Structure of the GCM domain-DNA complex: a DNA-binding domain with a novel fold and mode of target site recognition
2003; Springer Nature; Volume: 22; Issue: 8 Linguagem: Inglês
10.1093/emboj/cdg182
ISSN1460-2075
Autores Tópico(s)RNA modifications and cancer
ResumoArticle15 April 2003free access Structure of the GCM domain–DNA complex: a DNA-binding domain with a novel fold and mode of target site recognition Serge X. Cohen Serge X. Cohen European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Martine Moulin Martine Moulin European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Said Hashemolhosseini Said Hashemolhosseini Institut für Biochemie, Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany Search for more papers by this author Karin Kilian Karin Kilian Institut für Biochemie, Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany Search for more papers by this author Michael Wegner Michael Wegner Institut für Biochemie, Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany Search for more papers by this author Christoph W. Müller Corresponding Author Christoph W. Müller European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Serge X. Cohen Serge X. Cohen European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Martine Moulin Martine Moulin European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Said Hashemolhosseini Said Hashemolhosseini Institut für Biochemie, Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany Search for more papers by this author Karin Kilian Karin Kilian Institut für Biochemie, Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany Search for more papers by this author Michael Wegner Michael Wegner Institut für Biochemie, Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany Search for more papers by this author Christoph W. Müller Corresponding Author Christoph W. Müller European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble, Cedex 9, France Search for more papers by this author Author Information Serge X. Cohen1, Martine Moulin1, Said Hashemolhosseini2, Karin Kilian2, Michael Wegner2 and Christoph W. Müller 1 1European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble, Cedex 9, France 2Institut für Biochemie, Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:1835-1845https://doi.org/10.1093/emboj/cdg182 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Glia cell missing (GCM) transcription factors form a small family of transcriptional regulators in metazoans. The prototypical Drosophila GCM protein directs the differentiation of neuron precursor cells into glia cells, whereas mammalian GCM proteins are involved in placenta and parathyroid development. GCM proteins share a highly conserved 150 amino acid residue region responsible for DNA binding, known as the GCM domain. Here we present the crystal structure of the GCM domain from murine GCMa bound to its octameric DNA target site at 2.85 Å resolution. The GCM domain exhibits a novel fold consisting of two domains tethered together by one of two structural Zn ions. We observe the novel use of a β-sheet in DNA recognition, whereby a five- stranded β-sheet protrudes into the major groove perpendicular to the DNA axis. The structure combined with mutational analysis of the target site and of DNA-contacting residues provides insight into DNA recognition by this new type of Zn-containing DNA-binding domain. Introduction GCM proteins form a small family of transcriptional regulators involved in fundamental developmental processes (Wegner and Riethmacher, 2001; Van de Bor and Giangrande, 2002). In Drosophila, where it was first identified, GCM directs the development of neuronal precursor cells into glial cells, acting as a master regulator of gliogenesis (Hosoya et al., 1995; Jones et al., 1995; Vincent et al., 1996). In contrast, neither of the two GCM homologs present in mammals appears to be involved in gliogenesis. Instead, GCMa regulates labyrinth formation in the developing placenta (Anson-Cartwright et al., 2000; Schreiber et al., 2000), while GCMb is involved in the development of the parathyroid gland (Gunther et al., 2000). Accordingly, inactivation of these genes leads to placental malfunction or parathyroid loss and hypoparathyroidism, respectively (Ding et al., 2001; Wegner and Riethmacher, 2001). GCM homologs have also been identified in fish and sea urchins (Figure 1A), but no homologs have yet been detected in the sequenced genomes of fungi (Saccharomyces cerevisiae), plants (Arabidopsis thaliana) or nematodes (Caenorhabditis elegans). Figure 1.(A) Alignment of the GCM domains from mouse (mGCMa, mGCMb), Drosophila melanogaster (dGCM, dGlide2), sea urchin (spGCM) (Ransick et al., 2002) and the pufferfish fugu (fuGCM). Conserved residues and conservatively substituted residues are drawn on a yellow background. Secondary structure elements are shown above the mGCMa sequence. Regions indicated by broken lines are disordered and have not been included in the final model. Magenta dots indicate DNA-contacting residues; light green and dark green triangles indicate residues coordinating the first and second Zn ions, respectively. (B) Sequence of the 13mer DNA duplex present in crystal forms A and A′. The octameric target site is numbered from 1 to 8 (1′ to 8′ for the opposite strand) and boxed. Flanking base pairs upstream and downstream of the target site are numbered −1 to 0 and 9 to 11, respectively. (C) Stereo diagram of the final 2Fo − Fc electron density map contoured at 1.5σ. Strands S2 and S3 and the contacted DNA target site are shown. The figure was produced using the program BOBSCRIPT (Esnouf, 1999). Download figure Download PowerPoint GCM transcription factors consist of ∼500 amino acid residues. The N-terminal moiety contains a DNA-binding domain of ∼150 residues. Sequence conservation is highest in this so-called GCM domain (Figure 1A). In contrast, the C-terminal moiety contains one or two transactivating regions and is only poorly conserved. In murine GCMb, an inhibitory region located between the two transactivating regions leads to decreased stability and lower transcriptional activity compared with other GCM transcription factors (Tuerk et al., 2000). GCM proteins bind their target sites as monomers. DNA selection experiments identified an 8 bp motif, 5′-ATGCGGGT-3′, as the optimal sequence; this is present with slight variations or in conserved form in potential target genes (Akiyama et al., 1996; Schreiber et al., 1997). As expected from their high degree of sequence similarity, the DNA-binding characteristics of different GCM homologs are very similar. Alanine mutations have identified a number of residues with critical roles in DNA recognition and stabilization of the GCM domain (Schreiber et al., 1998). Sequence conservation also indicated the importance of several conserved cysteine and histidine residues. EXAFS and microPixe analyses have demonstrated that most of these residues are involved in ligating two Zn ions required for the stability of the GCM domain (Cohen et al., 2002). A detailed structural and functional analysis of the GCM domain has been hampered by the lack of a crystallographic structure. Here we present the crystal structure of the GCM domain of murine GCMa bound to a 13 bp DNA duplex containing its octameric target site (Figure 1B) at 2.85 Å resolution. Our results identify the GCM domain as a new class of Zn-containing DNA-binding domain with no similarity to any other DNA-binding domain. The GCM domain consists of a large and a small domain tethered together by one of the two Zn ions present in the structure (Figure 2). The large and the small domains comprise five- and three-stranded β-sheets, respectively, with three small helical segments packed against the same side of the two β-sheets. The GCM domain exercises a novel mode of sequence-specific DNA recognition, where the five-stranded β-pleated sheet inserts into the major groove of the DNA. Residues protruding from the edge strand of the β-pleated sheet and the following loop and strand contact the bases and backbone of both DNA strands, providing specificity for its DNA target site. Figure 2.Structure of the GCM domain. (A) Ribbon representation of the GCM domain bound to its cognate DNA. The β-sheets of the large and small domains are depicted in dark blue and light blue, respectively. Helices H1, H2 and H3 are shown in red, and the DNA is shown in yellow. The two Zn ions and their coordinating ligands are depicted. Figures 2A and B, 3B, 4A and 6 were produced using the program RIBBONS (Carson, 1991). (B) View of the GCM domain with the DNA axis running vertically. DNA bases are numbered according to Figure 1B. (C) Topology diagram of the GCM domain. DNA-contacting residues and the first and second Zn ion coordinating residues are marked as dots. The color code corresponds to Figures 1A and 2A. Download figure Download PowerPoint Results and discussion Overall structure The GCM domain–DNA complex structure was solved by the multiple isomorphous replacement method using three iodinated DNA derivatives (Table I). The crystal contains one complex in the asymmetric unit. The current model contains 153 amino acid residues, 26 nucleotides, two Zn ions and four water molecules, and has been refined to a crystallographic R factor of 21.8% (Rfree = 28.3%) using all data from 20 to 2.85 Å. The final 2Fo − Fc electron density is well defined for the DNA, the polypeptide main chain and most of the protein side chains (Figure 1C). The highest mobility of the polypeptide chain is observed at the N- and C-terminal ends. N-terminal residues 1–13 are disordered and have not been included in the model. For the following residues 14–30 the main chain can be unambiguously followed but for most side chains the electron density is missing. The C-terminal residues 171–175 are also disordered. Table 1. Structure determination of the GCM domain–DNA complexa Dataset Crystal form A Crystal form A′ Nat-1 Nat-41 IU16 IU25 IU34 Processing statistics Resolution range (Å) 30–2.85 40.0–2.9 40–3.05 40–3.15 40–2.82 Wavelength (Å) 0.931 0.933 0.933 0.933 0.933 Completeness (%) 97.9 (95.6) 91.9 (78.4) 90.8 (72.7) 91.3 (78.8) 91.3 (74.6) Multiplicity 2.9 (2.4) 4.1 (3.6) 3.4 (2.7) 3.4 (2.9) 3.4 (2.7) Rmeas (%)b 4.9 (28.2) 5.6 (27.4) 8.6 (25.4) 7.5 (19.5) 7.4 (29.4) I/σ (I) > 3 79.2 (34.8) 81.8 (49.7) 79.2 (46.4) 82.0 (55.8) 79.9 (42.2) Phasing statistics No. of iodine atoms 3 2 3 Isomorphous difference (%) 19.4 15.1 21.7 Phasing power Isomorphous – 1.61 1.81 1.44 Anomalous 0.94 0.85 0.84 0.73 Refinement Resolution range (Å) 20–2.85 Rwork (%)c 21.8 (5714 reflections) Rfree (%)c 28.3 (516 reflections) Total no. of non-hydrogen atoms 1810 No. of protein atoms 1277 No. of DNA atoms 527 No. of water molecules 4 No. of Zn ions 2 R.m.s.ds Bond lengths (Å) 0.008 Bond angles (degrees) 1.31 a Nat-1 (crystal form A) was used for the final refinement, whereas dataset Nat-41 (crystal form A′) was used as native data for the MIRAS phasing. For Nat-41, the anomalous signal of the Zn ions was included in the heavy-atom parameter refinement. Values in the highest resolution shell are given in parentheses. b Rmeas = ΣhklΣi|Ii(hkl) − |/ΣhklΣiIi(hkl), where Ii is the ith measurement of reflection I(hkl). c Rfree was calculated using 8.1% of the data. No σ cut-off was applied to the data. The GCM domain has a roughly parallelepiped shape with dimensions of 60 × 30 × 30 Å. The longest dimension runs along the major groove at an angle of ∼45° with respect to the DNA axis (Figure 2B). The GCM domain can be divided into two domains. The large domain consists of an N-terminal extension, a five- stranded antiparallel β-sheet (strands S1, S2, S3, S6 and S7) and a short helix H1. Residues 31–39 of the N-terminal extension, helix H1 and the following linker residues 56–61 pack against the β-pleated sheet. Residues 31–39 and the linker residues 56–61 almost form the second layer of a β-barrel. However, only one main-chain hydrogen bond connects these two stretches of residues and therefore the β-barrel is only partially closed. The small domain contains a three-stranded β-pleated sheet (strands S3′, S4 and S5), helix H2 and the C-terminal helix H3. Helix H2 contains mostly polar residues and connects strand S4 with strand S5. A search for structurally similar proteins with the program DALI (Holm and Sander, 1993) did not find any high-scoring hits. The top hits matched the five-stranded β-sheet of the GCM domain with the seven-stranded β-sheet of bovine profilin (Cedergren-Zeppezauer et al., 1994) (Z score of 3.5) and with the six-stranded β-sheet formed by the C-terminal 100 residues of the mouse ap2 clathrin adaptor α-subunit (Traub et al., 1999) (Z score of 3.0). The overall similarities are low, as indicated by the Z scores, although the β-sheets in these two proteins share the same topology with the GCM domain, except for the insertion of the smaller domain between GCM domain strands S3 and S6 (Figure 2C). Despite the division of the GCM domain into two domains we do not consider them to form independent folding units. In fact, the two domains share a large hydrophobic interface and are probably unable to move independently with respect to each other. Furthermore, one of the two Zn coordination centers plays an important role in tethering the two domains together by coordinating Cys76, Cys125, His152 and His154. The residues following the two histidines fill a groove between the two domains and also contribute to connecting the two domains. DNA recognition Both domains of the GCM domain are involved in DNA recognition, forming a clamp that seizes the substrate from two sides of the major groove (Figure 2A). The β-sheet of the large domain forms the upper jaw of the clamp, with its strands oriented orthogonally to the DNA axis (Figure 2A and B). At the edge of this sheet, the β-hairpin formed by strands S2 and S3 constitutes the most important recognition element within the GCM domain. This hairpin inserts into the major groove and contacts four backbone phosphates (positions 3, 5, 6′ and 8′) and three bases (Cyt4, Gua6 and Gua7) (Figure 3). Polar backbone contacts are made by residues Arg62, Ser69, Lys73 and Lys74; the last two residues point their side chains in opposite directions, bridging across the entire major groove to contact phosphates Gua3 and Ade8′ from complementary DNA strands. In addition, Leu72 forms a hydrophobic contact with the deoxyribose of Gua3 (Figures 1C and 3). Base-specific contacts are mediated by residues Asn63, Asn65 and His67 from strand S2 and the loop following it. The side chain OD1 and ND2 atoms of Asn63 point towards the exocyclic N4 atom of Cyt4 and the N7 atom of Gua3, respectively. However, both interatomic distances exceed 3.3 Å, which is too much to form direct hydrogen bonds. The ND2 atom of Asn65 forms a hydrogen bond with the exocyclic O6 of Gua6, while its backbone carbonyl contacts the exocyclic N4 of Cyt7′ from the complementary DNA strand. The side chain NE2 atom of His67 forms a hydrogen bond with the O6 of Gua7. Figure 3.DNA recognition by the GCM domain. (A) Protein–DNA interactions between the GCM domain and its DNA target site. Arrows and dotted lines indicate polar and hydrophobic interactions, respectively. Residues involved in polar and hydrophobic interactions are drawn on blue and magenta backgrounds, respectively. (B) Ribbon representation of the interactions between the GCM domain and its DNA target site Upper and lower strands as shown in Figure 1B are depicted in yellow and orange, respectively. Broken lines indicate polar interactions. Download figure Download PowerPoint The lower jaw of the clamp is formed by helix H2 of the small domain. Within this helix, Lys107 contacts the phosphate group of Gua0, while at its N-terminus Ile100 and the backbone atoms of Cys101 form a hydrophobic barrier buttressing the exocyclic methyl group of Thy2. Cys101 is the only strongly conserved cysteine in the GCM domain that does not coordinate Zn (Figure 1A). Its sulfhydryl group points towards DNA bases Gua0 and Ade3, explaining mutagenesis results whereby Cys101 was shown to confer redox sensitivity to DNA binding (Schreiber et al., 1998). In addition to the two jaw regions, DNA binding also involves residues His55 and Lys160 from helices H1 and H3 and Phe131 in the linker between strands S5 and S6. His55 and Lys160 contact the phosphate groups of Gua3 and Thy2, respectively (Figure 3A), whereas Phe131 packs against the deoxyribose of Ade8′. Arg167 in helix H3 points towards the Gua0 phosphate. This is probably also an important contact, although in the crystal structure the Arg167 side chain is highly mobile and appears to be influenced by a phosphate group from a neighboring DNA strand in the crystal lattice. GCM domain residues contact both DNA strands, but it is worth noting that 12 residues contact one strand and only four residues (including Asn65) contact the other (Figure 3B). Almost all the DNA-contacting residues are conserved between different species (Figures 1A and 3). Subtle differences in the DNA-binding requirements of mGCMa and mGCMb (Tuerk et al., 2000) are probably not caused by differences in direct protein–DNA interactions but, rather, are indirect effects resulting from slight differences in the overall structure of both orthologs. Conformation of the DNA The overall conformation of the DNA in the GCM domain–DNA complex resembles B-form DNA, although its helical axis is highly distorted. These distortions consist of a central bend of ∼30° at bp 6 and two kinks of ∼25° between bp 2/3 and 7/8 (Figure 4A). These kinks direct the DNA axis in opposite directions, above and below the plane defined by the central bend. As a result the DNA axis has an S-like shape. Figure 4.DNA bending observed in the GCM domain–DNA complex. (A) Two orthogonal views of the 13mer DNA duplex in the GCM domain–DNA complex superimposed with canonical B-form DNA. Strands of the GCM-bound DNA are colored in blue. Helical axes were calculated using the program CURVES (Lavery and Sklenar, 1988). (B) The consensus GCM binding site (gbs) was inserted between the XbaI and SalI sites of pBEND2 (Kim et al., 1989) and retrieved with flanking sequences using the restriction enzymes BglII (1), XhoI (2), XmaI (3), Asp718 (4) and BamHI (5). This generates fragments of identical size with circular permutations of the same sequence and the GCM binding site at varying positions. (C) Circular permutation analyses of DNA bending by electrophoretic mobility shift assays with fragments 1–5 from (A) as probes and the GCM domains of Drosophila GCM (dGCM), mouse GCMa (GCMa) and mouse GCMb (GCMb) expressed in transiently transfected COS cells. (D) Calculation of bending angle for GCMa as described previously (Scaffidi and Bianchi, 2001). The mobility of the protein–DNA complexes (Rbound) was normalized to the mobility of the corresponding free probe (Rfree). The distance of the center of the GCM binding site from the 5′ end of the fragment was divided by the total length of the probe (flexure displacement D/L). The plotted points were interpolated with quadratic functions y = 0.207x2 − 0.203x + 0.813 (r2 = 0.987). The first- and second-order parameters are in close agreement and yield an estimate of 37° for the flexure angle. Similar calculations lead to flexure angles of 34° for Drosophila GCM and 35° for GCMb. Download figure Download PowerPoint This overall curvature allows the DNA to form favorable hydrophobic and polar contacts with the protein. In the center of the binding site, the DNA curves around the five-stranded β-sheet that sticks into the major groove (Figure 4A, left panel). One important contact point is formed by the side chain and main chain carbonyl of residue Asn65 and bases Gua6 and Cyt7′. These interactions cause the base of Cyt7′ to rotate out of plane, leading to a considerable buckle and propeller twist of bp 7, which is propagated along the DNA duplex and contributes to the overall bend observed. A combination of polar and hydrophobic contacts is also responsible for the two kinks in opposite directions orthogonal to the central bend (Figure 4A). At one end of the duplex, one strand forms hydrophobic contacts with residues of helix H2 assisted through polar interactions with His55, Lys107 and Lys160 (see above) and leans towards the smaller domain, while at the other end the opposite strand passes through a cleft between the β-hairpin S2/S3 and the bulge between strands S5 and S6 with main contact points formed by Arg62, Lys73 and Phe131 protruding from the bulge (Figure 3B). The two kinks in opposite directions allow the 13mer DNA duplexes to pack continuously along the crystallographic b axis. However, even though the DNA stacks end to end, the polyphosphate backbone is discontinuous in the crystal. Adjacent DNA duplexes are rotated by ∼35° in opposite directions to the helical twist of the DNA. Therefore, the first base pair of each DNA duplex and the penultimate base pair of the neighboring duplex show the same twist angles. In order to assess whether the observed DNA bending was due primarily to GCM domain binding or merely to crystal packing effects, we performed an electrophoretic mobility shift assay designed to measure the degree of DNA bending in solution. As probes, we used five DNA duplexes of identical length but with different permutations of the nucleotide sequence such that the GCM binding site was positioned differently within each probe (Figure 4B). Protein-induced DNA bending causes a probe with a centrally located binding site to be retarded more than one with a binding site near the end, and the magnitude of this effect can be used to estimate the bending angle (Scaffidi and Bianchi, 2001). When we performed the assay with the GCM domain of murine GCMa, the degree of retardation of the five probes differed significantly, corresponding to an estimated bending angle of 37° (Figure 4C and D). Similar bending angles were also obtained when the assay was performed with the GCM domains of murine GCMb and the Drosophila homolog dGCM. Therefore the solution studies also support a considerable bending of the DNA upon binding of the GCM domain. Thus the considerable deformation of DNA observed in our structure appears to be due primarily to the binding of the GCM domain, with at most only a minor contribution from the crystal packing. Specificity of the DNA recognition Experiments on DNA binding of mouse and Drosophila GCM domains to consensus and mutated DNA recognition sequences identified bp 2, 3, 6 and 7 as the strongest determinants of specificity (Schreiber et al., 1998). In accordance, we observe important hydrophobic contacts to Thy2 (Ile100, Cys102) and hydrogen bonds to Gua6, Cyt7′ (Asn65) and Gua7 (His67). The importance of bp 3 is less obvious from the crystal structure as Asn63 only indirectly contacts Gua3. However, changing Gua3 into Thy3 in bp 3 completely abolishes GCM binding (Schreiber et al., 1998). The sequence-dependent conformation of the bound DNA, which is often referred to as ‘indirect readout’, might specify this base pair. Indeed, at this position we see strong deviations of the DNA from the canonical B-form: the DNA is bent between bp 2 and 3 (see above), which accounts for a roll angle of 13° between them. In addition, bp 2 shows a strong buckle of ∼10° with Thy2 leaning towards Gua3. To investigate the indirect recognition of bp 3 we also replaced guanine by adenine, cytosine and uracil. All these mutations lead to stronger GCM binding compared with the initial M3 mutant site (Figure 5C). Our results correlate well with the conformational mobility of dinucleotide steps deduced from the comparison of DNA duplex crystal structures (El Hassan and Calladine, 1996). This analysis identified TG/CA (present in the consensus GCM binding site) and TA/TA steps (3A site) as particularly flexible and often found in ‘hinges’ in DNA duplexes, whereas TT/AA steps (as present in the M3 site) are very rigid. Our results suggest that only certain base pairs are flexible enough to allow the pronounced roll between bp 2 and 3. The exocyclic 5-methyl group of thymine appears particularly unfavorable. Changing thymine into uracil (3U site) restores ∼50% of the wild-type DNA-binding affinity either because removing the 5-methyl group allows more conformational flexibility (El Hassan and Calladine, 1996) or because it prevents a clash with the adjacent 5-methyl group of Thy2. Figure 5.DNA-binding properties of mutant GCM domains. (A) Expression of T7-epitope tagged wild-type (WT) and mutant (N63A, N63Q, N65A, N65D, K74M, K74I) GCM domains was verified by western blot of nuclear extracts from transfected COS cells with a monoclonal antibody against the tag. (B) Electrophoretic mobility shift assay with the consensus GCM binding site as probe and extracts from transfected COS cells expressing the wild-type and mutant GCM domains. Equal amounts of each GCM domain were used. (C) Comparative DNA- binding analysis of wild-type GCMa and GCM protein mutants by competition analyses. Electrophoretic mobility shift assays were performed with the consensus GCM binding site as probe and extracts expressing the wild-type and mutant GCM protein in the absence and presence of increasing amounts of competitor (5-, 10-, 25-, 50- and 100-fold molar excess). Oligonucleotides containing the consensus GCM binding site (WT) and its variants (M1–M8, 3A, 3U, 3C) were used as competitors. Conditions were such that in the absence of competitor, 20–30% of the radioactively labeled probe was in complex with the GCM domain. The competitor-dependent reduction of probe in the complex was determined by phosphoimager analysis. The graph summarizes the relative level of competition obtained with a 10-fold excess of each competitor (WT, M1–M8, 3A, 3U, 3C) for wild-type GCMa (open bars) and GCM mutants (black bars). WT and mutant target sites (M1–M8, 3A, 3U, 3C) are listed. Directly and indirectly contacted bases as observed in the crystal structure are marked with filled and open circles, respectively. Download figure Download PowerPoint To gain further insight into GCM domain DNA recognition we mutated a number of residues of the DNA-contacting β-hairpin. We mutated three residues involved in base-specific contacts (mutations N63A, N63Q; N65A, N65D; H67A) and one residue contacting the DNA backbone (K74I, K74M, K74A). Expression of the mutated proteins in transiently transfected COS cells was verified by western blots, and their ability to bind to the consensus and mutated DNA target sites was tested by electrophoretic mobility shift assays (Figure 5A and B); DNA binding of the H76A and K74A mutants was analyzed earlier (Schreiber et al., 1998). Our results show distinct roles for Asn63 and Asn65 in site-specific DNA recognition. Mutant protein N63A binds with slightly lower affinity, which agrees with the crystal structure where Asn63 does not form direct hydrogen bonds with DNA bases. In contrast, mutant N65A shows greatly reduced DNA affinity because it can no longer contact Gua6 and Cyt7′. DNA binding is completely abolished in the N65D mutant, probably because the mutation introduces a carboxy group that points towards the Gua6 O6 atom. Our experiments also show the importance of the polar contact formed between Lys74 and the DNA backbone. Changing this residue into a leucine, methionine or alanine residue completely abolishes DNA binding (Figure 5B; Schreiber et al., 1998). We also performed a series of competitive binding assays in which we assessed the ability of nine different DNA probes, comprising either the natural target site sequence or eight mutated variants (M1–M8), to displace wild-type and mutant GCM domains from the target site (Figure 5C). We observed considerable changes in the site specificity of the N63Q and N65A mutants. Mutant protein N63Q shows reduced binding affinity for the wild-type DNA sequence (Figure 5B) and instead preferentially binds DNA sites M4 and M5, whereas mutant protein N65A preferentially binds to the M6 site (Figure 5C). The crystal structure suggests that the slightly longer glutamine side chain of the N63Q mutant could fill a cavity in the major groove (indicated by an asterisk in Figure 3B), which would allow the N63Q mutant to form favorable interactions with the A–T and T–A base pairs of the M4 and M5 sites. However, the glutamine side chain probably does not form direct interactions with bp 3 as mutant N63Q (like N65A and the wild type) does not clearly distinguish between guanine, adenine, uracil and cytosine in bp 3 (Figure 5C). For the N65A mutant, model building suggests that the alanine CB atom forms a hydrophobic contact with the exocyclic methyl group of Thy6 in the M6 site, which could compensate for the loss of the polar interaction between Asn65 and the Gua6 O6. The H67A mutant shows similar DNA binding to the wild type but a strongly reduced binding to sites M4 a
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