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Metal Ion Binding to a Zinc Finger Peptide Containing the Cys-X2-Cys-X4-His-X4-Cys Domain of a Nucleic Acid Binding Protein Encoded by theDrosophilaFw-Element

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Metal Ion Binding to a Zinc Finger Peptide Containing the Cys-X2-Cys-X4-His-X4-Cys Domain of a Nucleic Acid Binding Protein Encoded by theDrosophilaFw-Element
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  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  242,  385–389 (1998)  ARTICLE NO.  RC977974 Metal Ion Binding to a Zinc Finger Peptide Containing theCys-X 2 -Cys-X 4 -His-X 4 -Cys Domain of a Nucleic AcidBinding Protein Encoded by the  Drosophila   Fw-Element  A. Bavoso,* A. Ostuni,* G. Battistuzzi,† L. Menabue,† M. Saladini,† and M. Sola† ,1 *  Department of Chemistry, University of Basilicata, Via N. Sauro, 85, 85100 Potenza, Italy; and †  Department of Chemistry, University of Modena, Via Campi 183, 41100 Modena, Italy Received November 17, 1997 recognition (1-3). Nucleocapsid proteins of retroviruses The metal binding properties of a 18-residue zinc are basic species of low-molecular weight (from about 6 finger peptide containing a CCHC box which repro- to9kDa)containingoneortwocopiesofthezinc-binding  duces one of the cysteine-rich domains of a putative motif Cys-X  2 -Cys-X  4 -His-X  4 -Cys (called ‘‘CCHC box’’) nucleic acid binding protein encoded by the Fw trans- which, at variance with above, bind to single stranded posable element from  Drosophila melanogaster   were nucleic acids (4-11). Proteins containing the same bind- investigated through electronic and  1 H NMR spectros- ing motif are also encoded by  Drosophila melanogaster copy. Dissociation constants of 2( { 1) 1 10 0 12 M and transposable elements (4, 12-16). 4( { 1) 1 10 0 7 M were determined for the Zn 2 / and Co 2 /  A number of investigations have focused on the bind- adduct, respectively. These values are similar to those ing properties of synthetic peptides containing zinc for other CCHC-peptides investigated previously, al- finger sequence(s) toward spectroscopically active met- though the length of the spacer between the second als such as Co 2 / , Ni 2 / , Fe 2 / and Cd 2 / and on the struc- cysteine and the histidine apparently exerts some in- tural features of the metal adducts (4, 17-23). The fluence on the spectral properties and on the stability metal binding affinities of peptides containing the of the Co 2 / -peptide adduct. The  1 H NMR spectrum of  CCHH and CCHC box were found to differ to some the present Co 2 / -derivative contains a number of well extent (17). Moreover, the peptide domain containing  resolved hyperfine-shifted resonances between 350 the CCHC box was shown to fold into a globular struc- and 0 50 ppm which arise from the metal binding resi- ture (4, 7, 24-26) which differs significantly from the dues and nearby groups. These peaks can in principle antiparallel  b  hairpin followed by a helix typical of  beprofitablyexploitedtomonitorprotein-nucleicacid ‘‘classical’’ CCHH zinc finger species (27). interactions.    1998 Academic Press Here, we report on the metal binding properties of a18-residuefingerpeptidecontainingaCCHCboxwhichreproduces one of the cysteine-rich domains of a puta-tive nucleic acid binding protein encoded by a 366-bp A wide variety of nucleic acid-binding and gene regu-open reading frame present at the truncated 5   termi-latory proteins contains a number of small domainsnus of the  Drosophila  Fw element (12). This peptide,structurally organized around tetrahedral Zn(II) ion(s) ValGln Cys ThrAsn Cys GlnGluTyrGly His ThrArgSer-coordinated by Cys and His residues, termed ‘‘zinc fin-Tyr Cys ThrLeu (DF hereafter), has X spacers scarcelyger’’ domains(1-3). Anincreasingly large numberof pro-sequence related with those of the peptides from ret-teins of this class binding to double-stranded nucleicroviral species studied previously (4, 24, 25) and aacidsarebeingrecognized.Themostextensivelycharac-lower isoelectric point (pI  Å  6.7 vs. 7.9-9.7) (4). Weterized species are eukaryotic transcription factors con-determined the binding affinity of DF toward Zn 2 / andtainingrepeatedmononuclearZndomainswithaCCHHCo 2 / through electronic spectroscopy and compared itbinding set (C  Å  cysteine, H  Å  histidine), also termedwith that of other zinc finger peptides. Moreover, we‘‘classical’’ zinc finger domains. However, other zinchave detected the hyperfine-shifted  1 H NMR reso-binding motifs, such as CCCC centers and Zn 2 (Cys) 6 nances arising from the metal-binding and contiguousclusters are known to beinvolved in protein-nucleic acidresidues for the Co 2 / -DF adduct. Location of these sig-nals, which are highly informative of the structuraland electronic properties of the metal site (28, 29), is 1 Corresponding author. Fax:  // 39-59-373-543. E-mail: sola@unimo.it.  unprecedented for this class of metal centers. 0006-291X/98 $25.00 Copyright    1998 by Academic Press All rights of reproduction in any form reserved. 385   Vol. 242, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS FIG. 1.  Absorption spectra of 2 1 10 0 4 M DF in 0.1 M phosphate buffer, pH 8, titrated with Co 2 / dichloride. Titration was performedunder strictly anaerobic conditions. Spectra were corrected for the absorption due to the free peptide. T  Å  25   C.TFAin CH 3 CN).Thepurepeptide waslyophilizedunder vacuumand MATERIALS AND METHODS stored in the presence of 5 equivalents of solid DTT under anaerobicconditions. The peptide was sequenced on an Applied Biosystem  Reagents.  N-9-fluorenylmethoxycarbonyl (Fmoc) aminoacids and491A instrument. A molecular mass of 2106.8 Da, consistent with4-hydroxymethylphenoxy-methyl-copoly(styrene-1%divinylbenzene)the theoretical value of 2106.3 Da was obtained by electrospray mass(HMP) resin were purchased from Nova Biochem (Switzerland). N-spectrometry.methylpyrrolidone (NMP), dicycloexylcarbodiimide (DCC), 4-dimeth-ylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), 1,2-  Metalbindingstudies.  Allpeptidemanipulationsdescribedbelowethanedithiol (EDT), dithiothreitol (DTT) and trifluoroacetic acid were performedunder anatmosphere of85% nitrogen,10% hydrogen(TFA) were from Applied Biosystem. and 5% CO 2  in an anaerobic chamber (Plas-Lab, Lansing, MI, USA)to avoid peptide oxidation. DTT was separated from the reduced  Peptide synthesis and purification.  The peptide was synthesizedpeptide through gel-filtration chromatography on a Sephadex G-15with the stepwise solid method on a Applied Biosystems 431A pep-column (5 1 80 mm) in 0.1 M phosphate buffer, pH 8. The reducedtide synthesizer using Fmoc chemistry. The C-terminal aminoacidpeptide showed an extinction coefficient of 2450 M 0 1 cm 0 1 at 280 nm.wasattachedtothe HMPresinwithDCCinthe presenceofDMAPasThe dissociation constant for the Co 2 / adduct was determined bycatalyst. The subsequent Fmoc aminoacids were bound using DCC/ titrating the reduced peptide in 0.1 M phosphate buffer at pH 8 withHOBt chemistry. Deprotection of the Fmoc group was obtained withthe metal ion and monitoring the absorption spectrum. This pH was20% (w/w) piperidine in NMP. The peptide was cleaved from thechosen in order to increase the solubility of the peptide, which isresin upon treatment with TFA (88%), phenol (4%), EDT (2%), thio-almost uncharged at pH 7. At higher pH values, the formation of anisole (3%) and H 2 O (3%) for three hour at ambient temperature;the metal-peptide adduct suffers competition with metal hydroxidethis procedure also allows detachment of the protecting groups of precipitation. The constant for Zn 2 / binding was obtained by follow-the side chains. The mixture was then filtered and the solution wasing the bleaching of the absorption spectrum of the Co 2 / -adduct uponconcentrated under vacuum. The peptide was then precipitated withaddition of the metal, as reported elsewhere (21) The electronic spec-cold ether, suspended in 0.1% TFA and lyophilized. The peptide,tra were recorded on a Perkin-Elmer Lambda 9 spectrophotometer.dissolved in Tris-HCl buffer in the presence of 50 equivalents of DTT,was purified on an analytical and semipreparative scale by reverse-  NMR spectra.  NMR samples of the Co 2 / -DF adduct were pre-pared by adding one equivalent of cobalt dichloride to 500  m L of thephase high performance liquid chromatography on a Beckman Model110B apparatus with a Bondclone C18 column 3.9  1  300 mm and reduced peptide (about 0.1 mM) in 0.1 M phosphate buffer at pH 8. 1 H NMR measurements were carried out on Bruker AMX-400 andon a Vydac C18 column 10 1 250 mm, respectively. The mobile phaseconsisted of solution A (0.1% TFA in H 2 O) and solution B (0.05% DPX-200spectrometersat400.13and200.03MHz,respectively.Typ-386   Vol. 242, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONSical acquisition parameters were as follows: spectral width, 125 kHz;pulse width, 6  m s (90   pulse); pulse delay, 0.2-0.4 s. Spectra werereferenced to tetramethylsilane (TMS) after calibration against thewater peak, set at 4.78 ppm from TMS (at 20   C). Suppression of thewater peak was achieved using the super-WEFT pulse sequence (30).Longitudinal relaxation times were determined with the inversionrecovery sequence preceded by a presaturation pulse. RESULTS AND DISCUSSION  Addition of Co 2 / to the reduced peptide in anaerobicconditions at pH 8 srcinates absorption bands at 310( e  Å  3100 M 0 1 cm 0 1 ), 355 (shoulder), 612 (shoulder),645 ( e  Å  460 M 0 1 cm 0 1 ) and 697 ( e  Å  380 M 0 1 cm 0 1 )nm (Fig. 1). The first two bands at higher energy canbe attributed to cysteinate to metal charge transfertransitions,whilethethreecomponentsofthespectralenvelope between 600 and 700 nm can be assigned to 4  A  2  to  4 T 1 (P) ligand field transitions typical of syn-thetic tetrahedral Co 2 / complexes with thiolate andimidazole ligands (4, 31, 32). The spectrum is almostidentical to those of the Co 2 / adducts of the nucleocap-sid protein p10 from Rauscher murine leukemia virus(RaMLV) (33) and of the 18 aminoacid fragment of thesame protein containing the CCHC binding motif Cys- X  2 -Cys-X  4 -His-X  4 -Cys (4). Titration of reduced DFwith Co 2 / was followed spectrophotometrically (Fig.2A). Band intensities increase linearly with increas-ing metal concentration and level off at the stoichio-metric Co 2 /  /peptide ratio of 1:1. The data could befitted by non linear least-squares methods whichyielded a dissociation constant of K  Cod  Å  4( { 1) 1 10 0 7 M. This value compares reasonably well with that de- FIG. 2.  Plot of the absorbance at 645 nm for (A) 2 1 10 0 4 M DF termined for the RaMLV peptide (K  Cod  Å  1 1 10 0 6 M) in 0.1 M phosphate buffer, pH 8, in the presence of added Co 2 / (see (4) (at least part of the difference may arise from the Fig. 1), as a function of the [Co 2 / ]/[DF] ratio, and (B) 2 1 10 0 4 M Co 2 / - fact that the two constants refers to different condi- DF in 0.1 M phosphate buffer, pH 8, in the presence of added Zn 2 / tions of ionic composition and pH). Instead, the Co 2 / -  as a function of the [Zn 2 / ]/[DF] ratio. T  Å  25   C. adduct of the His 24  to Cys variant of CP-1 [CP-1(CCHC)] (where CP-1 is is the consensus zinc fingerpeptide ProTyrLysCys 4 ProGluCys 7 GlyLysSerPheSer- of the peptide, while residue substitutions within theconserved retroviral sequence motif Cys-X  2 -Cys-X  4 -GluLysSerAspLeuValLysHis 20 GlnArgThrHis 24 Thr-Gly prototypic of the zinc binding domains of eukary- His-X  4 -Cys have little effect. The affinity of DF forZn 2 / wasdeterminedby titratingtheCo 2 / adductwithotic transcription factors (20)) has a lower dissociationconstant (K  Cod  Å 6.3 1 10 0 8 M) (17). It may be proposed the above metal in the presence of a known excess of Co 2 / ion (50 fold), and following the disappearance of that the lower affinity of the peptides of retroviralsrcin for Co 2 / is due to some constraints to the coordi- the absorption bands due to the displacement of Co 2 / by Zn 2 / , which is known to bind more tightly to thesenation geometry imposed by the only four-residuespacing between the second Cys and the His ligands peptides(17,20,21)(Fig.2B).TheZn 2 / ioninfactdoesnot undergo loss in ligand field stabilization energy oninstead of the 12-residue spacing in CP-1(CCHC).Moreover,thethreed-dbandsintheelectronicspectra passing from the aquoion to a tetrahedral coordina-tion, as does Co 2 / , as pointed out elsewhere (17). Theof the Co 2 / -CP-1(CCHC) adduct are spread over awider region (from 580 to 724 nm) and are better re- dissociation constant was determined to be: K  Znd  Å 2( { 1) 1 10 0 12 M, which is close to that determined forsolved than those of the Co 2 / adducts of the RaMLV and DF peptides (17, 20, 22). Thus, it appears that CP-1(CCHC) (K  Znd  Å  3.2( { 1) 1 10 0 12 M) (17).The paramagnetic Co 2 / ion is profitably exploited asthe length of the spacer between the second cysteineand the histidine in the sequence of the CCHC domain NMR probe for the investigation of metal sites in metal-loproteins, due to the contact and pseudocontact contri-exerts some influence on the ligand field transitionsof the metal chromophore and on the metal affinity butions to the chemical shift and nuclear relaxation of  387   Vol. 242, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS FIG. 3.  1 H NMR spectrum of 2 1 10 0 4 M Co 2 / -DF in 0.1 M phosphate buffer, pH 8. (A) 200 MHz spectrum showing the details of thehigh- and low-frequency paramagnetic region. Number of scans, 16k. (B) 400 MHz spectrum showing the hyperfine-shifted signals close tothe diamagnetic region. Number of scans, 8k. T 1  values (ms) are indicated below each peak. Shaded signals are those which disappear inthe spectrum recorded in D 2 O, and correspond to exchangeable amide protons. T  Å  27   C. the protons in the surroundings of the metal, which pro- above systems, six of the eight nonexchangeable signalswhich fall above 40 ppm ( a-g, i ) should correspond tovide valuable structural information (28, 29). The 200MHz  1 H NMR spectrum of the Co 2 / -DF adduct contains the  b -CH 2  groups of the three cysteine ligands. The re-maining two peaks reasonably arise from  a -CH pro-a number of strongly hyperfine-shifted resonances be-tween 350 and 0 50 ppm (Fig. 3A). T 1  vales are submilli- ton(s) of binding cysteine(s) and/or ring protons of thehistidine ligand which likely binds through the N e 2second (from below 0.1 to 0.3 ms), except peak  h  (T 1  Å 15 ms). Other well resolved and more slowly relaxing atom (24). The exchangeable peak  h  at 63 ppm can beattributed to the N d 1 H proton of the above histidine.resonances are located from 10 to 25 and from 0 to 0 15ppm (Fig. 3B). Similar spectral patterns were observed This  1 H NMR spectrum of a Co 2 / -substituted zinc fingerpeptide containing the complete set of hyperfine-shiftedfor a Co 2 / -substituted rubredoxin which possesses a tet-rahedral Co-S(Cys) 4  site (29, 34), and for Co 2 / -metallo- resonances arising from the metal binding residues isunprecedented. Previous  1 H NMR work on the Co 2 / -thioneinswhichcontainpolynuclearCo-thiolateclustersin which each metal is again tetrahedrally coordinated CP-1 adduct focused only on the resonances affected bypseudocontactshift,allowingdeterminationoftheorien-by four cysteine residues (35, 36). By reference to the 388   Vol. 242, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS9. Karpel, R. L., Henderson, L. E., and Oroszlan, S. (1987)  J. Biol. tation and anisotropy of the magnetic susceptibility ten- Chem.  262,  4961–4967. sor (18). 10. Prats, A. C., Housset, V., De Billy, G., Cornille, F., Prats, H.,  Assignment of the hyperfine-shifted resonances of  Roques, B. P., and Darlix, J.-L. (1991)  Nucleic Acids Res.  19, the Co 2 / -DF adduct could not be achieved due to sev- 3533–3541. eral unfavorable factors, namely:  i)  the small stead 11. Prats, A. C., Sarih, L., Gabus, C., Litvak, S., Keith, G., and Dar- state NOEs (most likely below detection) which are ex-  lix, J. L. (1988)  EMBO J.  7,  1777–1783. pectedtocorrelate thesesignals,dueto theremarkably  12. Di Nocera, P. P., and Casari, G. (1987)  Proc. Natl. Acad. Sci.USA  84,  5843–5847. low T 1  values and the low molecular weight of the ad- 13. Di Nocera, P. P. (1988)  Nucleic Acids Res.  16,  4041–4053. duct (28, 29);  ii)  the scarce solubility of DF in aqueous 14. Fawcett, T., Lister, C. K., Kellett, E., and Finnegan, D. J. (1986) solutionatneutralorslightly alkalinepH(max0.3mM Cell  47,  1007–1015. at pH 8);  iii)  the instability of the Co 2 / -DF derivative 15. Mount, S. M., and Rubin, G. M. (1985)  Mol. Cell. Biol.  5,  1630– which, in inert atmosphere, decomposes appreciably 1638. already after 6-8 hours after metal addition. Neverthe- 16. Rubin, G. M. (1983)  in  Mobile Genetic Elements (Shapiro, J. A., less, the present work shows that, despite tetrahedral Ed.) pp. 329–361. Academic Press, New York. four-coordination of the high-spin Co 2 / ion induces a 17. Krizek, B. A., Merkle, D. L., and Berg, J. M. (1993)  Inorg. Chem. larger broadening of the paramagnetic resonances as  32,  937–940. compared to five or six-coordination (28, 29), a number  18. Harper, L. V., Amann, B. T., Kilfoil, V. J., and Berg, J. M. (1993) of well resolved  1 H NMR peaks due to the protons of   J. Am. Chem. Soc.  115,  2577–2580.19. Krizek, B. A., and Berg, J. M. (1992)  Inorg. Chem.  31,  2984– the metal site experiencing contact and pseudocontact 2986. shiftcanbe detectedfortheseadducts. Similarspectral 20. Krizek, B. A., Amann, B. T., Kilfoil, V. J., Merkle, D. L., and features should also characterize the Co 2 / -derivatives Berg, J. M. (1991)  J. Am. Chem. Soc.  113,  4518–4523. of the whole proteins for which measurable NOEs 21. Berg, J. M., and Merkle, D. L. (1989)  J. Am. Chem. Soc.  111, should in principle be observed especially at high mag- 3759–3761. netic fields. Thus, paramagnetic  1 H NMR can in princi- 22. Blasie, C. A., and Berg, J. M. (1997)  Biochemistry  36,  6218– ple be successfully exploited for obtaining structural 6222. information in solution for Co 2 / -substituted zinc finger 23. South, T. L., Kim, B., and Summers, M. F. (1989)  J. Am. Chem. proteins and for the investigation of their interaction  Soc.  111,  395–396. with nucleic acids.  24. Summers, M. F., South, T. L., Kim, B., and Hare, D. R. (1990)  Biochemistry  29,  329–340.25. South, T. L., Blake, P. R., Hare, D. R., and Summers, M. F. ACKNOWLEDGMENTS (1991)  Biochemistry  30,  6342–6349.26. Omichinski, J. G., Clore, G. M., Sakaguchi, K., Appella, E., andThis study was supported by the University of Modena (Finanzia-Gronenborn, A. M. (1991)  FEBS Lett.  292,  25–30.mento Ricerca Avanzata) and by the National Research Council of 27. Kim, C. A., and Berg, J. M. (1996)  Nat. Struct. Biol.  3,  940–945.Italy (Progetto Strategico Tecnologie Chimiche Innovative).28. Bertini, I., and Luchinat, C. (1996)  Coord. Chem. Rev.  150,  1–296. REFERENCES 29. 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