0013-7227/91/1296-3027$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 6 Printed in U.S.A.

Effects of Zinc and Other Divalent Metals on Deoxyribonucleic Acid Binding and Hormone-Binding Activity of Human al Thyroid Hormone Receptor Expressed in Escherichia coli* TAKAHIDE MIYAMOTO, AKIHIRO SAKURAI, AND LESLIE J. D E G R O O T Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, Illinois 60637

ABSTRACT. Full-length human thyroid hormone receptor a l (hTRal) was expressed in Escherichia coli using a T7 expression system. While present in large amounts, the receptor was highly enriched in the insoluble fraction after cell lysis. We describe here the successful solubilization and refolding of the expressed receptor in a functional form in the presence of Zn2+. Using a DNA-cellulose binding assay and gel shift assay, we found that treatment of expressed receptor with 1 mM EDTA in the denaturing agent (5 M guanidine-HCl) results in the formation of aporeceptor that does not specifically recognize target DNA,

T

while it does retain T3-binding activity. This aporeceptor recovered DNA-binding activity by adding Zn2+ during refolding. Zinc-induced restoration of DNA-binding activity occurred in a dose- and time-dependent manner. Moreover, once recovered, this DNA-binding activity persisted without Zn2+, even in the presence of 1 mM EDTA. These data indicate that the hTRal molecule has a high affinity for Zn2+, and this metal coordination is essential for proper folding of TR protein into its native active structure. (.Endocrinology 129: 3027-3033,1991)

protein in E. coli using a T7 RNA polymerase system (14). In E. coli, as commonly found for other expressed proteins, the overexpressed TR protein accumulates intracellularly in an insoluble form as inclusion bodies (14). To recover biologically active receptor in high yield, the receptor protein in the inclusion bodies must be solubilized and correctly refolded. The DNA-binding domain of the glucocorticoid receptor expressed in E. coli requires Zn2+ or Cd2+ for specific binding (15), in common with other v-er6A-related nuclear receptors. We report here successful solubilization and an essential metal coordination for proper folding of the TR molecule expressed in E. coli.

HYROID hormone, T3, has profound effects on growth, development, and metabolism, which are mediated by binding to nuclear thyroid hormone receptors (TR) (1). Recently, several isoforms of TR and TR variants have been isolated (2-6) and shown to be closely related to the oncogene v-erbA and to be members of a gene superfamily that includes steroid, retinoic acid, and vitamin D receptors (7). A near N-terminal region of these receptor molecules specifies DNA-binding activity and contains two sequence motifs that are believed to form fingers through coordination of Zn2+ to four cysteine residues (8, 9). TR enhances or represses transcription of thyroid hormone-responsive genes by binding to specific DNA sequences termed thyroid hormone response elements (TREs) through this domain (10,11). Since large quantities of TR protein of high purity are extremely difficult to obtain from tissues (12, 13), and the multiple forms of TR cannot be separated, a system that supplies a sufficient amount of homogenous TR protein is necessary for structural and functional analysis. For this purpose, we decided to express the TR

Materials and Methods Construction ofplasmid vector

Received June 4,1991. Address all correspondence and requests for reprints to: Leslie J. DeGroot, M.D., Thyroid Study Unit, Box 138, University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637. • This work was supported by USPHS Grants DK-13377 and DK27384, March of Dimes Birth Defects Foundation Grant 1-1166, the Boots Pharmaceutical Co., and the David Wiener Research Fund.

Using pET-3a, a T7 RNA polymerase-dependent expression vector, a nonfusion protein can be obtained by insertion of the cDNA to be expressed into the Ndel-BamHl site (14). The strategy for construction of the human TRal (hTRal) expression plasmid (pETal) is shown in Fig. 1. To introduce the Ndel and BarnHl sites into the 5' and 3' ends of the coding sequence of hTRal, respectively, pMe21, which contains the entire coding region of hTRal cDNA (4), was amplified by polymerase chain reaction (PCR) using the following synthetic oligonucleotide primers: A, 5'-TACATATGGAACAGAAGCCAAGCAA-3'; and B, 5'-CGGATCCTTTAGACTTCCT-

3027

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 01 November 2015. at 00:59 For personal use only. No other uses without permission. . All rights reserved.

EFFECTS OF DIVALENT METALS ON TR

3028 [Primer A] 5 1 - TACAT

hTRai cDNA

*>

-CEB>

[RTG}—

AAAA..

CCTAGGC-51

I

[Primer B]

PCR

PCR product

TAI&S TAW

Nde I

Endo«1991 Voll29»No6

or without 1 mM EDTA] for each gram of cells. After incubation on ice for 60 min, the solution was centrifuged for 30 min at 14,000 X g. The supernatant that contains receptor was saved, diluted 1:10 with refolding buffer (20 mM Tris-HCl, pH 8.0, containing 200 mM NaCl, 10% glycerol, 1 mM dithiothreitol (DTT), and 0-100 fiM ZnCl2), and incubated overnight on ice. The protein solution was cleared additionally by centrifugation at 14,000 X g for 30 min and dialyzed against the refolding buffer. This solution contains renatured hTRal. Western blotting

Proteins resolved by sodium dodecyl sulfate-polyacrylamide gel were electrophoretically transferred overnight at 30 V onto nitrocellulose sheets in 0.025 M Tris-0.193 M glycine buffer, pH 8.35, containing 20% methanol, using a Bio-Rad Trans Blot Cell (Richmond, CA). After transfer, the nitrocellulose sheets were washed with distilled water, stained with Ponceou red, and destained with 0.5 M NaCl and 10 mM Tris-Cl, pH 7.5, FIG. 1. Construction of hTRal expression vector pETal. The coding followed by 1-h incubation with blocking buffer [0.15 M NaCl region of hTRal was amplified by PCR, as described in Materials and and 10 mM Tris-Cl, pH 7.5 (TBS), containing 5% nonfat milk Methods, and inserted into the Ndel-BamHI site of pET-3a. The bold and 0.05% Tween-20] at 22 C. The nitrocellulose sheets were line denotes the amino acid coding region. The translation initiation then incubated with primary antibody at a final dilution of codon (ATG) and termination codon (TAA) are boxed. 1:250 in TBS containing 0.05% Tween-20 at 22 C for 1 h. After GATCCTCA-3'. Primer A contains the Ndel site (CATATG) washing with TBS-Tween-20 three times (10 min each), the followed by the coding strand sequence at the N-terminus of blots were immediately incubated with the secondary antibody hTRal. Primer B includes the BamHl site (GGATCC) and the (biotinylated goat antirabbit immunoglobulin G (Vector Labnoncoding strand sequence at the C-terminus of hTRal. One oratories, Burlingame, CA) in TBS-Tween-20 at 22 C for 1 h, hundred picomoles each of two primers were hybridized to 50 followed by washing three times (10 min each) with TBS. ng pMe21 and amplified for 40 cycles by the PCR using a Nitrocellulose sheets were then incubated with avidin-biotinPerkin-Elmer Cetus thermocycler (Norwalk, CT) (16). One peroxidase complex in TBS at 22 C for 1 h and washed with cycle consisted of denaturation for 1 min at 94 C, annealing for TBS three times (10 min each time). 4-Chloro-l-napthol (0.25 2 min at 55 C, and extension for 3 min at 72 C. After digestion mg/ml in TBS) was added in 0.005% H2O2 to visualize the with iVdel and BamHI, PCR products were inserted into the specific immunoglobulin G bound. Ndel-BamHI site of pET3a. The presence of the correct insert T3 binding assay was confirmed by dideoxy sequencing (17). This expression vector was expected to produce a full-length nonfused hTRal Assay of binding of [125I]T3 to hTRal was performed as protein. previously described (12). Thirty microliters of solubilized Expression and refolding of hTRal protein BL21 (DE3)-pLysS cells were transformed with pETal. These cells carry a stable integrant of the T7 RNA polymerase gene under control of the lac UV5 promoter (9). Cells were propagated in LB broth in the presence of 100 fig/ml ampicillin at 37 C. T7 RNA polymerase was induced when cell density reached an absorbance (600 nM) of 0.6-1.0 by the addition of isopropyl-jS-D-thiogalactoside (IPTG) to 1 mM, resulting in expression of hTRal protein. Three hours after induction, cells were collected by centrifugation and suspended in 3 ml lysis buffer [20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 200 mM NaCl, 1 mM phenylmethanesulfonylfluoride, and 0.27 mg/ml lysozyme] for each gram (wet weight) of cells. After incubation for 20 min on ice, deoxycholic acid was added to the suspension to a concentration of 1.5 mg/ml during continuous stirring. The material was then placed at 37 C for 30 min. The cell lysate was treated with DNase-I (50 fig/ml) for 30 min at 22 C and centrifuged at 12,000 X g for 15 min at 4 C. The pellet was washed three times with 9 vol lysis buffer containing 0.5% Triton X-100, and the pellet was suspended in 5 ml extraction buffer [20 mM Tris-HCl (pH 8.0) and 5 M guanidine-HCl with

hTRal were incubated with 50 pM [125I]T3 (DuPont-New England Nuclear, Boston, MA) in 300 fi\ 20 mM KPO4, pH 8.0, 50 mM KC1, 1 mM EDTA, 1 mM DTT, and 1 mM MgCl2 at 22 C for 2 h. Dowex anion exchange resin was used for separation of bound T 3 from free T 3 (18). Binding of hTRal to DNA DNA cellubse binding assay. Thirty microliters of solubilized hTRal were labeled as described using 50 pM [125I]T3. [125I]T3labeled hTRal was added to 10 /il DNA cellulose (Sigma Chemical Co., St. Louis, MO; 50% slurry) and incubated on ice for 1 h. Cellulose was collected by centrifugation at 3000 X g for 5 min and washed with 20 mM KPO4 (pH 8.0), 50 mM KC1, 0.5 mM EDTA, 1 mM MgCl2, and 1 mM DTT. The radioactivity of cellulose was counted, and receptor bound to DNA was calculated by subtracting nonspecific binding to an equivalent amount of cellulose without DNA. Gel shift assay. Gel shift assay was performed as described by Lavin et al. (19). Synthetic oligonucleotides representing each strand of a consensus palindromic TRE (5'-GATCCAGGTCATGACCTG-3') (20) were purified and annealed. Double

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 01 November 2015. at 00:59 For personal use only. No other uses without permission. . All rights reserved.

EFFECTS OF DIVALENT METALS ON TR stranded oligonucleotides were end labeled with [7-32P]ATP (>4000 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) by T4 polynucleotide kinase to a specific activity of 1 X 107 cpm/^g. Labeled oligonucleotides were separated from unincorporated nucleotides by centrifugation through a Sephadex G-50 column, which was equilibrated with 10 mM Tris-Cl (pH 8.0), 1 mM EDTA, and 150 mM NaCl. Refolded hTRal (20-30 fmol) was incubated with labeled DNA (1 X 104 cpm) and 1 ng poly(dldC) in 30 n\ 20 mM KPO4 (pH 7.5), 0.5 mM EDTA, 0.5 mM MgCl2, 5% glycerol, 1 mM DTT, and 50 mM KC1 for 20 min at 22 C. Protein-DNA complexes were separated from proteinfree DNA by nondenaturing electrophoresis in 5% polyacrylamide gels. Gels were run at 4 C in 50 mM Tris-boric acid, pH 8.0, and 1 mM EDTA, at a constant voltage of 200 V, dried, and exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) overnight.

Results

M

kD

3029 12

3 4 5 6 7

97-^ 68-*.f— ~+- hTRal

45 - •

30

-= H'

14-

B

1

2

3

4

5 6

7

Expression and solubilization of hTRal A protein with molecular mass of 46 kDa was produced (Fig. 2) when E. coli transformed with pETal was induced by 1 mM IPTG. Cell fractionation studies showed that the expressed hTRal was strongly enriched in the insoluble fraction and present in cytoplasmic inclusion bodies (Fig. 2A, lines 5 and 6). Immunoblotting was employed to confirm the expression of hTRal. a-17, which recognizes amino acid residue 17-33 (21), specifically reacted with the expressed protein (Fig. 2B). To solubilize expressed proteins at the highest efficiency, we compared three denaturing agents (guanidine-HCl, urea, and KC1) at various concentrations. Inclusion bodies were treated with those denaturants, and solubilized hTRal was refolded overnight, as described in Materials and Methods. Efficiency of solubilization was monitored by T3-binding activity of the refolded protein. As shown in Fig. 3, either 3 M guanidine-HCl, or 6 M urea was effective, while 3 M KC1 failed to solubilize the protein. Most of the hTRal still remained in the insoluble fraction after KC1 treatment, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown). Accordingly, we used 5 M guanidine-HCl for solubilization in subsequent studies. Guanidine-HCl (5 M) solubilized 20-50% of hTRal from the inclusion bodies (Fig. 2). By washing the inclusion bodies with buffer containing 0.5% Triton X-100 before extraction, more than 70% purity of hTR was obtained (Fig. 2A, lane 7). We recovered 1-5 mg hTRal from 1 liter E. coli culture. The expressed hTRal bound T 3 with an association constant (Ka) of 6.1 ± 2.6 X 109 M"1 (mean ± SD; n = 7), which is similar to the reported affinity constant (4.26). The maximal binding capacity was 1.5 nmol/mg protein in the best preparation, although it varied among preparations.

hTRal

FIG. 2. Expression of hTRal in E. coli. Cells bearing pET-3a and pETal were grown and induced by IPTG and analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A, Coomassie bluestained gel of uninduced (-) and induced (+) cultures of pET-3a (lanes 1 and 2) and pETal (lanes 3 and 4), soluble fraction (lane 5), insoluble fraction (lane 6) from induced pETal culture, and solubilized inclusion body (lane 7). Protein standards are shown by the arrows on the left. T7 represents the position of T7 RNA polymerase. M, Mol wt markers. B, Western immunoblot analysis of expressed protein. The same samples used in A were blotted onto a nitrocellulose filter and immunostained with hTRa-specific antibody a-17. Lane numbers are identical to those in A.

Time and Zn2+ dependency of refolding of the DNAbinding domain DNA cellulose binding assay was employed to evaluate the DNA-binding activity of refolded hTRal (Fig. 4A). When hTRal was solubilized in the absence of EDTA, refolded hTRal showed high DNA-binding activity regardless of the absence or presence of Zn2+. On the other hand, hTRal extract in the presence of 1 mM EDTA

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 01 November 2015. at 00:59 For personal use only. No other uses without permission. . All rights reserved.

EFFECTS OF DIVALENT METALS ON TR

3030 3.0

A

Endo• 1991 Voll29«No6

401

x E

Q. O

T Q 3