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• W2018329608 abstract "Poly(ADP-ribose) polymerase-1 (PARP-1) is a chromatin-associated enzyme with multiple cellular functions, including DNA repair, transcriptional regulation, and cell signaling. PARP-1 has a modular architecture with six independent domains comprising the 113-kDa polypeptide. Two zinc finger domains at the N terminus of PARP-1 bind to DNA and thereby activate the catalytic domain situated at the C terminus of the enzyme. The tight coupling of DNA binding and catalytic activities is critical to the cellular regulation of PARP-1 function; however, the mechanism for coordinating these activities remains an unsolved problem. Here, we demonstrate using spectroscopic and crystallographic analysis that human PARP-1 has a third zinc-binding domain. Biochemical mutagenesis and deletion analysis indicate that this region mediates interdomain contacts important for DNA-dependent enzyme activation. The crystal structure of the third zinc-binding domain reveals a zinc ribbon fold and suggests conserved residues that could form interdomain contacts. The new zinc-binding domain self-associates in the crystal lattice to form a homodimer with a head-totail arrangement. The structure of the homodimer provides a scaffold for assembling the activated state of PARP-1 and suggests a mechanism for coupling the DNA binding and catalytic functions of PARP-1. Poly(ADP-ribose) polymerase-1 (PARP-1) is a chromatin-associated enzyme with multiple cellular functions, including DNA repair, transcriptional regulation, and cell signaling. PARP-1 has a modular architecture with six independent domains comprising the 113-kDa polypeptide. Two zinc finger domains at the N terminus of PARP-1 bind to DNA and thereby activate the catalytic domain situated at the C terminus of the enzyme. The tight coupling of DNA binding and catalytic activities is critical to the cellular regulation of PARP-1 function; however, the mechanism for coordinating these activities remains an unsolved problem. Here, we demonstrate using spectroscopic and crystallographic analysis that human PARP-1 has a third zinc-binding domain. Biochemical mutagenesis and deletion analysis indicate that this region mediates interdomain contacts important for DNA-dependent enzyme activation. The crystal structure of the third zinc-binding domain reveals a zinc ribbon fold and suggests conserved residues that could form interdomain contacts. The new zinc-binding domain self-associates in the crystal lattice to form a homodimer with a head-totail arrangement. The structure of the homodimer provides a scaffold for assembling the activated state of PARP-1 and suggests a mechanism for coupling the DNA binding and catalytic functions of PARP-1. Poly(ADP-ribose) polymerase-1 (PARP-1) 3The abbreviations used are: PARPpoly(ADP-ribose) polymerasePARpolymers of ADP-riboseDBDDNA-binding domainBRCTBRCA1 C terminusTCEPTris[2-carboxyethyl] phosphineSeMetselenomethionineSADsingle-wavelength anomalous dispersion. is a chromatin-associated enzyme involved in multiple cellular processes including DNA repair, cell cycle control, apoptotic signaling, and transcriptional regulation (1D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar, 2Schreiber V. Dantzer F. Ame J.C. de Murcia G. Nat. Rev. Mol. Cell. Biol. 2006; 7: 517-528Crossref PubMed Scopus (1589) Google Scholar, 3Kim M.Y. Zhang T. Kraus W.L. Genes Dev. 2005; 19: 1951-1967Crossref PubMed Scopus (660) Google Scholar). Using NAD+ as a precursor, PARP-1 creates polymers of ADP-ribose (PAR) that can have long and branched structures with up to 200 units (4Alvarez-Gonzalez R. Jacobson M.K. Biochemistry. 1987; 26: 3218-3224Crossref PubMed Scopus (188) Google Scholar, 5Kawaichi M. Ueda K. Hayaishi O. J. Biol. Chem. 1981; 256: 9483-9489Abstract Full Text PDF PubMed Google Scholar). PAR synthesis is initiated on glutamate residues of target proteins, and subsequent polymerization of ADP-ribose units extends from the site of initiation. As a post-translational modification, PAR substantially changes the biophysical and electrostatic properties of a protein and can alter the DNA binding functions, the protein interaction properties, and the cellular location of target proteins (2Schreiber V. Dantzer F. Ame J.C. de Murcia G. Nat. Rev. Mol. Cell. Biol. 2006; 7: 517-528Crossref PubMed Scopus (1589) Google Scholar, 6Kanai M. Hanashiro K. Kim S.H. Hanai S. Boulares A.H. Miwa M. Fukasawa K. Nat. Cell Biol. 2007; 9: 1175-1183Crossref PubMed Scopus (165) Google Scholar). PAR is also a signaling molecule that can initiate a caspase-independent cell death program (7Andrabi S.A. Kim N.S. Yu S.W. Wang H. Koh D.W. Sasaki M. Klaus J.A. Otsuka T. Zhang Z. Koehler R.C. Hurn P.D. Poirier G.G. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 18308-18313Crossref PubMed Scopus (520) Google Scholar). Humans have as many as 18 PARP enzymes, but PARP-1 is the most abundant and active member of the PARP family (8Ame J.C. Spenlehauer C. de Murcia G. Bioessays. 2004; 26: 882-893Crossref PubMed Scopus (1239) Google Scholar). poly(ADP-ribose) polymerase polymers of ADP-ribose DNA-binding domain BRCA1 C terminus Tris[2-carboxyethyl] phosphine selenomethionine single-wavelength anomalous dispersion. Although PARP-1 modifies several nuclear proteins, the major in vivo target is PARP-1 itself (automodification activity) (1D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar). Automodification modulates the cellular activities of PARP-1 (1D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar, 2Schreiber V. Dantzer F. Ame J.C. de Murcia G. Nat. Rev. Mol. Cell. Biol. 2006; 7: 517-528Crossref PubMed Scopus (1589) Google Scholar, 3Kim M.Y. Zhang T. Kraus W.L. Genes Dev. 2005; 19: 1951-1967Crossref PubMed Scopus (660) Google Scholar). For example, PARP-1 has a structural role in promoting the compaction of chromatin to repress transcription (9Kim M.Y. Mauro S. Gevry N. Lis J.T. Kraus W.L. Cell. 2004; 119: 803-814Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). Automodification of PARP-1 releases the enzyme from chromatin, allowing transcriptional machinery to access DNA and thereby controlling gene expression (9Kim M.Y. Mauro S. Gevry N. Lis J.T. Kraus W.L. Cell. 2004; 119: 803-814Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). PARP-1 also plays a role in DNA damage repair. DNA strand breaks stimulate PARP-1 automodification, and activated PARP-1 recruits DNA repair factors to the site of DNA damage to facilitate repair (10Masson M. Niedergang C. Schreiber V. Muller S. Menissier-de Murcia J. de Murcia G. Mol. Cell. Biol. 1998; 18: 3563-3571Crossref PubMed Scopus (833) Google Scholar, 11El-Khamisy S.F. Masutani M. Suzuki H. Caldecott K.W. Nucleic Acids Res. 2003; 31: 5526-5533Crossref PubMed Scopus (527) Google Scholar). There are several factors that control PARP-1 activity, including self-association, interaction with histones and nucleosomes, NAD+ concentrations, structure-specific binding to DNA, and automodification (1D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar, 2Schreiber V. Dantzer F. Ame J.C. de Murcia G. Nat. Rev. Mol. Cell. Biol. 2006; 7: 517-528Crossref PubMed Scopus (1589) Google Scholar, 3Kim M.Y. Zhang T. Kraus W.L. Genes Dev. 2005; 19: 1951-1967Crossref PubMed Scopus (660) Google Scholar, 12Bauer P.I. Buki K.G. Hakam A. Kun E. Biochem. J. 1990; 270: 17-26Crossref PubMed Scopus (58) Google Scholar). However, there are few molecular level insights into mechanisms that control PARP-1 activity. PARP-1 is 113 kDa (in human) and has a modular architecture composed of multiple, independently folded domains (Fig. 1A). The PARP-1 polypeptide is generally described in three major segments that represent the biochemical activities and functional roles of the enzyme: the DNA-binding domain (DBD; residues 1–374), the automodification domain (residues 375–525), and the catalytic domain (residues 526–1014). The catalytic domain of PARP-1 is located at the C-terminal end of the protein. It is highly conserved in the PARP superfamily, particularly in a region called the PARP signature that is responsible for binding NAD+ (8Ame J.C. Spenlehauer C. de Murcia G. Bioessays. 2004; 26: 882-893Crossref PubMed Scopus (1239) Google Scholar, 13Ruf A. Mennissier de Murcia J. de Murcia G. Schulz G.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7481-7485Crossref PubMed Scopus (222) Google Scholar). The automodification domain has a BRCT fold (BRCA1 C terminus). This fold is present in several DNA repair factors and is frequently found to mediate protein-protein interactions (14Bork P. Hofmann K. Bucher P. Neuwald A.F. Altschul S.F. Koonin E.V. FASEB J. 1997; 11: 68-76Crossref PubMed Scopus (660) Google Scholar). Indeed, the BRCT domain of PARP-1 is responsible for the protein-protein interaction that recruits DNA repair enzymes during strand break repair (10Masson M. Niedergang C. Schreiber V. Muller S. Menissier-de Murcia J. de Murcia G. Mol. Cell. Biol. 1998; 18: 3563-3571Crossref PubMed Scopus (833) Google Scholar, 11El-Khamisy S.F. Masutani M. Suzuki H. Caldecott K.W. Nucleic Acids Res. 2003; 31: 5526-5533Crossref PubMed Scopus (527) Google Scholar). The DNA-binding domain is located at the N terminus of PARP-1. The DBD contains two zinc fingers that bind to various DNA structures (15Rolli V. Ruf A. Augustin A. Schulz G.E. Ménissier-de Murcia J. de Murcia G. de Murcia G. Shall S. From DNA Damage and Stress Signalling to Cell Death: Poly ADP-ribosylation Reactions. Oxford University Press, New York2000: 35-79Google Scholar), a nuclear localization signal (16Schreiber V. Molinete M. Boeuf H. de Murcia G. Menissier-de Murcia J. EMBO J. 1992; 11: 3263-3269Crossref PubMed Scopus (97) Google Scholar), and a caspase-3 cleavage site (17Kaufmann S.H. Desnoyers S. Ottaviano Y. Davidson N.E. Poirier G.G. Cancer Res. 1993; 53: 3976-3985PubMed Google Scholar). NMR and x-ray models have structurally defined each of the independent domains of PARP-1 (Fig. 1A), except for the C-terminal region of the DBD, between the first two zinc fingers and the BRCT domain (Fig. 1B). We therefore undertook a structural analysis of this region to understand its role in PARP-1 function and to build upon our understanding of the multidomain structure of PARP-1 and how these domains assemble into an active DNA-dependent enzyme. The two N-terminal zinc fingers of PARP-1 bind to DNA structures to trigger activation of the C-terminal catalytic domain of PARP-1 (18Gradwohl G. Menissier de Murcia J.M. Molinete M. Simonin F. Koken M. Hoeijmakers J.H. de Murcia G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2990-2994Crossref PubMed Scopus (228) Google Scholar, 19Ikejima M. Noguchi S. Yamashita R. Ogura T. Sugimura T. Gill D.M. Miwa M. J. Biol. Chem. 1990; 265: 21907-21913Abstract Full Text PDF PubMed Google Scholar). The molecular mechanism for coupling the DNA binding and catalytic functions of PARP-1 remains an open question. Based on our structural and biochemical analysis, we show that the DBD of human PARP-1 contains yet a third zinc-binding domain. One function of the new zinc-binding domain is to couple the DNA binding and catalytic activities of PARP-1. Gene Cloning and Mutagenesis–The gene coding for full-length human PARP-1 (residues 1–1014) and for PARP-1 residues 1–234 were placed in the pET28 expression vector (Novagen) using restriction sites NdeI/XhoI such that full-length PARP-1 (PARP-1wt) and PARP-11–234 were each produced with an N-terminal hexahistidine tag and associated linker. The portions of the PARP-1 gene coding for residues 216–366, residues 1–366, and residues 379–1014 were placed in the pET24 expression vector (Novagen) using restriction sites NdeI/XhoI such that the protein fragments PARP-1216–366, PARP-11–366, and PARP-1379–1014 were each produced with a C-terminal hexahistine tag and associated linker. Mutagenesis was performed using the QuikChange Protocol (Stratagene). Protein Expression and Purification–PARP-1wt, PARP-1216–366, and PARP-11–366 were expressed in Escherichia coli strain BL21(DE3)RIPL (Stratagene). PARP-1C298A, PARP-11–234, and PARP-1379–1014 were expressed in E. coli strain BL21(DE3)Rosetta2 (Novagen). The cells were grown in LB medium containing 100 μm ZnCl2 and induced with 200 μm isopropyl β-d-thiogalactopyranoside at 16 °C for 20 h. The cells were resuspended in 20 mm Hepes, pH 8.0, 500 mm NaCl, 0.5 mm Tris[2-carboxyethyl] phosphine (TCEP), 0.1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride (and protease inhibitors) and lysed using a cell disrupter (Avestin). Cell debris were pelleted for 2 h at 40,000 × g, and the supernatant was then loaded onto a 5-ml HP chelating column (GE Healthcare) charged with Ni(II) and pre-equilibrated in lysis buffer without Nonidet P-40. The column was washed in lysis buffer containing 20 mm imidazole and eluted with 400 mm imidazole. The proteins were then loaded onto a 5-ml HP heparin column (GE Healthcare), except for PARP-1379–1014 (see below). For full-length PARP-1 constructs and the PARP-11–366 and PARP-11–234 fragments, the heparin column was equilibrated at 50 mm Tris-HCl, pH 7.0, 150 mm NaCl, 0.1 mm TCEP, and 1 mm EDTA and eluted with a gradient from 150 mm to 1 m NaCl. For PARP-1216–366, the heparin column was equilibrated with only 50 mm NaCl, and the elution gradient was 50 mm to 500 mm NaCl. PARP-1379–1014 was loaded onto a 5-ml HP Q column (GE Healthcare) equilibrated in 50 mm Tris-HCl, pH 7.0, 50 mm NaCl, 0.1 mm TCEP, and 1 mm EDTA and eluted with a gradient from 50 mm to 1 m NaCl. The proteins were next passed over a Sephacryl S200 gel filtration column (GE Healthcare) in 20 mm Hepes, pH 8.0, 150 mm NaCl, 0.1 mm TCEP, and 0.1 mm EDTA. Full-length PARP-1wt did not require the gel filtration purification step. Proteolytic degradation removed a small portion of the N terminus of PARP-11–366 and the C terminus of PARP-11–234 during protein purification. The degradation is noticed as a doublet of bands on SDS-PAGE. For Co(II)-substituted PARP-1216–366 (Co(II)-PARP-1216–366), E. coli were grown in minimal medium supplemented with 30 μm CoCl2. For selenomethionine (SeMet)-containing PARP-1216–366, BL21(DE3)RIPL E. coli were grown in defined medium (20Van Duyne G.D. Standaert R.F. Karplus P.A. Schreiber S.L. Clardy J. J. Mol. Biol. 1993; 229: 105-124Crossref PubMed Scopus (1091) Google Scholar). The purification protocol for Co(II)- and SeMet-PARP-1216–366 was as described for native PARP-1216–366. Atomic Absorption and Amino Acid Composition Analysis–Atomic absorption analysis of zinc content was performed at Galbraith Laboratories, Inc. (Knoxville, TN). Amino acid composition analysis was performed at the Molecular Biology Core Facilities of The Dana Farber Cancer Institute (Boston, MA). Absorbance Spectra of PARP-1216–366 and Co(II)-PARP-1216–366–Native PARP-1216–366 and Co(II)-PARP-1216–366 were diluted in 20 mm Tris, pH 8.0, and 100 mm NaCl to a final concentration of 35 μm. Absorbance spectra were recorded on a Shimadzu UV-2401PC spectrophotometer in the wavelength ranges of 800–500 and 500–250 nm. For metal competition experiments, 125 μm ZnCl2 or MgCl2 was added to 20 μm of Co(II)-PARP-1216–366. The mixture was incubated for 15 min and then rescanned from 500 to 250 nm. CD Spectroscopy–A JASCO J-810 spectropolarimeter was used to record CD spectra. Wavelength scans (280–200 nm) were performed at 4 °C in a quartz cuvette of 1-mm path length. The protein was at 5 μm in a buffer composed of 5 mm Na,KPO4, pH 7.5, 50 mm Na2SO4, and 0.1 mm TCEP. The final spectra represent the averages of three scans. The data were analyzed using the JFIT program (Dr. B. Rupp). Fluorescence Polarization DNA Binding Assay–A 42-base pair duplex DNA was assembled to contain the chicken cTnT promoter sequence from -102 to -59, including an MCAT-1 element (21Huang K. Tidyman W.E. Le K.U. Kirsten E. Kun E. Ordahl C.P. Biochemistry. 2004; 43: 217-223Crossref PubMed Scopus (35) Google Scholar). The MCAT DNA probe carried a 5′ FAM on the bottom strand. The binding reactions were performed in 12 mm Hepes, pH 8.0, 60 mm KCl, 0.12 mm EDTA, 5.5 μm β-mercaptoethanol, 8 mm MgCl2, 0.05 mg/ml bovine serum albumin, 4% glycerol. The reactions contained 5 nm DNA probe and various concentrations of protein. The reactions were incubated at room temperature (22 °C) for 30 min and then at 4 °C for 90 min. Fluorescence polarization data were collected on a Polarstar Optima microplate reader (BMG Labtech). The observed binding constant (KD) was obtained by fitting the data to a two-state binding model. DNA-dependent Automodification Assay–The automodification reactions were performed at room temperature in 20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 7.5 mm MgCl2, and 0.2 mm TCEP. PARP-1wt or PARP-1C298A (0.62 μm) was first preincubated with 1 μm nonfluorescent MCAT DNA (same sequence as referenced above (21Huang K. Tidyman W.E. Le K.U. Kirsten E. Kun E. Ordahl C.P. Biochemistry. 2004; 43: 217-223Crossref PubMed Scopus (35) Google Scholar)) for 20 min. 5 mm NAD+ was then added to the reaction, and the mixture was incubated for various times. Under these conditions, automodification results in a visible shift in the electrophoretic mobility of PARP-1 on SDS-PAGE (22Mendoza-Alvarez H. Alvarez-Gonzalez R. J. Biol. Chem. 1993; 268: 22575-22580Abstract Full Text PDF PubMed Google Scholar). For the complementation assay, PARP-1379–1014 (0.93 μm) was incubated with a 1:1 molar ratio (0.93 μm) or 2:1 molar ratio (1.85 μm) of PARP-11–366, or PARP-11–234. The reactions were incubated for 10 min, after the addition of DNA and NAD+ as specified above. In each of the experiments described above, the reactions were stopped by the addition of SDS-loading buffer containing 0.1 m EDTA. The samples were resolved on a 7.5% SDS-PAGE, and the gel was treated with Imperial protein stain (Pierce). Crystallization and Structure Determination of PARP-1216–366–Native and SeMet crystals of PARP-1216–366 were grown in sitting drops at 4 °C. The protein at 30–60 mg/ml was mixed with an equal volume of 20% ethanol and 100 mm Tris-HCl pH 8.5. Before flash-cooling in liquid nitrogen, the crystals were briefly (<1 min) transferred to 15% propanediol, 25% glycerol, 50 mm Tris-HCl, pH 8.5, 25 mm NaCl, and 0.5 mm TCEP. X-ray diffraction data were collected at Beamline X-12C at the National Synchrotron Light Source (Brookhaven National Laboratory, Upton, NY). X-ray data were collected at two wavelengths for both native and SeMet-PARP-1216–366 crystals, and the data were processed using HKL2000 (23Otwinowski Z. Minor W. Carter Jr., C.W. Sweet R.M. Methods in Enzymology. 276. Academic Press, New York1997: 307-326Google Scholar) (Table 1). Single-wavelength anomalous dispersion (SAD) experimental phases were calculated in SOLVE/RESOLVE (24Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar) using the data collected at the zinc and at the selenium absorption edge for SeMet-PARP-1216–366 crystals. The model was primarily built into experimental electron density maps calculated with phases from the zinc SAD experiment (supplemental Fig. S1). The SeMet SAD data were mostly used to locate methionine residues to confirm the register of the amino acid sequence. Even so, electron density maps calculated with SeMet SAD phases were of good quality and helped confirm difficult areas of the map. The model was constructed using the molecular graphics program COOT (25Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar), partial models output by RESOLVE (24Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar), and electron density maps constructed in ARP/wARP (26Morris R.J. Perrakis A. Lamzin V.S. Carter Jr., C.W. Sweet R.M. Methods in Enzymology. 374. Academic Press, New York2003: 229-244Google Scholar) using partially built models. The atomic model was refined using REFMAC (27Collaborative Computational Project N.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar, 28Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) (Table 1). The final model includes residues 225–359, one zinc atom, 160 water molecules, one ethanol molecule, and three glycerol molecules. N-terminal residues 216–224 and C-terminal residues 360–366 were poorly represented in electron density maps and therefore were excluded from the model. The side chain atoms of residues 225–228, 233, and 337 were not clearly represented in electron density maps; therefore the side chains of these residues were truncated after the β-carbon. The illustrations were made using Pymol (DeLano Scientific), Photoshop (Adobe Systems), and Illustrator (Adobe Systems).TABLE 1Crystallographic data collection and refinement statisticsData collectionaThe values in parentheses refer to data in the highest resolution shellBeamline X-12C (NSLS) Space groupC2221 (a = 71.8 Å, b = 85.7 Å, c = 67.8 Å, α = β = γ = 90°), 1 molecule/asymmetric unit CrystalNative PARP-1216–366SeMet-PARP-1216–366 Wavelength (Å)1.11.28 (Zn edge)1.28 (Zn edge)0.98 (Se edge) Resolution range (Å)40-1.7 (1.76-1.7)40-1.9 (1.97-1.9)40-2.5 (2.59-2.5)40-2.2 (2.28-2.2) Completeness (%)98.3 (84.6)98.9 (91.4)99.9 (100)99.8 (98.7) Redundancy8.0 (5.6)3.0 (2.5)3.7 (3.5)3.4 (3.2) Mean I/σ(I)34.8 (3.6)29.5 (3.5)25.9 (7.1)23.9 (4.5) Rmerge (%)bRmerge = ΣhklΣj|Ij – 〈I〉|/ΣhklΣjIj, where 〈I〉 is the mean intensity of j observations of reflection hkl and its symmetry equivalents4.8 (38.0)3.5 (23.4)4.3 (17.6)4.8 (25.2) FOM, SOLVE/RESOLVEcFigure of merit as reported in SOLVE/RESOLVE (24)0.367/0.700.43/0.75Model refinementaThe values in parentheses refer to data in the highest resolution shell Resolution Range (Å)20-1.7 (1.74-1.7) Number of reflections21,771 (1,311) RcrystdRcryst = Σhkl|Fobs – kFcalc|/Σhkl|Fobs|. Rfree = Rcryst for 5% of reflections excluded from crystallographic refinement0.181 (0.222) RfreedRcryst = Σhkl|Fobs – kFcalc|/Σhkl|Fobs|. Rfree = Rcryst for 5% of reflections excluded from crystallographic refinement0.230 (0.245) Number of atoms/average B-factor (Å2)1257/39.2Protein1064/39.3Zinc1/35.6Solvent192/44.7 Ramachandran, most favored (%)100 Root mean square deviationBond angles (deg.)1.46Bond lengths (Å)0.015a The values in parentheses refer to data in the highest resolution shellb Rmerge = ΣhklΣj|Ij – 〈I〉|/ΣhklΣjIj, where 〈I〉 is the mean intensity of j observations of reflection hkl and its symmetry equivalentsc Figure of merit as reported in SOLVE/RESOLVE (24Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar)d Rcryst = Σhkl|Fobs – kFcalc|/Σhkl|Fobs|. Rfree = Rcryst for 5% of reflections excluded from crystallographic refinement Open table in a new tab PARP-1216–366 Is a Zinc-binding Domain–We were interested in determining the functional role of the C-terminal region of the PARP-1 DBD, that is, the portion of the PARP-1 polypeptide between the N-terminal zinc fingers and the central BRCT domain (residues 200–400) (Fig. 1). Several PARP-1 fragments were produced that sampled potential domain boundaries at the N-terminal and C-terminal ends of this region. Limited proteolysis of the PARP-1 fragments and mass spectrometry of the proteolytic products were used to determine the minimal boundaries of a domain that was resistant to further proteolysis (data not shown). A PARP-1 fragment resulting from this analysis included residues 216–366 of PARP-1 with a C-terminal hexahistidine affinity tag, PARP-1216–366. CD analysis of PARP-1216–366 showed that this fragment of PARP-1 had secondary structure elements (peaks at 220 and 210 nm) (Fig. 2A, Native curve) (29Gratzer W.B. Cowburn D.A. Nature. 1969; 222: 426-431Crossref PubMed Scopus (76) Google Scholar) that underwent similar cooperative transitions during thermal melts (data not shown), consistent with the denaturation of a folded protein domain. The CD spectrum was analyzed using JFIT, which predicted that the domain is composed of α-helices and coils (52.1 and 47.9%, respectively; R factor: 16.5%). The N terminus of PARP-1216–366 was sensitive to trypsin digestion because of a patch of Lys residues between residues 221 and 226; therefore the true boundary of this domain of PARP-1 is likely to be after this Lys-rich region. However, other fragments that sampled the N-terminal boundary gave the same CD profile. Further structural analysis focused on the PARP-1216–366 fragment of PARP-1. There are no strong amino acid sequence homologs for PARP-1216–366 that can be identified through comparison with structure databases. Through visual inspection of the multiple sequence alignment of PARP-1 homologs (Fig. 1B), we noted four strictly conserved Cys residues (Cys295, Cys298, Cys311, and Cys321, in humans) within the PARP-1216–366 domain that had spacing reminiscent of the zinc finger protein fold (30Krishna S.S. Majumdar I. Grishin N.V. Nucleic Acids Res. 2003; 31: 532-550Crossref PubMed Scopus (659) Google Scholar). The spacing between the four Cys residues is strongly conserved among all organisms, following the pattern CX2CX11/12CX9C where C is cysteine and Xn is the number of amino acids between the Cys residues. We speculated that PARP-1216–366 might bind zinc as part of the structure of this domain. Atomic absorption analysis indicated that zinc co-purified with PARP-1216–366 through three chromatographic steps and extensive dialysis. Based on this analysis, there were 1.5 mol of zinc/mol of protein, whereas no zinc was detected in dialysis buffer. We therefore concluded that PARP-1216–366 binds at least one molecule of zinc. Co(II)-PARP-1216–366 was produced to gain more insight into the metal binding properties of the new zinc-binding domain. Co(II) can frequently substitute for zinc-binding sites, and the spectroscopic properties of a protein-Co(II) complex can provide structural insights into a metal-binding protein (31Jakob U. Eser M. Bardwell J.C. J. Biol. Chem. 2000; 275: 38302-38310Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 32Matthews J.M. Kowalski K. Liew C.K. Sharpe B.K. Fox A.H. Crossley M. MacKay J.P. Eur J Biochem. 2000; 267: 1030-1038Crossref PubMed Scopus (53) Google Scholar). The absorbance spectra of native PARP-1216–366 and Co(II)-PARP-1216–366 (both at 35 μm) were analyzed from 800 to 500 nm (Fig. 2B) and from 450 to 280 nm (Fig. 2C). The absorbance peak at 340 nm for Co(II)-PARP-1216–366 is consistent with a charge transfer between a thiol ligand and Co(II) (Fig. 2B). This absorbance peak is not present in native PARP-1216–366. The absorbance measurement at 340 nm estimated that there are four to six Co(II)-thiol bonds/molecule of PARP-1216–366 (A340 = 0.175; ϵ = 1300–900 m-1 cm-1 (33May S.W. Kuo J.Y. Biochemistry. 1978; 17: 3333-3338Crossref PubMed Scopus (90) Google Scholar)). Furthermore, the ligand field bands in the visible range of the absorbance spectrum (600–750 nm) (Fig. 2C) are characteristic of a tetrahedrally coordinated Co(II) (34Bertini I. Luchinat C. Adv. Inorg. Biochem. 1984; 6: 71-111PubMed Google Scholar), indicating that there are four ligands to Co(II). The absorbance at 685 nm estimated that there were 1.4 Co(II)/molecule of PARP-1216–366 (A685 = 0.038; ϵ = 780 m-1 cm-1). Collectively, the spectroscopic analysis of Co(II)-PARP-1216–366 indicated that there was one Co(II) coordinated by four Cys residues. The absorbance peak at 340 nm disappears with the addition of a 6-fold excess of Zn(II) to Co(II)-PARP-1216–366 (Fig. 2D). In contrast, the same concentration of Mg(II) did not change the absorbance spectrum between 450 and 300 nm (Fig. 2D, inset). This result indicated that PARP-1216–366 preferentially binds zinc and that Co(II) occupies the same metal-binding site as zinc. Importantly, the secondary structure of Co(II)-PARP-1216–366, before and after treatment with excess zinc, was observed to be the same as native PARP-1216–366 by CD analysis (Fig. 2A), indicating that Co(II)- and Zn(II)-bound PARP-1216–366 have the same overall fold. The Third Zinc-binding Domain Mediates Interdomain Contacts Important for PARP-1 Activation–Spectral analysis of the metal binding properties of PARP-1216–366 in solution strongly suggested that four Cys residues coordinated a zinc atom. The strictly conserved Cys residues (Cys295, Cys298, Cys311, and Cys321) were deemed the most likely zinc ligands. Each of these Cys residues was changed to Ala in PARP-1216–366 to test their potential roles as zinc-binding ligands and structural elements of the protein fold. However, each of these mutations made PARP-1216–366 difficult to produce in E. coli, and the extremely low yield of protein prevented further analysis of the mutants in this context. Wild-type PARP-1216–366 was abundantly produced in E. coli; therefore it is likely that the Cys to Ala mutations compromised the capacity of PARP-1216–366 to form a stable fold. The Cys to Ala point mutants were also created in full-length human PARP-1 to test the functional role of the third zinc-binding domain. Each of these single amino acid substitutions led to a drastic decrease in protein production in E. coli as compared with full-length PARP-1 (PARP-1wt), further suggesting that the conserved Cys residues are important for efficient" @default.
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• W2018329608 title "A Third Zinc-binding Domain of Human Poly(ADP-ribose) Polymerase-1 Coordinates DNA-dependent Enzyme Activation" @default.
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