NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+-binding motif

Masato Shimizu1,2,3, Hidekazu Hiroaki1,4, Daisuke Kohda1,5, Toshihiko Hosoya6, Yasuko Akiyama-Oda7, Yoshiki Hotta6, Eugene Hayato Morita3,8,9 and Kosuke Morikawa1,9

1 Biomolecular Engineering Research Institute, 6–2–3 Furue-dai, Suita, Osaka 560-0874, 2 Japan Science and Technology Corporation,4–1–8 Honcho, Kawaguchi, Saitama 332-0012, 3 Center for Gene Research, Ehime University, 3–5–7 Tarumi, Matsuyama, Ehime 790-8566, 4 Division of Biophysics, Graduate School of Integrated Sciences, Yokohama City University, 1–7–29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, 5 Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3–1–1 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, 7–3–1 Bunkyo-ku, Tokyo 113-0033 and 8 Venture Business Laboratory, Ehime University, 3–5 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


    Abstract
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 Materials and methods
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Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of glial precursors from the neural default to glia. The GCM protein contains the functional domain that is essential for recognition of the upstream sequence of the repo gene. In the DNA-binding region of this GCM protein, there is a cysteine-rich region with which divalent metal ions such as Zn2+ must bind and other proteins belonging to the GCM family have a corresponding region. To obtain a more detailed insight into the structural and functional features of this DNA-binding region, we have determined the minimal DNA-binding domain and obtained inductively coupled plasma atomic emission spectra and 1H–15N, 1H–15N–13C and 113Cd2+ NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn2+-binding sites, one of which is adjacent to the interface for DNA binding. Systematic mutational analyses of the conserved cysteine residues in the minimal DNA-binding domain revealed that one Zn2+-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.

Keywords: 113Cd2+ NMR/DNA-binding domain/glial cell missing/inductively coupled plasma atomic emission spectroscopy/transcription factor


    Introduction
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 Abstract
 Introduction
 Materials and methods
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During development of the Drosophila nervous system, the GCM (glial cell missing) protein was identified as a binary switch between neuronal and glial lineages (Hosoya et al., 1995Go; Jones et al., 1995Go). The Drosophila GCM protein serves as a transcriptional regulator of the target genes, pointed (pnt), reversed polarity (repo) and tramtrak (ttk), that determine a glial or neuronal cell fate. In recent studies, it was found that the gcm gene was expressed in the peripheral mechanosensory nerve system in adult flies and the mesodermal hematopoietic lineage (Wegner and Riethmacher, 2001Go). As mammalian homologs of GCM, only two related sequences were found, i.e. GCM1/GCMa and GCM2/GCMb (Akiyama et al., 1996Go; Schreiber et al., 1997Go; Kim et al., 1998Go). Degenerated PCR approaches, low stringency screening and database searches revealed no other additional gcm-related sequences in Saccharomyces cerevisiae, Arabidopsis thaliana or Ceanohabitis elegans. Interestingly, despite their high similarity and domain architecture, mammalian GCMs function in placental and parathyroid gland development rather than neuronal systems (Wegner and Riethmacher, 2001Go).

The N-terminal highly conserved region of the GCM protein called the ‘GCM-box’ acts as a DNA-binding domain [Figure 1aGo (Akiyama et al., 1996Go)]. 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., 2000Go) 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 protein–DNA complex through oxidative or reductive conditions (Schreiber et al., 1998Go). It was further mentioned that the DNA binding of mGCMa was not dependent on Zn2+. This suggests that Zn2+ binding does not stabilize the mGCMa–DNA 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., 2002Go). However, the residues coordinated with Zn2+ remain unclear.



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Fig. 1. (a) Multiple sequence alignment of the N-terminal domain from Drosophila and mouse GCM homologs. Conserved cysteines are boxed. (b) DNase I footprinting assay of MK243 and truncated Drosophila GCM DNA-binding regions. (c) Construction of plasmids expressing the Drosophila GCM DNA-binding region [GCM(243)] and truncated forms of it. Normalized DNA-binding activity (+, bound; -, unbound) is shown in the right column. All proteins were expressed as fused proteins with thioredoxin and the linker sequence could be digested with PreScission protease. (d) Gel retardation assaying of AS157 with truncated Drosophila GCM DNA-binding regions used in (b).

 
In this study, we identified the minimal DNA-binding domain of the GCM DNA-binding region and determined the structural features of this domain by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and heteronuclear NMR spectroscopy. In addition, the putative secondary structure and DNA-binding region of this domain were identified. From all the results, we concluded that the minimal DNA-binding domain of Drosophila GCM binds with Zn2+ in a Zn2+/protein ratio of 2. One of these two Zn2+ tightly binds at the Cysx4 type Zn2+-binding site adjacent to the DNA-binding region, as speculated and the binding of Zn2+ with this site stabilizes the higher order structure of this domain. The other Zn2+ binds weakly with this DNA-binding domain and this Zn2+-binding does not significantly affect the features of this DNA-binding domain.

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.


    Materials and methods
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 Materials and methods
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Expression and purification of a truncated form of Drosophila GCM

A plasmid harboring the gcm gene fragment coding for the N-terminal region of GCM (N243) (Akiyama et al., 1996Go) 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 Tris–HCl (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., 1986Go), 0.5x TBE (45 mM Tris–borate/1 mM EDTA) and run at 4°C. DNase I footprinting assay was performed as described elsewhere (Yamamoto et al., 1991Go) 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 [{alpha}-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, 1989Go) and the secondary structure of AS157 was determined by the method of Yang et al. (Yang et al., 1968Go).

NMR spectroscopy

One-dimensional 113Cd2+ NMR spectra were recorded at 25°C with a DMX-500 FT-NMR spectrometer (Bruker). 1H–15N HSQC and 1H–15N–13C 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% H2O–5% 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{alpha} 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., 1995Go). Analysis of 2D/3D NMR spectra was performed with XEASY (Bartels et al., 1995Go).

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., 1996Go).


    Results
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 Materials and methods
 Results
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 References
 
The DNA-binding region and truncated forms of the Drosophila GCM protein were overexpressed in E.coli BL21(DE3)/pLysS as insoluble forms and the proteins fused with thioredoxin were overexpressed as soluble forms only when the E.coli cells were cultured at 28°C. On DNase I footprinting assay, it was found that GCM243 (DNA-binding region of GCM) recognized the DNA sequence 5'-ACCCGCAT-3' (Figure 1bGo) derived from its biological target gene repo. Next, we designed 16 truncated Drosophila DNA-binding regions (Figure 1cGo) and overexpressed them in E.coli cells. On comparison of the affinities of MK243 (M1 to K243; whole DNA-binding region), AK219 (A25 to K243), GK199 (G45 to K243) and AK171 (A73 to K243) to the preferred DNA sequences, MK243 and AK219 showed almost the same binding affinities and the other proteins showed lower affinities. Furthermore, on comparison of the affinities of AK219, AR191 (A25 to R215), AA165 (A25 to A189) and AS157 (A25 to S181), all showed almost the same affinities. From these results, we concluded that AS157 is the minimal DNA-binding domain of the GCM DNA-binding region and therefore used it as a sample in this study (Figure 1dGo). Overexpressed AS157 was extracted as a soluble protein and purified step by step, as shown in Figure 2Go. The purity of the sample used in this study was more than 95%, as judged from the SDS–PAGE results (lane 4 in Figure 2Go).



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Fig. 2. Purification of overexpressed AS157 in E.coli BL21(DE3)/pLysS. (1) Crude soluble extract of E.coli cells obtained on disruption by sonication. (2) Proteins adsorbed on the Ni2+ charged HiTrap chelating column were purified by gel filtration column chromatography. (3) Proteins (2) were digested with PreScission protease overnight. (4) AS157 was purified with a HiTrap SP cation-exchange column.

 
The CD spectrum of AS157 is shown in Figure 3Go. As estimated by the method of Yang et al. (Yang et al., 1968Go), helices, ß-structure, turns and random coils comprised 11.9, 14.8, 29.0 and 44.8%, respectively.



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Fig. 3. CD spectrum of AS157. At 20°C, a 50 µM sample was placed in the quartz cell (1 mm thick) and 32 scans were averaged. The secondary structure of AS157 was determined by the method by Yang et al. (Yang et al., 1968Go).

 
Sequence-specific resonance assignment of uniformly 15N/13C-labeled GCM (AS157) was performed (Figure 4aGo) and a putative secondary structure of this domain was elucidated by the chemical shift index method (Figure 4bGo–d) (Wishart et al., 1995Go). The chemical shift values of backbone atoms have been deposited in the BioMagResBank under accession number 5626. The 1H–15N HSQC spectra of AS157 showed good chemical shift dispersion on both the 1H and 15N axes, which suggested that the minimal DNA-binding domain forms the folded domain of the {alpha}/ß-protein. Furthermore, AS157 contains nine short ß-strands and two {alpha}-helices.



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Fig. 4. Summary of the NMR data for assignments and secondary structure determination. (a) 1H–15N HSQC spectrum of 15N-labeled AS157. Assignments of the backbone NH signals are indicated. (b) Illustration of the putative secondary structure elements of AS157. Deviations of (c) 13C{alpha} and (d) 13C' chemical shift values from those of random coils are shown relative to the residue numbers of AS157.

 
The results of ICP measurement for AS157 are summarized in Table IGo. In the presence of sufficient amounts of Zn2+, the latter binds stoichiometrically with AS157 in a Zn2+/AS157 ratio of 2. However, on 0.1 mM EDTA treatment, weakly bound Zn2+ is washed out and the Zn2+/AS157 ratio is 1. These results indicate that there are two Zn2+-binding sites in AS157 and the binding affinities of these two Zn2+ to AS157 are different. To characterize the Zn2+-binding sites, we measured the 1H–15N NMR spectral differences between Zn2+- and Cd2+-bound AS157s and the 113Cd2+ NMR spectra for 113Cd2+-bound AS157. As shown in Figure 5aGo, only one 113Cd2+ signal at 702 p.p.m. was observed. Then, the 1H–15N HSQC spectrum of Cd2+-coordinating AS157 was compared with that of Zn2+-coordinating AS157. Whereas AS157 contains nine cysteine residues, the chemical shift values of amide protons around five cysteine residues (C99, C103, C118, C130 and C135) differed considerably between Zn2+-coordinating AS157 and Cd2+-coordinating AS157 (Figure 5bGo). The residue numbers of AS157 correspond to those of full-length Drosophila GCM protein.


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Table I. Molar ratio of bound Zn2+/AS157
 


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Fig. 5. (a) 113Cd2+ NMR spectrum of 113Cd2+-bound AS157. (b) The differences between the corresponding amide proton chemical shift values observed in 1H–15N HSQC spectra for Zn2+- and 113Cd2+-bound AS157.

 
In order to determine the ligand residues for Zn2+ coordination, we further designed five point-mutated AS157 proteins (C99A, C103A, C118A, C130A and C135A) in which each cysteine residue was replaced by an alanine residue and all but C118A were overexpressed as insoluble forms (Figure 6Go). Then, the 1H–15N HSQC spectra of the wild-type and C118A were compared (Figure 7a and dGo). The overall spectral patterns were very similar, suggesting that the folding of C118A is similar to that of the wild-type. In addition, after 12 h of treatment with 20 mM EDTA, the 1H–15N HSQC spectral patterns for both AS157 and C118A–AS157 had both drastically changed into the typical spectral pattern for unfolded proteins (Figure 7c and fGo).



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Fig. 6. Overexpression of point-mutated AS157. (1) C99A; (2) C103A; (3) C118A; (4) C130A; and (5) C135A. Only C118A was overexpressed as a soluble protein.

 


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Fig. 7. Changes observed in the 1H–15N HSQC spectral patterns of AS157 and C118A after removal of Zn2+. (a)–(c) AS157; (d)–(f) C118A. (a), (d) 0 h, (b), (e) 3 h and (c), (f) 12 h after addition of 20 mM EDTA.

 
Finally, chemical shift perturbation experiments on AS157 with its specific target DNA were performed (Figure 8Go). The largest chemical shift deviations were found at H72, R79 and N85. We concluded that the DNA-binding region is composed of the residues around 72–85.



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Fig. 8. The differences between the corresponding amide proton chemical shift values observed in the 1H–15N HSQC spectra of 15N-labeled AS157 in the presence and absence of DNA. There was a 1:1 molar ratio of the DNA 24-mer derived from specific repo sequences. The putative secondary structure and tightly bound cysteine residues are indicated in the top panel.

 

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Footprinting and gel retardation assay indicated that AS157 is a minimal fragment showing almost the same DNA-binding affinity as that of GCM(243). Furthermore, CD and 1H–15N NMR spectral analyses suggested that AS157 was the minimal core of the compactly folded domain of the {alpha}/ß-protein. From these results, we concluded that AS157 is the minimal DNA-binding domain of Drosophila GCM.

Although nine ß-strands and two {alpha}-helices were predicted, based on the chemical shifts of 13C{alpha} 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, 1998Go). 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 1H–15N 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 1H–15N HSQC spectral patterns of the wild-type and C118A were similar.

The changes observed in the 1H–15N 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 1H–15N 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., 1998Go). 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., 2002Go). 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 72–85. This region is predicted to comprise a short ß-strand and a loop. On the other hand, the two predicted {alpha}-helices, residues D67–K71 and C118–Q126, 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 –H–X12–H–X6–C–X5–C–X3–C–X14–C–X11–C–X24–C–X8–C–X2–H–X23–H–X2–H– (Cohen et al., 2002Go). Focusing on the tightly bound C4-type Zn2+, we propose a simplified signature of the GCM box, i.e. –C–X3–C–X26–C–X24–C–. However, this simplified GCM box signature is still not similar to other known Zn2+-binding motifs such as C2H2 Zn fingers (–C–X24–C–X12–H–X35–H–), GATA-1 (–C–X2–C–X17–C–X2–C–) and p53 (–C–X3–H–X59–C–X3–C–). 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.


    Acknowledgments
 
This work was partly supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (No. 12780498) and Core Research Science and Technology (CREST) of the Japan Scientific and Technology Cooperation (JST).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received December 24, 2002; revised February 4, 2003; accepted March 7, 2003.





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