James R. Hemmings A.M. Nat. Struct. Biol. 1999; 6: 243-252Crossref PubMed Scopus (157) Google Scholar, 15Kleanthous C. Walker D. Trends Biochem. Sci. 2001; 10: 624-631Abstract Full Text Full Text PDF Scopus (101) Google Scholar). The catalytic center of the E9 DNase domain contains the HNH motif, which is the site for both DNA and metal binding (16Walker D.C. Georgiou T. Pommer A.J. Walker D. Moore G.R. Kleanthous C. James R. Nucleic Acids Res. 2002; 30: 3225-3234Crossref PubMed Scopus (59) Google Scholar). The HNH motif is also found in a variety of endonucleases, including the caspase-activated DNase that is responsible for degradation of the chromosome during eukaryotic apoptosis (16Walker D.C. Georgiou T. Pommer A.J. Walker D. Moore G.R. Kleanthous C. James R. Nucleic Acids Res. 2002; 30: 3225-3234Crossref PubMed Scopus (59) Google Scholar, 17Nagata S. Nagase H. Kawane K. Mukae N. Fukuyama H. Cell Death Differ. 2003; 10: 108-116Crossref PubMed Scopus (369) Google Scholar, 18Scholz S.R. Korn C. Bujnicki J.M. Gimadutdinow O. Pingoud A. Meiss G. Biochemistry. 2003; 42: 9288-9294Crossref PubMed Scopus (24) Google Scholar). The E9 DNase binds Zn2+ ions with nm affinity, and this interaction considerably stabilizes the protein (19Pommer A.J. Kühlmann U.C. Cooper A. Hemmings A.M. Moore G.R. James R. Kleanthous C. J. Biol. Chem. 1999; 274: 27153-27160Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). However, for the DNase domain to be enzymatically active in vivo, Mg2+ ions are required, although these do not bind directly to the protein in the absence of DNA (16Walker D.C. Georgiou T. Pommer A.J. Walker D. Moore G.R. Kleanthous C. James R. Nucleic Acids Res. 2002; 30: 3225-3234Crossref PubMed Scopus (59) Google Scholar, 20Pommer A.J. Cal S. Keeble A.H. Walker D. Evans S.J. Kuhlmann U.C. Cooper A. Connolly B.A. Hemmings A.M. Moore G.R. James R. Kleanthous C. J. Mol. Biol. 2001; 314: 735-749Crossref PubMed Scopus (90) Google Scholar). Intoxication of E. coli cells by colicin E9 induces the SOS response, the characteristic response to DNA damage, prior to cell death (21Walker D. Rolfe M. Thompson A. Moore G.R. James R. Hinton J.C.D. Kleanthous C. J. Bacteriol. 2004; 186: 866-869Crossref PubMed Scopus (35) Google Scholar). Here we describe the interaction of the E9 DNase with phospholipid vesicles. Working predominantly with anionic phospholipids, we studied the effects of the protein-lipid interaction on the structure and stability of the protein using fluorescence spectroscopy in combination with chemical and thermal denaturation experiments. We also compared the accessibility of the protein tryptophans to quenching agents with and without lipids. Our data show strong similarities to those resulting from the global changes that occur to the structure of poreforming colicins during the initial stages of their association with negatively charged phospholipid membranes, and these are discussed in the paper. Protein Purification—The E9 DNase domain and Im9 with a C-terminal 6-histidine tag were co-expressed from BL21 (DE3) cells containing the plasmid pRJ353 (22Garinot-Schneider C. Pommer A.J. Moore G.R. Kleanthous C. James R. J. Mol. Biol. 1996; 260: 731-742Crossref PubMed Scopus (68) Google Scholar). The E9 DNase domain was purified by nickel-affinity chromatography, as described previously, with minor modifications (22Garinot-Schneider C. Pommer A.J. Moore G.R. Kleanthous C. James R. J. Mol. Biol. 1996; 260: 731-742Crossref PubMed Scopus (68) Google Scholar). To ensure that the protein was metal-free, after nickel affinity chromatography, EDTA was added to a final concentration of 10 mm, and the protein was dialysed against 50 mm KPi, pH 7.2 and desalted by gel filtration chromatography in the same buffer (Superdex-75). The protein was then dialysed against 3 × 5 liters of 50 mm Tris-HCl, pH 7.5 containing 500 mm NaCl, 1 × 5 liters of 50 mm Tris-HCl, pH 7.5 containing 200 mm NaCl, 5 liters of 50 mm Tris-HCl, pH 7.5, and 3 × 5 liters of dH2O. The protein was verified as being free of contamination by both metal and EDTA by its ability to bind a stoichiometric amount of zinc as determined by ANS binding, as described by Pommer et al. (19Pommer A.J. Kühlmann U.C. Cooper A. Hemmings A.M. Moore G.R. James R. Kleanthous C. J. Biol. Chem. 1999; 274: 27153-27160Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The protein was then aliquoted, lyophilized, and stored at -20 °C. The concentration of the E9 DNase was determined from the absorbance at 280 nm using a molar extinction coefficient of 17,550 m-1 cm-1 (19Pommer A.J. Kühlmann U.C. Cooper A. Hemmings A.M. Moore G.R. James R. Kleanthous C. J. Biol. Chem. 1999; 274: 27153-27160Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). For experiments in which the zinc-bound protein was used, ZnCl2 was added to give a Zn2+:E9 DNase ratio of 1.2:1. Lipid Vesicle Preparation—1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were supplied by Avanti Polar Lipids (Alabaster, AL) and used without further purification. Lipid vesicles were prepared by using a film hydration method followed by extrusion through polycarbonate filters (23Olson F. Hunt C.A. Szoka F.C. Vail W.J. Papahadjopoulos D. Biochim. Biophys. Acta. 1979; 557: 9-23Crossref PubMed Scopus (971) Google Scholar). Briefly, 2 mg of phospholipid in a 1:1 chloroform:methanol solution was dried by evaporation under nitrogen, and final traces of solvent were removed under vacuum. The lipid film was then hydrated with 1 ml of buffer and stirred at room temperature to form multilamellar vesicles. To form homogenous unilamellar lipid vesicles, the phospholipid solution was extruded 12 times through 0.2 μm polycarbonate filters. The presence of unilamellar vesicles was confirmed by electron microscopy after negative staining with ammonium molybdate (23Olson F. Hunt C.A. Szoka F.C. Vail W.J. Papahadjopoulos D. Biochim. Biophys. Acta. 1979; 557: 9-23Crossref PubMed Scopus (971) Google Scholar). Fluorescence Measurements—Fluorescence emission spectra were recorded on a Spex-FluoroMax-3 spectrofluorimeter (Jobin Yvon) equipped with a Neslab RTE-111 circulating water bath. Spectra were recorded in 10 mm KPi, pH 7.5 at a protein concentration of 0.2 or 1 μm using an excitation wavelength of 280 nm, with excitation and emission slits set to 3 nm. Thermal denaturation profiles were obtained by monitoring the λmax in the temperature range 15-80 °C. Tm values were obtained by fitting raw data to Eq. 1. y=y0+a/(1+exp(−(x−x0)/b))(Eq. 1) where y is the λmax, y0 is the starting value, x is the temperature, and x0 is the Tm. Fluorescence emission was used to monitor the E9 DNase denaturation with increasing concentrations of urea. Solutions of the E9 DNase (1 μm) with urea at concentrations from 0-5 m were prepared in 50 mm Tris-HCl, pH 7.5 and incubated for 1 h at room temperature. Phospholipids in the form of lipid vesicles (DOPG and DOPC) were included at a concentration of 30 μm where shown. Quenching experiments using acrylamide were carried out by using an excitation wavelength of 295 nm at a protein concentration of 1 μm in either 50 mm Tris-HCl, pH 7.5, or 50 mm Tris acetate, pH 4.0, at a phospholipid: protein ratio of 150:1, where stated. Acrylamide, from a 5.63 m stock dissolved in water (Fluka Biochemika), was titrated into the protein or protein-lipid solution up to a final concentration of 0.2 m. All of the spectra were buffer-subtracted and corrected for dilution. The Stern-Volmer equation Fo/F=1+Ksv[Q](Eq. 2) was used to determine the Stern-Volmer quenching constant Ksv. Fo and F are the fluorescence intensity in the absence and presence of the quencher (Q). Where this equation did not represent the data well, the modified Stern-Volmer equation (Eq. 3) was used to determine the accessibility of the tryptophan residues to the quencher. Fo/(Fo−F)=1/faKa[Q]+1/fa(Eq. 3) Therefore, a plot of Fo/(Fo - F) against 1/(Q) has a slope equal to 1/faKa and an intercept equal to 1/fa. In this case, Ka is the Stern-Volmer quenching constant for the accessible fraction of the tryptophan residues, and fa is the fraction of the initial fluorescence, which is accessible to the quencher. Circular Dichroism—Circular dichroism (CD) spectra of the E9 DNase were recorded on a Jasco J-810 spectropolarimeter equipped with a Jasco Peltier temperature controller (PFD-4255). Spectra were recorded in a 10-mm path length cuvette at a scan speed of 100 nm min-1, with a response time of 1 s and with the spectral bandwidth set to 1 nm. Measurements in the far-UV (190-300 nm) were recorded in 10 mm KPi, pH 7.5 at a protein concentration of 0.5 μm. The spectra obtained were the average of 10 scans with baseline subtraction. Proteolysis—Tryptic digests of the E9 DNase domain were performed in 50 mm Tris-HCl, pH 7.5, at an E9 DNase concentration of 1 mg ml-1 and a trypsin concentration of 20 μg ml-1 at 37 °C. Zn2+ was present in slight molar excess (1.2:1) with DOPC or DOPG vesicles at 330 μm where indicated. Samples were removed at the times indicated, and proteolysis was stopped by the addition of an excess of trypsin inhibitor (Sigma). The products of proteolysis were analyzed by SDS-16% PAGE. E9 DNase Specifically Interacts with Negatively Charged Phospholipid Vesicles—In aqueous solution close to neutral pH, the intrinsic fluorescence emission spectrum of the E9 DNase shows a maximum value (λmax) of 333 nm, indicating that its two tryptophan residues are substantially buried in the interior of the protein. The intrinsic fluorescence of the E9 DNase is very sensitive to ligand-binding events, to the extent that binding at the immunity protein exosite and active site can be distinguished (12Wallis R. Moore G.R. James R. Kleanthous C. Biochemistry. 1995; 34: 13743-13750Crossref PubMed Scopus (146) Google Scholar, 19Pommer A.J. Kühlmann U.C. Cooper A. Hemmings A.M. Moore G.R. James R. Kleanthous C. J. Biol. Chem. 1999; 274: 27153-27160Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Complex formation with immunity protein at the exosite gives rise to an enhancement in its intrinsic fluorescence, whereas binding of either transition metals or DNA at the active site of the protein quench the intrinsic fluorescence. Hence, fluorescence was used to monitor the effects of lipids on the E9 DNase. Fluorescence spectra of the E9 DNase in the presence and absence of DOPG are shown in Fig. 1A. The spectra were recorded at a protein concentration of 1 μm and a lipid:protein ratio of 150. We found that the addition of negatively charged DOPG phospholipid vesicles to the E9 DNase at pH 7.5 gave rise to a significant red-shift in the λmax of the fluorescence of the protein that was not observed with neutral DOPC vesicles (data not shown). Interestingly, in addition to the shift in λmax, a reduction in the fluorescence signal was also observed after the addition of DOPG. The change in λmax for DOPG-treated DNase to 346 nm indicated a significant change in the environment of the tryptophan residues, as observed in protein denaturation. However, DOPG-treated DNase is not the unfolded state of the protein because it differs significantly from that induced by pH or urea denaturation (Fig. 1A). The shift in λmax was found to be approximately linearly dependent upon the molar lipid:protein ratio (RL-P) with DOPG vesicles, up to an RL-P value of around 100, with the addition of further DOPG having little effect on the value of the λmax (Fig. 1B). Increasing the E9 DNase concentration 5-fold to 1 μm showed that the change in λmax is independent of the total lipid concentration and dependent only upon the lipid:protein ratio (data not shown). With neutral DOPC vesicles, no change in λmax was observed up to a lipid:protein ratio of 500, indicating a strong electrostatic contribution to the protein-lipid interaction with DOPG. Using mixed phospholipid vesicles consisting of equimolar amounts of DOPC and DOPG, we observed a similar dependence of the λmax upon the value of RL-P with DOPG alone, but with a reduced effect upon the total change of the λmax (Fig. 1B). Probing the Structure of the Protein-lipid Complex by Fluorescence Quenching—At pH 4.0, near-UV CD indicates that the E9 DNase does not possess a well defined tertiary structure, whereas far-UV CD indicates only subtle changes in secondary structure compared with the protein at neutral pH (24van den Bremer E.T.J. Jiskoot W. James R. Moore G.R. Kleanthous C. Heck A.J.R. Maier C.S. Protein Sci. 2002; 11: 1738-1852Crossref PubMed Scopus (54) Google Scholar). The absence of persistent tertiary structure for the E9 DNase at pH 4.0 is confirmed by fluorescence spectroscopy, where λmax is red-shifted from 333 to 354 nm, indicating complete solvent exposure of the two tryptophans (Fig. 1A). Upon the addition of DOPG phospholipids at pH 4.0 (RL-P = 150), we found that there was a significant blue-shift to ∼341 nm, but there was no shift in the λmax upon addition of DOPC (data not shown). Not only does this illustrate that complexation between the E9 DNase and phospholipids does not require defined tertiary structure, but it implies that electrostatics are the likely basis for this association. We return to this issue in “Discussion.” The λmax in the presence of DOPG at pH 4.0 is similar to that observed upon addition of DOPG to the E9 DNase at pH 7.5 (Fig. 1A), suggesting that the solvent accessibility of the tryptophans in the final protein-lipid complex is similar at both pHs. This is supported by fluorescence-quenching experiments in the presence and absence of DOPG vesicles with the water-soluble quencher acrylamide at pH 4.0 and 7.5. Fig. 2 shows Stern-Volmer plots for the E9 DNase under these conditions. In the case of E9 DNase in solution at pH 4.0, the Stern-Volmer plot displayed a downward curvature (Fig. 2A); therefore, a modified Stern-Volmer plot was used (Fig. 2B). As expected, the accessibility of the E9 DNase tryptophan residues to acrylamide at pH 4.0 (Ka = 11 m-1), where the protein is unfolded, is much greater than in the folded state at pH 7.5 (Ksv = 2.7 ± 0.1 m-1). However, in the presence of DOPG vesicles at pH 4.0, the accessibility to acrylamide decreases to a value similar to that of the folded protein (K = 2.3 ± 0.1 m-1) and is very similar to that of the E9 DNase in the presence of DOPG vesicles at pH 7.5 (Ksv = 2.1 ± 0.1 m-1). These data are consistent with the E9 DNase forming non-voltage gated channels in planar lipid bilayers both at pH 4.0 and pH 7.5 and indicates that the protein does not have to be in its native conformation to interact with membranes. 2K. Mosbahi and C. Kleanthous, unpublished results. Effect of Ligand Binding and Disulfide Bond Formation on the Ability of the E9 DNase to Interact with Lipids—The experiments described above were performed on protein preparations of the E9 DNase in the absence of bound metal. However, it has been shown previously that binding of a stoichiometric amount of Zn2+ causes a considerable increase in the conformational stability of the E9 DNase (19Pommer A.J. Kühlmann U.C. Cooper A. Hemmings A.M. Moore G.R. James R. Kleanthous C. J. Biol. Chem. 1999; 274: 27153-27160Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). This is manifest by an increase of 22 °C (from 37 to 59 °C) in the melting temperature of the protein at pH 7.5, a considerable decrease in the susceptibility of the protein to proteolysis, and a decrease in affinity of the protein for the hydrophobic dye ANS (19Pommer A.J. Kühlmann U.C. Cooper A. Hemmings A.M. Moore G.R. James R. Kleanthous C. J. Biol. Chem. 1999; 274: 27153-27160Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In view of these differences, we performed a similar experiment to that described in Fig. 1B in the presence of zinc (Fig. 3A). We found no difference in the change in λmax between the zinc-bound form and the metal-free protein upon association with DOPG vesicles, showing that the ability of the E9 DNase to bind negatively charged phospholipids is not affected by the conformational stability of the protein. This result is also consistent with the observation that the presence of bound zinc has no effect upon the ability of the E9 DNase to form channels in planar lipid bilayers (10Mosbahi K. Lemaître C. Keeble A.H. Mobasheri H. Morel B. James R. Moore G.R. Lea E.J. Kleanthous C. Nat. Struct. Biol. 2002; 9: 476-484Crossref PubMed Scopus (48) Google Scholar). The E9 DNase forms a high affinity complex with its cognate immunity protein Im9 (12Wallis R. Moore G.R. James R. Kleanthous C. Biochemistry. 1995; 34: 13743-13750Crossref PubMed Scopus (146) Google Scholar). The formation of this heterodimeric complex has been shown previously to prevent channel formation by the E9 DNase in planar lipid bilayers (10Mosbahi K. Lemaître C. Keeble A.H. Mobasheri H. Morel B. James R. Moore G.R. Lea E.J. Kleanthous C. Nat. Struct. Biol. 2002; 9: 476-484Crossref PubMed Scopus (48) Google Scholar). Consistent with this, we observed no change in the λmax of the E9 DNase-Im9 complex (∼330 nm) upon addition of DOPG vesicles at an RL-P = 150 (Fig. 3B). Therefore, we infer that the presence of the immunity protein prevents the formation of a protein-lipid complex. We have shown previously that the ability of the DNase to form channels in planar lipid bilayers can be abolished by the formation of an artificial intramolecular disulfide bond between residues 20 and 66 (10Mosbahi K. Lemaître C. Keeble A.H. Mobasheri H. Morel B. James R. Moore G.R. Lea E.J. Kleanthous C. Nat. Struct. Biol. 2002; 9: 476-484Crossref PubMed Scopus (48) Google Scholar). This loss of channel activity is accompanied by a loss of cytotoxicity for the intact toxin, but there is little effect upon its endonuclease activity. Therefore, the formation of this disulfide bond affects the ability of the E9 DNase to translocate across E. coli cellular membranes. Because this disulfide-containing mutant of the E9 DNase differs from the wild-type protein in its ability to form channels in planar lipid bilayers, we tested its ability to interact with DOPG vesicles using fluorescence. Monitoring the change in the λmax at an increasing lipid-protein ratio (as in Fig. 1B) gave an essentially identical profile to that observed for the wild-type protein (data not shown). Thus, the ability of the E9 DNase to interact with phospholipid vesicles, as monitored in this work, is not on its own sufficient for the protein to form ion channels in planar lipid bilayers. Negatively Charged Phospholipids Decrease the Stability of the E9 DNase—Proteins such as the pore-forming domain of colicin A have been shown to interact with phospholipid vesicles in a manner that leads to destabilization of the protein (5Muga A. González-Mañas J.M. Lakey J.H. Pattus F. Surewicz W.K. J. Biol. Chem. 1993; 268: 1553-1557Abstract Full Text PDF PubMed Google Scholar). The E9 DNase has been shown previously to unfold reversibly in a cooperative manner with a melting temperature (Tm) of 37 °C for the metal-free protein, which increases to 58 °C upon binding Zn2+, as determined by differential scanning calorimetry (19Pommer A.J. Kühlmann U.C. Cooper A. Hemmings A.M. Moore G.R. James R. Kleanthous C. J. Biol. Chem. 1999; 274: 27153-27160Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Unfolding of E9 DNase can also be monitored by a change in the λmax of the intrinsic fluorescence with temperature (Fig. 4A). In the absence of a boun
Colicin
Inhibitor protein
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Homing endonuclease
Intein
Homing (biology)
Protein splicing
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Recognition sequence
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During genetic crosses between the interfertile green algae Chlamydomonas eugametos and Chlamydomonas moewusii , the I‐ Ceul endonuclease encoded by the fifth group I intron (CeLSU · 5) in the C. eugametos chloroplast large subunit rRNA gene mediates the mobility of this intron by introducing a double‐strand break near the insertion site of the intron in the corresponding C. moewusii intronless allele. To characterize the biochemical properties of this endonuclease, we have purified I‐ Ceu I and determined the optimal reaction conditions for cleavage. I‐ Ceu I activity is maximal at 50°C, pH 10.0, 2.5 mM MgCl 2 and in the absence of NaCl. Unlike the well‐characterized I‐ Sce I endonuclease, I‐ Ceu I remains stable following preincubation in the absence of substrate. We discuss the role that homing endonucleases may have played in the evolution of Chlamydomonas chloroplast group I introns.
Homing endonuclease
Chlamydomonas
Cleavage (geology)
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Homing endonuclease
Antiparallel (mathematics)
Linker
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Homing endonuclease
Homing (biology)
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Intein
Homing endonuclease
Protein splicing
Protein tag
Homing (biology)
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