1 Biomolecular Engineering Research Institute, 623 Furue-dai, Suita, Osaka 560-0874, 2 Japan Science and Technology Corporation,418 Honcho, Kawaguchi, Saitama 332-0012, 3 Center for Gene Research, Ehime University, 357 Tarumi, Matsuyama, Ehime 790-8566, 4 Division of Biophysics, Graduate School of Integrated Sciences, Yokohama City University, 1729 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, 5 Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 311 Maidashi, Higashi-ku, Fukuoka 812-8582, 6 National Institute of Genetics, Mishima, Shizuoka 411-8540, 7 Department of Physics, Graduate School of Science, University of Tokyo, 731 Bunkyo-ku, Tokyo 113-0033 and 8 Venture Business Laboratory, Ehime University, 35 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan
To whom correspondence should be addressed. E-mail: ehmorita{at}dpc.ehime-u.ac.jp; morikawa{at}beri.or.jp
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Abstract |
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Keywords: 113Cd2+ NMR/DNA-binding domain/glial cell missing/inductively coupled plasma atomic emission spectroscopy/transcription factor
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Introduction |
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The N-terminal highly conserved region of the GCM protein called the GCM-box acts as a DNA-binding domain [Figure 1a (Akiyama et al., 1996
)]. This domain recognizes 5'-AT(G/A)CGGGT-3' preferentially and no structural similarity has yet been found between the GCM family and other DNA-binding proteins. Furthermore, this domain contains nine cysteine residues, four of which are aligned in a manner reminiscent of a Zn-finger motif. However, previous mutational work on mouse GCM (Tuerk et al., 2000
) suggested that the seven conserved cysteine residues played an important role in the recognition of DNA. In particular, two of the seven cysteine residues (C76 and C125) interact with the target DNA sequence directly, and the others stabilize the proteinDNA complex through oxidative or reductive conditions (Schreiber et al., 1998
). It was further mentioned that the DNA binding of mGCMa was not dependent on Zn2+. This suggests that Zn2+ binding does not stabilize the mGCMaDNA complex. In EXAFS and microPIXE studies, Cohen et al. recently found that the two Zn2+ were coordinated with cysteine and histidine residues (Cohen et al., 2002
). However, the residues coordinated with Zn2+ remain unclear.
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This is the first experimental evidence of the occurrence of a direct interaction between Zn2+ and the Drosophila GCM DNA-binding domain and the functional meaning of this interaction.
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Materials and methods |
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A plasmid harboring the gcm gene fragment coding for the N-terminal region of GCM (N243) (Akiyama et al., 1996) was used as a template and further truncated fragments coding truncated GCM DNA-binding regions were amplified by PCR and then transferred to the NdeI and HindIII sites of pGEMEX-1 (Promega, Madison, WI). With these plasmids, truncated GCM-binding regions were expressed as fusion proteins with the His-patched thioredoxin gene (Invitrogen) followed by the PreScission protease recognition sequence.
The constructed plasmids were transformed into Escherichia coli BL21(DE3)/pLysS. The transformants were grown at 28°C in LB or M9 medium containing ampicillin (0.1 mg/ml). At OD530 = 0.5, protein expression was induced by the addition of isopropyl-ß-D-thiogalactopyranoside (IPTG) and Zn2+, to a final concentration of 0.5 mM and 50 µM, respectively. Then, the transformants were grown further for 6 h. Cells were collected (6000 g for 5 min) and the expressed proteins were extracted by sonication [TOMY UD-201; 30 W (50% duty cycle) for 25 min]. The supernatants containing the expressed proteins were collected by high-speed centrifugation (20 000 g for 20 min) and then further purified on an Ni2+ charged HiTrap chelating column (Amersham Pharmacia Biotech, Uppsala, Sweden) followed by HiLoad (26/60) 75pg gel filtration chromatography. These partially purified fusion proteins were truncated with PreScission protease (Amersham Pharmacia Biotech). The fragments containing truncated GCM DNA-binding domains were further purified with a HiTrap SP (Amersham Pharmacia Biotech) column.
For NMR studies, the minimal DNA-binding region of Drosophila GCM (AS157), containing the residues from A25 to S181, was used. A 15N-uniformly labeled protein sample was prepared by growing cells in M9 minimal medium containing 15NH4Cl as a sole nitrogen source. A 13C/15N-uniformly labeled protein sample was prepared by growing cells in BioExpress-Min medium (Cambridge Isotope Laboratory, Bethesda, MD). A 113Cd2+-containing GCM (AS157) sample was prepared by growing cells on M9 minimal medium containing 10 µM 113CdCl2.
Site-directed mutagenesis
Alanine substitutions of cysteine residues were made with a QuikChange site-directed mutagenesis kit (Stratagene).
Gel retardation and DNase I footprinting assay
Gel retardation assays were performed under the following conditions: 50 mM TrisHCl (pH 7.48), 50 mM KCl, 1 mM DTT, 10% glycerol. Different concentrations of truncated GCM DNA-binding regions (2.5, 25 and 250 nM) were incubated with 10 000 c.p.m. of 32P-labeled DNA [d(GG-CCTACCCGCATTACGC*C*)/d(GGCGTAATGCGGGTAG-GC*C*)] (where * represents 32P-labeled base) at 25°C for 30 min. The reaction mixtures were loaded on an 8% polyacrylamide gel (acrylamide:bisacrylamide = 75:1) (Liu-Johnson et al., 1986), 0.5x TBE (45 mM Trisborate/1 mM EDTA) and run at 4°C. DNase I footprinting assay was performed as described elsewhere (Yamamoto et al., 1991
) using the 32P-end-labeled DNA as follows. The upstream region of the repo gene was cloned into the BamHI and SalI sites of pUC19, then the plasmid was cleaved with EcoRI and HindIII. The filled-in reaction involving the Klenow fragment labeled with [
-32P]dATP was performed for the EcoRI terminus of the DNA fragment.
CD spectrometry
A circular dichroism (CD) spectrum of AS157 was obtained with a J-720 spectrometer (JASCO), equipped with a Peltier thermo controller. At 20°C, a 50 µM sample was placed in the quartz cell (1 mm thick) and 32 scans were averaged.
Protein concentrations were determined by measuring the optical absorption at 280 nm. The molar extinction coefficient of the GCM DNA-binding region was determined as 25 800, according to the method of Gill and von Hippel (Gill and von Hippel, 1989) and the secondary structure of AS157 was determined by the method of Yang et al. (Yang et al., 1968
).
NMR spectroscopy
One-dimensional 113Cd2+ NMR spectra were recorded at 25°C with a DMX-500 FT-NMR spectrometer (Bruker). 1H15N HSQC and 1H15N13C triple-resonance NMR experiments were performed at 20°C with DMX-600 and DMX-750 FT-NMR spectrometers (Bruker) equipped with TXI three-axis gradient 1H {13C, 15N} probes (Bruker).
For the NMR signal assignments and structural studies, extensive examination of solution conditions revealed that GCM (AS157) was most stable in the presence of 300 mM KCl and 5% glycerol. However, even under these conditions, the samples gradually started to precipitate at protein concentrations higher than 0.6 mM. NMR samples containing up to 0.6 mM protein were prepared in 99.9% D2O or 95% H2O5% D2O buffer containing 20 mM potassium phosphate, pH 6.5, 300 mM KCl and 10% glycerol. The details of the NMR signal assignments have been given elsewhere (Shimizu et al., 2002). A putative secondary structure of GCM (AS157) was determined on the basis of the deviations of 13C and 13C' chemical shift values from the corresponding values for random coils. 13Cß chemical shift values were not included, since some 13Cß signals were not detected because of the poor signal-to-noise ratio.
For NMR titration experiments on GCM (AS157) involving DNA, a cognate DNA 19-mer containing a GCM-recognized sequence, dGACTTTGCCCGCATTTCGG/dCCGAAATGCGGGCAAAGTC, was used; 0.1 mM 15N-labeled AS157 and 5 mM DNA were dissolved in the aforementioned buffer and then a small amount of DNA was added.
All NMR data were processed with NMRPipe software (Delaglio et al., 1995). Analysis of 2D/3D NMR spectra was performed with XEASY (Bartels et al., 1995
).
Inductively coupled plasma atomic emission spectrometry
The zinc content of the truncated GCM DNA-binding region was determined with an Optima3000 ICP-AES instrument (Perkin-Elmer) at the Advanced Instrumentation Center for Chemical Analysis, Ehime University (Morita et al., 1996).
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Results |
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Discussion |
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Although nine ß-strands and two -helices were predicted, based on the chemical shifts of 13C
and 13C' of AS157, the CD spectrum showed a lower secondary structure content in AS157. Underestimation of ß-strands on secondary structural analysis of the CD spectrum is explained as being due to the shortness of most of the ß-strands in AS157 and the linkage of each strand by a loop, turn or Zn2+-binding cysteine residues. This is a characteristic of Zn2+-coordinating protein domains, such as the RING finger or FYVE domain.
ICP spectral measurements indicated that in the presence of sufficient Zn2+, two Zn2+ bind with AS157 and only one Zn2+ binds with AS157 in the absence of Zn2+ in the solution. This means that AS157 has two Zn2+-binding sites, and at one site Zn2+ binds tightly and at the other loosely.
In the 113Cd2+ NMR spectrum of 113Cd2+-bound AS157, only one peak located at 702 p.p.m. was observed. 113Cd2+ NMR spectral measurement is one of the best ways of characterizing the coordination environments around Zn2+-binding sites. The chemical shift value of this peak is typically responsible for 113Cd2+ in C4-type coordination (Summers, 1998). From this, we concluded that at the tightly Zn2+-bound site, four cysteine residues are ligated to Zn2+. Furthermore, the observation of only one peak in the 113Cd2+ NMR spectrum means that another 113Cd2+ binds loosely with AS157, and this indicates that another Zn2+-binding is not so stable.
The presence of a C4-type Zn2+-binding site is also indicated by the 1H15N NMR spectral differences between Zn2+- and Cd2+-bound AS157. In this experiment, the chemical shift values of amide protons around C99, C103, C118, C130 and C135 were drastically changed when AS157 was prepared from medium containing an excess amount of Cd2+ instead of Zn2+. Note that there was no additional step of exchanging bound cations during the purification of AS157. The radii of the two metal ions are different and this means that these five cysteine residues are located fairly close to Zn2+. AS157 has nine cysteine residues, seven of which are well conserved in all GCM homologs. To identify the ligand for this Zn2+, five point-mutated AS157s (C99A, C103A, C118A, C130A and C135A) were designed and only C118A was overexpressed as a soluble protein. This means that the lack of the cysteine residues other than C118 disrupts the Zn2+-binding site and that the loss of this Zn2+-binding site destabilizes the higher order structure of AS157. This further means that the ligand residues comprising this Zn2+-binding site are C99, C103, C130 and C135, i.e. C118 does not function in this site. The above conclusion is also supported by the result that the overall 1H15N HSQC spectral patterns of the wild-type and C118A were similar.
The changes observed in the 1H15N HSQC spectral patterns for both AS157 and C118A during 12 h of treatment with 20 mM EDTA also suggest that the presence of this C4-type Zn2+-binding site is indispensable for maintaining the stability of the higher order structure of AS157. Note that the time courses of the removal of Zn2+ shown by the spectral changes on 1H15N HSQC were almost the same for the wild-type and C118A. Again, C118 does not coordinate to Zn2+ in the same manner as the other four cysteines. The affinity of this C4-type Zn2+-binding site is relatively tighter than those of known Zn2+-coordinating DNA-binding domains. In a previous study, Schreiber et al. concluded that mGCMa did not bind with Zn2+, because brief exposure of mGCMa to 1 mM EDTA did not have any effect of the DNA-binding activity of mGCMa (Schreiber et al., 1998). In the recent study by Cohen et al., the Zn2+-binding site was found to be buried in the core of mGCMa and this Zn2+ could only be removed under the denaturing conditions with a large excess of 1,10-phenanthroline (Cohen et al., 2002
). In our case, the tightly bound Zn2+ in AS157 could only be removed slowly in the presence of 20 mM (200-fold excess) of EDTA. Although some difference in the speculation exists, our observation on the Drosophila AS157 is consistent with the reported features of mGCMa.
The binding site of the other weakly bound Zn2+ remained unclear in our study. Cohen et al. concluded that two Zn2+ ions were liganded by conserved Cys and His residues in mGCM. Here, we illustrated that one of the two Zn2+ was ligated with cysteine residues, such as C99, C103, C130 and C135. Thus, the additional Zn2+ is probably coordinated with other cysteine and histidine residues.
Finally, we identified the DNA-binding site of the AS157 by means of NMR titration experiments. Upon titration of AS157 with DNA, most of the NH signals were detectable and mainly the chemical shifts rather than the intensity of the NH signals changed (data not shown). This suggests that the binding of the AS157 to GCM-recognized DNA sequences occurs in a fast-exchanging manner. The DNA-binding region is around residues 7285. This region is predicted to comprise a short ß-strand and a loop. On the other hand, the two predicted -helices, residues D67K71 and C118Q126, seemed not to be responsible for the DNA binding. The largest chemical shift deviations of NH signals were observed for residues H72, R79 and N85. These three residues are critically conserved in all five known GCM-related sequences. In addition, the residues between W76 and N85 are in the highly conserved region in GCM-box proteins, which strongly suggests that the conservation of the amino acid sequence of this region is also important for its biological functions, such as specific recognition of the target DNA sequence, 5'-AT(G/A)CGGGT-3'. Interestingly, this DNA-binding region is not included in, but followed by, the Zn2+-coordinating cysteine-rich region. This observation constitutes additional support for our conclusion, i.e. that the Zn2+-binding site is important for stabilizing the overall folding of the AS157 rather than direct binding to DNA.
The alignment of the four Zn2+-bound cysteine residues, as well as the relative distance between the DNA-binding region and the Zn2+-binding site, is reminiscent of a typical C4-type zinc-finger motif. Initially, the Zn2+-coordinating sequence feature of the GCM domain was reported to be HX12HX6CX5CX3CX14CX11CX24CX8CX2HX23HX2H (Cohen et al., 2002). Focusing on the tightly bound C4-type Zn2+, we propose a simplified signature of the GCM box, i.e. CX3CX26CX24C. However, this simplified GCM box signature is still not similar to other known Zn2+-binding motifs such as C2H2 Zn fingers (CX24CX12HX35H), GATA-1 (CX2CX17CX2C) and p53 (CX3HX59CX3C). From the weak sequence similarity between GCM and GATA-1, resemblance of the DNA-binding domain was predicted. The results of our secondary structure elucidation ruled out this possibility.
From all the results, we drew the following conclusions. (1) AS157 has two Zn2+-binding sites. The Zn2+-binding site at which one Zn2+ binds tightly is of the C4 type, which does not function in the DNA binding. (2) This C4-type Zn2+ binding site is indispensable for maintaining the higher order structure of AS157. (3) Another Zn2+-binding site, whose structural features and functional importance remain unclear, may comprise histidine residues. (4) Judging from the topology of metal-coordinating residues, the secondary structure and the DNA-binding interface, the GCM box is a novel DNA-binding domain that is only found in higher eukaryotes. Although further investigation is now in progress, the present results are the first direct evidence of the presence of Zn2+-binding sites in the Drosophila GCM DNA-binding domain and the first to reveal the structural features and functional importance of the tightly Zn2+-bound site.
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Acknowledgments |
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Received December 24, 2002; revised February 4, 2003; accepted March 7, 2003.