Yonago Acta medica 2010;53:25–28

Circular Dichroism Studies on C-terminal Zinc Finger Domainof Transcription Factor GATA-2
Ojeiru F. Ezomo, Kazuya Takahashi, Yuki Horie, Mohammed S. Mustak and Shunsuke
Meshitsuka
Division of Integrative Bioscience, Institute of Regenerative Medicine and Biofunction, Graduate
School of Medical Science, Tottori University, Yonago 683-8503, JapanThe C-terminal zinc finger domain of GATA-2 transcription factor from Rattus norvegicus has been expressed and purified to elucidate its secondary structure using circular dichroism spectroscopy. Circular dichroism spectra showed that native GATA-2 C-terminal
domain of (Cys)4 type zinc finger has 12% -helix, 36% -sheet and 52% random coil content. The estimated structure was compared with predicted structures by sequence based
prediction software SOPMA and found to be similar. Furthermore, the effect of pH on
the secondary structure of GATA-2 C-finger was examined. This study provides an insight
into the understanding of the structure and function of transcription factor GATA-2.
Key words: circular dichroism; GATA-2; transcription factor; Zn finger

GATA transcription factors comprise a family of
six zinc finger proteins (GATA-1 to 6) which regulate cell differentiation and proliferation (Hoene
et al., 2001; Ferreira et al., 2005). GATA-2 isan essential transcription factor for the regulation of multiple aspects of hematopoiesis and expressed within the same hematopoietic lineages
as GATA-1 and also in several nonhematopoietic
cell types (Yamamoto et al., 1990; Arceci et al.,
1993; Pandolfi et al., 1995; Dasen et al., 1999;
Lilleväli et al., 2006). The most notable featuresof the GATA proteins are the two adjacent zincfinger domains referred to as the N-terminal zincfinger (N-finger) and the C-terminal zinc finger(C-finger) which have highly conserved amino acidsequences (Lowry and Atchley, 2000; Vonderfecht
et al., 2008). In the case of GATA-2, the N-finger
and C-finger amino acid residues reside between
295-319 and 349-373 respectively. Members of
the family bind to the DNA consensus sequence
WGATAR; (W = A/T; R = A/G) by two character-istic C4 (Cys-X2-Cys-X17-Cys-X2-Cys) zinc finger
domains (Orkin, 1992; Ko L et al., 1993). Studies
have shown that the C-terminal zinc finger and its
adjacent C-terminal basic tail are necessary for
GATA to bind to its cognate sequence (Evans etal., 1988; Ko and Engel, 1993; Merika and Orkin,
1993; Omichinski et al., 1993; Visvader et al., 1995;
Ferreira et al., 2005). It has been shown that the
N-finger of GATA-2 and GATA-3 bind to DNA
independently with slightly different sequence
preferences while contributing to the stabilizationand specificity of DNA binding (Martin and Orkin,
1990; Merika and Orkin, 1993; Pedone et al., 1997;
Newton et al., 2001). It has also been shown thatzinc finger plays a crucial role in the ability of
GATA-1 to induce terminal erythroid differentiation (Wiess et al., 1997). In a recent study a crosstalk between GATA-1 and GATA-2 during the proliferation and differentiation stages of erythropoiesis was reported (Huang et al., 2009). It is known
that GATA-2 gene is upregulated in the absence ofAbbreviations: CD, circular dichroism; DTT, dithiothreitol; GST, glutathione S-transferase

25

O. F. Ezomo et al.

GATA-1, in other words high levels of GATA-2 areexpressed in the proliferating erythroid progenitors.
Upon differentiation, GATA-1 expression increases
whereas GATA-2 is repressed. In another study
by Visvader et al., it was shown that the GATA2 C-terminal zinc finger can induce megakaryocytic differentiation of an early myeloid cell line
(Visvader et al., 1995). Therefore, the structureof C-finger is very crucial in its function for DNA
binding and protein-protein interaction. Although
the structure and function of GATA-1 have beenreported (Omichinski et al., 1993; Bates et al.,2008), there are so far no studies on the structure of
GATA-2. GATA-1 C-finger and GATA-2 C-finger
of Rattus norvegicus have a sequence homology
of about 87% (United States National Center for
Biotechnology Information Basic Local Alignment
Search) but they differ in their functions during
erythropoesis (Huang et al., 2009).
The present study aimed to investigate thesecondary structure of GATA-2 C-finger using
circular dichroism (CD) spectroscopy. GATA-2
C-finger (amino acid residues 324–387) was subcloned into the expression vector pGEX4T-2 (GEHealthcare, Little Chalfont, Buckinghamshire,
United Kingdom) as a glutathione S-transferase(GST) tag fusion protein. The sub-cloned constructs were transformed into BL21(DE3) E. coli
for protein expression. The expression of the
GATA-2 C-finger was induced by IPTG for 6 h at
37˚C. The cells were harvested by centrifugation
and resuspended in lysis buffer and stored at –20˚C
until protein extraction. The cell suspension was
thawed and cell lysis was performed by ultrasonication, five times for 30 s. The supernatant after
centrifugation at 12,000 rpm was used for the purification. The protein was first purified by affinity
chromatography using glutathione sepharose beads
and then digested by thrombin protease for 1 h atambient temperature to remove the GST tag. The
protein was further purified through gel chromatography using a column HiLoad 16/60 superdex
30 pg, AKTA prime (GE Healthcare) and stored at
4˚C in the presence of 1 mM dithiothreitol (DTT).
The protein sample was analyzed by tricine-SDS
PAGE and confirmed to be single band.

Fig. 1. The effect of pH on the secondary structure of
GATA-2 C-finger: CD spectra were recorded for 30 µM
GATA-2 C finger protein in 20 mM Tris-HCl buffer (pH
7.4, solid line), 20 mM phosphate buffer (pH 6.0, dashedline) and 20 mM acetate buffer (pH 4.0, dotted line) containing 150 mM NaCl and 1 mM DTT. The spectra wererecorded in 0.1 nm intervals at 50 nm/min at 25˚C.

In the present study the CD spectra were recorded from 250 nm to 190 nm using a Jasco J-720
spectrometer (Jasco, Tokyo) purged with nitrogen.
The spectra were acquired using a 0.1 cm pathlength cuvette at 25˚C with a resolution of 0.1 nm
and a scanning speed of 50 nm/min. Spectra presented were averages of ten consecutive measurements. CD signals were converted to molar ellipticity ( ) using the equation;=[ ]obs • 10 −3 • MW
deg • dmol–1 • cm2
C • l • n • 10 –2

where [ ]obs is the observed ellipticity, MW is molecular weight, C is concentration (in mg/mL), l is
the path length of the cuvette in centimeters, n is
the number of residues, and deg is degrees (Das etal., 2004).
The experiments were carried out separately
using five independently purified protein. Our result showed that all the GATA-2 C-fingers display anegative peak near 200 nm and a shoulder around222 nm, suggesting that the protein is largely in arandom coil conformation with some helical content under physiological conditions (20 mM TrisHCl, 150 mM NaCl, 1 mM DTT, pH 7.4) (Fig. 1).
Furthermore, we examined the effect of pH on the26Structure of GATA-2 C-terminal finger

Table 1. Comparison of the secondary structures of GATA-2 C-finger fromCD analysis and sequence based prediction software SOPMA
Sample-Helix (%)-Sheet (%)

Coil (%)

MethodGATA-2 C-finger

12.6 ± 7.2
12.5

36 ± 5.0
29.7

52.1 ± 6.0
57.8

CD analysis*
SOPMA prediction†*Dichroweb software package.
†http://npsa-pbil.ibcp.fr/cgi-bin/secpred_sopma.pl

understanding of the secondary structure and function of (Cys)4 type zinc finger proteins. Further
work needs to be done on the structure of GATA2 C-finger in relation to the metal binding, thermal
stability and DNA and protein interactions.

secondary structure of GATA-2 C-finger. The estimated secondary structure of GATA-2 C-finger at
pH 6.0 was not significantly different from pH 7.4.
We estimated the helical, -sheet and random coilcontent by CD spectrum using K2D algorithm from
Dichroweb software package (Andrade et al., 1993;
Whitmore and Wallace, 2008). We demonstratein the present study that the secondary structureof GATA-2 C-finger deduced from CD analysis isconsistent with that obtained from the sequencebased secondary structure prediction software
SOPMA presented in Table 1. At low pH the secondary structure of GATA-2 C-finger was alteredresulting in a decrease in -helical content and anincrease in -sheet compared to the native structure. The -helix content is essential for the zincfinger type protein to carry out certain functions
such as DNA binding. The secondary structure
estimate showed that GATA-2 C-finger is rich inrandom coil while it has less -sheet and -helicalcontent. This result strongly agrees with other zincfinger domain secondary structures (Lachenmann
et al., 2002; Negi et al., 2004; Sakai-Kato et al.,2009). In contrast, the secondary structure of
ZIC3 which has a (Cys)2-(His)2 type Zinc finger
was reported to have a high -sheet content (Miuraet al., 1998). In another study it was reported that
histidine residues play an important role in stabilizing the -helical structure when Zn2+ is introduced(Nomura and Sugiura, 2003). However, GATA-2
C-terminal zinc finger is a (Cys)4 type zinc finger,
which has no histidine residues. This might be oneof the reasons for the low helical content.
In summary, CD experiments demonstratethat GATA-2 C-finger has 13% -helix, 36%-sheet and remaining random coil in its native
form. This study provides insight into a better

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Received December 8, 2009; accepted December 28, 2009
Corresponding author: Shunsuke Meshitsuka, PhD

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