Effects of zinc and other divalent metals on deoxyribonucleic acid binding and hormone-binding activity of human alpha 1 thyroid hormone receptor expressed in Escherichia coli.
Full-length human thyroid hormone receptor alpha 1 (hTR alpha 1) 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, 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 hTR alpha 1 molecule has a high affinity for Zn2+, and this metal coordination is essential for proper folding of TR protein into its native active structure.
Miyamoto
T
T
Department of Medicine, University of Chicago, Illinois 60637.
Sakurai
A
A
DeGroot
L J
LJ
D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent; D004247:DNA / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D014284:Triiodothyronine / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology*
D002413:Cations, Divalent; D006146:Guanidines; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D014284:Triiodothyronine; D004247:DNA; D004492:Edetic Acid; D015032:Zinc; D019791:Guanidine