Structure of the Dimeric Exonuclease TREX1 in Complex with DNA Displays a Proline-rich Binding Site for WW Domains
2007; Elsevier BV; Volume: 282; Issue: 19 Linguagem: Inglês
10.1074/jbc.m700236200
ISSN1083-351X
AutoresMarina Brucet, Jordi Querol‐Audí, Maria Paola Serra, Ximena Ramirez‐Espain, Kamila Bertlik, Lidia Ruíz, Jorge Lloberas, María J. Macías, Ignacio Fita, Antonio Celada,
Tópico(s)Ubiquitin and proteasome pathways
ResumoTREX1 is the most abundant mammalian 3′ → 5′ DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif "PPPVPRPP" does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Co-immunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought. TREX1 is the most abundant mammalian 3′ → 5′ DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif "PPPVPRPP" does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Co-immunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought. When DNA replicates, recombines, or is repaired, its ends often require remodeling to prevent the formation of aberrant structures, which might result in serious cell dysfunctions. Among these DNA remodeling enzymes the 3′ → 5′ exonucleases act by removing nucleotides at the 3′ termini and were initially found to perform proofreading functions associated with DNA polymerases. However, in the last few years, several eukaryotic 3′ → 5′ exonucleases with functions unrelated to proofreading activities have been reported. Alterations in the genes encoding these autonomous exonucleases lead to dramatic consequences, such as strong mutator phenotypes, premature aging, susceptibility to cancer, and even lack of viability (1Shevelev I.V. Ramadan K. Hubscher U. Scientific World J. 2002; 2: 275Crossref Scopus (20) Google Scholar). Despite unquestionable evidence about the in vivo importance of autonomous exonucleases, the molecular mechanisms and biological roles of these enzymes are only now beginning to be elucidated. First detected in mammalian liver and thymus extracts (2Lindahl T. Gally J.A. Edelman G.M. J. Biol. Chem. 1969; 244: 5014-5019Abstract Full Text PDF PubMed Google Scholar, 3Perrino F.W. Miller H. Ealey K.A. J. Biol. Chem. 1994; 269: 16357-16363Abstract Full Text PDF PubMed Google Scholar, 4Perrino F.W. Mazur D.J. Ward H. Harvey S. Cell Biochem. Biophys. 1999; 30: 331-352Crossref PubMed Google Scholar), TREX1 is the most abundant mammalian 3′ → 5′ exonuclease. The inactivation of the Trex1 gene in mice revealed the relevance of this exonuclease. Trex1 knock-out mice develop inflammatory myocarditis, resulting in progressive cardiomyopathy that leads to circulatory failure and a dramatic reduction in survival (5Morita M. Stamp G. Robins P. Dulic A. Rosewell I. Hrivnak G. Daly G. Lindahl T. Barnes D.E. Mol. Cell. Biol. 2004; 24: 6719-6727Crossref PubMed Scopus (292) Google Scholar). Recently, TREX1 has been implicated in the Aicardi-Goutières syndrome, a severe neurological brain disease that mimics a viral infection acquired in the uterus (6Crow Y.J. Hayward B.E. Parmar R. Robins P. Leitch A. Ali M. Black D.N. van Bokhoven H. Brunner H.G. Hamel B.C. Corry P.C. Cowan F.M. Frints S.G. Klepper J. Livingston J.H. Lynch S.A. Massey R.F. Meritet J.F. Michaud J.L. Ponsot G. Voit T. Lebon P. Bonthron D.T. Jackson A.P. Barnes D.E. Lindahl T. Nat. Genet. 2006; 38: 917-920Crossref PubMed Scopus (690) Google Scholar). TREX1, together with some members of the SET complex, has recently been implicated in DNA degradation during granzyme A-mediated cell death (7Chowdhury D. Beresford P.J. Zhu P. Zhang D. Sung J.S. Demple B. Perrino F.W. Lieberman J. Mol. Cell. 2006; 23: 133-142Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Given that TREX1 is an autonomous non-processive robust exonuclease, several biological roles have been proposed for this enzyme, including participation in proofreading functions. In fact, in reconstituted systems with exonuclease-deficient DNA polymerases, TREX1 shows 3′-editing activity, in particular for (i) the nuclease-deficient replicative DNA polymerase α (3Perrino F.W. Miller H. Ealey K.A. J. Biol. Chem. 1994; 269: 16357-16363Abstract Full Text PDF PubMed Google Scholar), (ii) the repair DNA polymerase β (8Hoss M. Robins P. Naven T.J. Pappin D.J. Sgouros J. Lindahl T. EMBO J. 1999; 18: 3868-3875Crossref PubMed Scopus (149) Google Scholar), and (iii) the DNA lesion bypass polymerase η (9Bebenek K. Matsuda T. Masutani C. Hanaoka F. Kunkel T.A. J. Biol. Chem. 2001; 276: 2317-2320Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Similar activity has been observed for the closely related TREX2, which interacts with the exonuclease-deficient DNA polymerase δ, thereby increasing its fidelity under adverse conditions (1Shevelev I.V. Ramadan K. Hubscher U. Scientific World J. 2002; 2: 275Crossref Scopus (20) Google Scholar). On the basis of its sequence, TREX1 has been classified as a member of the 3′ → 5′ exonuclease family DnaQ. This family includes the proofreading exonuclease fragments Klenow of polymerase I and ɛ186 of polymerase III in Escherichia coli and is characterized by the presence of four non-contiguous acidic residues, three aspartates, and one glutamate, which play a crucial role by binding the two catalytic metal ions (10Derbyshire V. Pinsonneault J.K. Joyce C.M. Methods Enzymol. 1995; 262: 363-385Crossref PubMed Scopus (70) Google Scholar). A fifth residue, either a tyrosine or a histidine, completes the family catalytic motif (represented as DEDDy or DEDDh, respectively) distributed among three separate sequence segments named Exo I, II, and III (11Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 5110-5118Crossref PubMed Scopus (205) Google Scholar). The information available to date indicates that the structure is well preserved among DnaQ family members, although some have very low sequence similarities outside the Exo segments (11Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 5110-5118Crossref PubMed Scopus (205) Google Scholar, 12Zuo Y. Deutscher M.P. Nucleic Acids Res. 2001; 29: 1017-1026Crossref PubMed Scopus (408) Google Scholar). Members of the family are monomeric, except for TREX1 and TREX2 proteins, which are the only DnaQ deoxyribonucleases characterized as dimers (13Mazur D.J. Perrino F.W. J. Biol. Chem. 2001; 276: 17022-17029Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). TREX2 is the only known homologue of TREX1 in mammals (44% sequence identity). TREX-related proteins have been identified in a number of insects and in baculovirus (14Yang D.H. de Jong J.G. Makhmoudova A. Arif B.M. Krell P.J. J. Gen. Virol. 2004; 85: 3569-3573Crossref PubMed Scopus (12) Google Scholar) but no homologous genes have been found in yeast. During the submission of this article, a complementary work showing the structure of TREX1 has been reported providing the molecular basis for understanding the mutations that lead to Aicardi-Goutières syndrome (15de Silva U. Choudhury S. Bailey S.L. Harvey S. Perrino F.W. Hollis T. J. Biol. Chem. 2007; 10.1074/jbcM700039200Google Scholar). To better understand the relationship between the structure and function of TREX1, we determined the crystal structures of the binary complexes with a nucleotide product of the exonuclease reaction and with a single-stranded DNA substrate, which we identified using binding site selection experiments. By means of nuclear magnetic resonance experiments, we demonstrated that a group 2 WW domain can interact with a peptide selected from the polyproline-rich region in TREX1. In addition, using confocal microscopy, we also analyzed the role of the C-terminal region in regulating the subcellular localization of the TREX1 protein. To our knowledge, TREX1 interaction with its substrate (DNA) and with its product (single nucleotide) represents the only structures of these complexes described to date from a mammalian deoxyribonuclease of the DnaQ family. Protein Preparation—A murine Trex1 cDNA construct (residues 9-245) was cloned into the expression vector pETM-10 (pETM-10-Trex1). The protein was overexpressed in the E. coli strain Rosetta (DE3) (Novagen) by adding 1 mm isopropyl 1-thio-β-d-galactopyranoside at a A595 = 0.6 and culturing the cells overnight at 15 °C. Pelleted cells were resuspended in buffer containing 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm MgCl2, 10 mm imidazole, 1 mm dithiothreitol, and 8% glycerol. A cell lysate was obtained after mechanical lysis using a French Press. The protein was affinity purified from the supernatant of the cell lysate by nickel-nitrilotriacetic acid-agarose resin (Qiagen), with an elution buffer containing 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm MgCl2, 200 mm imidazole, 1 mm dithiothreitol, and 8% glycerol, and a second purification step was carried out by a Superdex-75 gel filtration column (GE Healthcare), in a Tris-based buffer (30 mm Tris-HCl, pH 8.0, 200 mm NaCl, 5 mm MgCl2, and 1 mm dithiothreitol). The purified protein was concentrated to 7 mg/ml in the same buffer using Centricon centrifugal filter units (Millipore). The cDNA for the mutant Trex1 H195A was produced from the pETM-10-TREX1 vector, using a PCR site-directed mutagenesis strategy with the QuikChange site-directed mutagenesis kit (Stratagene), and the mutant protein was purified by the same protocol. Crystallization, Data Collection, and Structure Refinement—Purified TREX1 protein solution was mixed with 2 mm dTMP, left overnight at 4 °C and used for crystallization. 1 μl of this solution was mixed with 1 μl of reservoir solution (22% PEG3350, 100 mm MES, 2The abbreviations used are: MES, 4-morpholineethanesulfonic acid; dTMP, deoxythimidine monophosphate; EGFP, enhanced green fluorescent protein; LRR3, leucine-rich repeat 3; MBP, maltose-binding protein; PPII, polyproline type II helix conformation; HPLC, high pressure liquid chromatography; SH3, Src homology domain 3. pH 6.0, 200 mm Li2SO4) and crystallization was carried out by the hanging drop vapor diffusion method at 20 °C. Single crystals were soaked for 24 h in the same crystallization solution, except that it contained 200 mm MgSO4 and 50 mm Li2SO4. Crystals were then flash-frozen with 30% ethylene glycol as cryoprotectant. For the complex with DNA, a 25-mer single-stranded oligonucleotide was used (5′-GCTAGGCAGGAACCCCTCCTCCCCT-3′, derived from a binding site selection technique). It was mixed with the purified protein at stoichiometric concentrations and incubated overnight at 4 °C. 1 μl of this preparation was mixed with 1 μl of reservoir solution (20% PEG2KMME, 100 mm imidazole, pH 6.5, 300 mm Li2SO4) and crystallized at 20 °C. These crystals were cross-linked with 25% glutaraldehyde for 30 min and then frozen with 28% glycerol as cryoprotectant. Diffraction data were collected at the European Synchrotron Radiation Facility (Grenoble, France), and processed using DENZO and SCALE-PACK (16Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38782) Google Scholar). A first model was obtained for TREX1-dTMP by molecular replacement from the structure of the TREX2 protein (17Perrino F.W. Harvey S. McMillin S. Hollis T. J. Biol. Chem. 2005; 280: 15212-15218Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and the model was completed and refined using the CCP4 package and manually refined with the graphic programs Turbo and Coot. Residues 166-174 were disordered and were therefore excluded from the model. The first model for TREX1-DNA was obtained by molecular replacement from the TREX1-dTMP structure, and the model was refined by the same method. The disordered loop in TREX1-DNA was partially resolved. Crystallographic data collection, processing, and refinement statistics are summarized in Table 1. Structure representations were made with Pymol, and electrostatic potential surfaces were calculated with GRASP.TABLE 1Data collection, structure solution, and refinement statisticsTREX1-nucleotideTREX1-DNAData collectionSpace groupP21P43212Cell parametersa, b, c (Aå)67.18, 81.46, 93.1380.76, 80.76, 171.22α, β, γ (°)90, 103.22, 9090, 90, 90Resolution limits (Aå)30.0-2.3525.0-3.5Multiplicity3.9 (3.9)6.9 (6.0)Number of unique reflections40,389 (4,047)6,708 (623)Completeness (%)98.2 (87.6)87.9 (83.6)Average 1/σ (I)10.0 (3.7)3.7 (1.1)Rsym (%)6.8 (23.2)15.6 (44.0)Crystallographic refinementResolution range (Aå)30.0-2.35 (2.41-2.35)24.85-3.5 (3.59-3.5)R factor20.33 (21.2)24.33 (29.6)Rfree factor25.46 (33.4)28.34 (35.7)Number of reflections38,687 (2,870)6,402 (321)Final model parametersNumber of monomers42Residues (each monomer)217218Hetero groups (each monomer)2 Magnesium ions 1 dTMP4-mer ssDNANumber of water molecules1870Average B-factor, protein (Aå2)19.996.2Average B-factor, water molecules (Aå2)25.6Root mean square deviationCovalent bond lengths (Aå)0.0100.005Bond angles (°)1.361.007Quiral centers (°)0.0740.050 Open table in a new tab NMR Experiments—A DNA fragment encoding the WW2 motif of mouse CA150 (corresponding to residues 442-479) was produced as described (18Macias M.J. Gervais V. Civera C. Oschkinat H. Nat. Struct. Biol. 2000; 7: 375-379Crossref PubMed Scopus (194) Google Scholar). This domain is the same as that of FBP28_WW2, and shares the same amino acid sequence in human and mouse. NMR samples of the WW2 domain had a concentration of ∼0.2 mm for structure determination and were dissolved in 100 mm sodium phosphate buffer, 20 mm NaCl, 1 mm NaN3, and 10% D2O at pH 6.0. For binding studies (or titrations) synthetic peptides corresponding to the prolinerich sequence of murine TREX1 (HPPPVPRPPRV) and human TREX1 (PPPTVPPPPRV) were synthesized in-house using the Fmoc (N-(9-fluorenyl)methoxycarbonyl) strategy with a rink-amide matrix (Novabiochem). The final peptides were cleaved with 95% trifluoroacetic acid and 5% H2O and then precipitated in cold ether. The crude material was purified by preparative HPLC using a C18 column to 90% purity (as characterized by HPLC-mass spectrometry). For the binding studies, we titrated the CA150_WW2 domain with increasing amounts of TREX1-derived peptides. Peptide was added to the 15N-labeled CA150_WW2 up to a molar peptide:protein ratio of ∼9:1. The NMR data corresponding to the titration were acquired on a Bruker DRX-800 NMR spectrometer at 285 K. Protein assignment was performed using published homonuclear data as the starting point. The same experiment was carried out with the human and murine TREX1 proline-rich peptides. Co-immunoprecipitation—Cell extracts were prepared as described (19Xaus J. Cardo M. Valledor A.F. Soler C. Lloberas J. Celada A. Immunity. 1999; 11: 103-113Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), mixed with pre-immune or CA150-specific antiserum and TrueBlot anti-rabbit Ig IP Beads (eBioscience) overnight at 4 °C. After three washes, Western blotting was performed with primary antibodies (CA150 or TREX1 specific serums) and secondary antibody (Rabbit ExactaCruz, Santa Cruz Biotechnology). Binding Site Selection—The binding site selection was made as described (20Tang W. Perry S.E. J. Biol. Chem. 2003; 278: 28154-28159Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Trex1 was cloned into pMal-C2 vector (Promega), and overexpressed in E. coli XL1Blue cells. After lysis, proteins were purified by an amylose resin (New England Biolabs). Maltose-binding protein (MBP) was prepared with the same protocol and used for controls. Randomized oligonucleotides (5′-CGACTCTAGAGGATCC(N)24GAATTCAAGCTTCACG-3′) were made double-stranded using Taq polymerase and [α-32P]dCTP. The probes (200,000 cpm) were used for electrophoretic mobility shift assay with 2-0.5 μg of MBP-TREX1. The purified DNA was PCR amplified with [α-32P]dCTP (primers: 5′-CGACTCTAGAGGATCC-3′, 5′-CGTGAAGCTTGAATTC-3′). The recovered DNA was used in 3 subsequent rounds and the affinity-selected oligonucleotides were cloned into a pCR2.1 vector (Invitrogen). Positive clones were sequenced, the sequences were aligned using DNAStar and the consensus sequences were derived after analysis. SYBR Green-based Exonuclease Activity Assay—Exonuclease reactions were performed in a 96-well plate. First, 10× the indicated double-stranded DNA oligonucleotide (50 μm) was incubated with 10× SYBR Green (Invitrogen), heated for 10 min at 95 °C, and left for 30 min at room temperature so as to anneal the DNA probe and allow the SYBR Green to incorporate into the DNA. For each well, the reaction mixture was prepared in a final volume of 14 μl containing 20 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 2 mm dithiothreitol, and 100 μg/ml bovine serum albumin and 1 ng of MBP-TREX1. 3 μl of the SYBR Green/DNA mixture was then added to the wall of each well and, after a spin of the plate, the reaction was real time followed with an ABI Prism 7700 sequence detection system (Applied Biosystems) (program: 25 °C for 5 min, 25 °C for 1 min for 90 times, and 4 °C for 2 min). TREX1 dilutions were prepared at 4 °C in 100 μg/ml bovine serum albumin. Each DNA probe was annealed with its complementary oligonucleotide by heating at 95 °C for 10 min. Subcellular Localization Experiments—A20 cells were permeabilized with 0,2% Triton X-100. After blocking with 3% bovine serum albumin in Tris-buffered saline, samples were incubated with TREX1 antiserum and secondary antibody (Cy3-Goat anti-rabbit IgG, Amersham Biosciences). DNA was labeled with 4′,6-diamidino-2-phenylindole (Sigma). Several Trex1 cDNA constructs were cloned into a pEGFP-N3 vector (Clontech) and transfected into the murine fibroblast cell line L929: pEGFP-Trex1 wt (residues 1-314), pEGFP-Trex1Δ1 (42-314), pEGFP-Trex1Δ12 (85-314), pEGFP-Trex1Δ123 (85-280), and pEGFP-Trex1Δ3 (1-280). Empty pEGFP-N3 was used as control (EGFP alone). 100,000 cells were plated into each well of a 24-well plate with each well containing a coverslip. After 24 h, cells were transfected with 1 μg of each construct using the transfection kit Lipofectamine™ 2000 (Invitrogen), following the manufacturer's instructions. Cells were incubated for 24 h more, fixed with 3% paraformaldehyde, and visualized by confocal microscopy. The Optimal DNA Sequences That Bound to TREX1 Correlate with the Exonuclease Activity—TREX1 was initially cloned by exploiting its capacity to recognize a given DNA motif (21Klemsz M.J. McKercher S.R. Celada A. Van Beveren C. Maki R.A. Cell. 1990; 61: 113-124Abstract Full Text PDF PubMed Scopus (868) Google Scholar). To further explore whether TREX1 displays DNA-binding specificity or selectivity toward certain motifs, we used the PCR binding site selection method (22Pollock R. Treisman R. Genes Dev. 1991; 5: 2327-2341Crossref PubMed Scopus (337) Google Scholar). We thus generated a pool of degenerated oligonucleotides and performed binding selection cycles with recombinant TREX1 protein. Three sets of oligonucleotide sequences were selected by the protein: in 26% of the cases the consensus showed GAG (sequence A), in 25% ACA (B), and in 18% CTGC (C). To examine the potential preference of TREX1 for these sequences, we performed binding assays by incubating the probes (oligonucleotides containing each of the three motifs in tandem) with increasing amounts of protein. Binding was detected by gel retardations. Under these experimental conditions, the affinity differed depending on the DNA sequence (Fig. 1A). Interestingly, when we replaced the Gly by Cys in motif A, binding was abolished. To correlate the binding with the functional activity of TREX1, we also performed a sensitive and quantitative exonuclease activity test, using SYBR Green as indicator. Detection was made by a real time PCR assay (Fig. 1B). We found a clear correlation between binding and exonuclease activity of TREX1. This observation indicates that the specificity of TREX1 for some sequences is correlated with its functional activity, a new feature in the field of deoxyribonucleases. Regions Responsible for TREX1 Nucleus-Cytoplasm Localization—TREX1 is involved in the SET complex that degrades DNA during granzyme A-mediated cell death. This observation implies an initial cytoplasmic localization for the protein and a stimulus-mediated translocation to the nucleus (7Chowdhury D. Beresford P.J. Zhu P. Zhang D. Sung J.S. Demple B. Perrino F.W. Lieberman J. Mol. Cell. 2006; 23: 133-142Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The observation that TREX1 binds proteins involved in nucleic acid polymerization (3Perrino F.W. Miller H. Ealey K.A. J. Biol. Chem. 1994; 269: 16357-16363Abstract Full Text PDF PubMed Google Scholar, 8Hoss M. Robins P. Naven T.J. Pappin D.J. Sgouros J. Lindahl T. EMBO J. 1999; 18: 3868-3875Crossref PubMed Scopus (149) Google Scholar, 9Bebenek K. Matsuda T. Masutani C. Hanaoka F. Kunkel T.A. J. Biol. Chem. 2001; 276: 2317-2320Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) suggests that TREX1 is involved in a range of activities in the cell nucleus. In fact, when we localized TREX1 using polyclonal antibodies we found that present in both the nucleus and cytoplasm (Fig. 2A). Both TREX proteins contain a pair of leucine-rich repeats at their N termini, which in TREX2 were incorporated in the protein fold. However, TREX1 contains a highly hydrophobic C-terminal region, absent in TREX2, which we named leucine-rich repeat 3 (LRR3). Indeed, bioinformatic tools predict that this region forms a putative transmembrane helix (supplemental Fig. S1). Furthermore, protein constructs lacking the LRR3 region were expressed in the soluble fractions and in higher amounts than the full-length protein. In addition, the TREX1 construct lacking the LRR3 segment maintained full catalytic capacity because it was able to bind and degrade DNA (supplemental Fig. S2). On the basis of these observations, we hypothesized that this region influences the subcellular localization of TREX1 in vivo. To test this hypothesis, cDNAs coding for distinct fragments of TREX1 were cloned in an expression vector fused to enhanced green fluorescent protein (EGFP). Four fragments were prepared, TREX1Δ1, TREX1Δ12, TREX1Δ123, and TREX1Δ3 (Fig. 2B). L929 fibroblasts were transfected with these constructs, and subcellular localization of the fusion proteins was visualized by confocal microscopy. Results showed that the entire TREX1 protein was located in the cytoplasm. The protein translocated to the nucleus when it lacked the LRR3 at the C terminus (TREX1Δ3 and TREX1Δ123), but not when it lacked any other region (TREX1Δ1 and TREX1Δ12). The pEGFP control (empty vector pEGFP-N3) showed the typical nuclear-cytoplasmic distribution for the EGFP protein. These results indicate that the LRR3 region is involved in the nucleus-cytoplasm localization of TREX1. Furthermore, TREX1Δ3 and TREX1Δ123 fused to two EGFPs presented the same nuclear localization pattern as when fused to a single EGFP. These observations discard the possibility that the nuclear localization was caused by passive transport (data not shown). Second, Trex1 wt cloned into a pEGFP-C1 vector, where TREX1 is at the C terminus of the EGFP, showed the same localization pattern as when cloned into pEGFP-N3, thereby demonstrating that the position of the EGFP does not determine the localization of the fusion protein (Data not shown). Thus our data showed that the presence of the C-terminal LRR3 region of TREX1 is involved in cytoplasmic retention of the protein and its elimination allows the protein to be translocated to the nucleus. On the basis of the data obtained from the solubility and localization experiments and also from the DNA selection technique, we designed a number of oligonucleotides and prepared several TREX1 constructs and mixtures with DNA, which were later used for crystallization trials. The mixtures contained either double-stranded or single-stranded DNAs corresponding to both the forward and reverse sequence of each oligonucleotide selected. In parallel, trials with 2 mm deoxythymidine 5′-monophosphate (dTMP) were also attempted. TREX1 Overall Structure—Despite a huge number of trials, only one construct lacking the LRR3 segment was crystallized. The protein crystals belong to space group P21, and the asymmetric unit contains four protein subunits organized as two molecular dimers, two magnesium ions, and a dTMP molecule per active center. This is consistent with the results obtained for the protein in solution (13Mazur D.J. Perrino F.W. J. Biol. Chem. 2001; 276: 17022-17029Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) (supplemental Fig. S3). The final model was refined at 2.35-Aå resolution (Table 1) and includes 190 solvent molecules. Both the global structure and the dimeric molecular organization of TREX1 are closely related to those in TREX2 (about 40% sequence identity) (17Perrino F.W. Harvey S. McMillin S. Hollis T. J. Biol. Chem. 2005; 280: 15212-15218Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Despite low sequence identity with respect to monomeric exonucleases of the DnaQ family, such as the Klenow fragment of E. coli DNA polymerase I (about 20%) (23Shevelev I.V. Hubscher U. Nat. Rev. Mol. Cell. Biol. 2002; 3: 364-376Crossref PubMed Scopus (216) Google Scholar), their overall fold is quite conserved. TREX subunits consist of a central five-stranded anti-parallel β-sheet surrounded by nine α-helices (Fig. 3A). The structures of the four subunits are very similar, with an averaged root mean square deviation between Cα atoms of 0.32 Aå. The sheet extends throughout the molecular 2-fold axis to the second subunit in the molecule, thereby giving a continuous 10-stranded anti-parallel sheet. Both polar and hydrophobic interactions act between monomers, mainly because of residues from the β3 strand and the α4 helix. TREX1 has a proline-rich segment (residues 54-63), which is absent in TREX2. This segment is located on the protein surface not far from the molecular 2-fold axis (closest distance 10.19 Aå, from Cα of Pro60) and adopts a polyproline type II helix conformation (PPII). There is a disordered segment in TREX1 adjacent to the active site (residues 166-174). The corresponding loop in TREX2, also disordered, appears to play an important role in DNA binding (17Perrino F.W. Harvey S. McMillin S. Hollis T. J. Biol. Chem. 2005; 280: 15212-15218Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Overall, the main differences between the two TREX structures are the additional helix α1 and a long loop, which, in TREX1, corresponds to residues 47-65 including the proline-rich segment (Fig. 3B). Active Site—The electron density map confirmed the binding to each protein subunit of one dTMP nucleotide and two magnesium ions (named MgA and MgB), defining an active site located away from the molecular 2-fold axis, in agreement with what was proposed for TREX2 (17Perrino F.W. Harvey S. McMillin S. Hollis T. J. Biol. Chem. 2005; 280: 15212-15218Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) (Fig. 3A). The dTMP molecule would correspond to the product of the exonuclease reaction that remains bound by hydrophobic and hydrogen-bond interactions with the protein and also by interactions with magnesium ions. MgA, with an approximate trigonal bipyramidal geometry, coordinates with oxygen molecules O2P and O3P from the dTMP phosphate group and with the carboxylate oxygen molecules from Asp18, Glu20, and Asp200. In turn, MgB, with nearly perfect octahedral geometry, coordinates with the dTMP oxygen O3P, with the second carboxylate oxygen from Asp18 and with four molecules of water, one of them hydrogen-bond
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